Lessons from Fungal F-Box Proteins · Dia2, Grr1, Hrt3, Mdm30, Rcy1, Ufo1, and ﬁve uncharacter-ized F-box proteins. In another study investigating the binding partners of Skp1 and

EUKARYOTIC CELL, May 2009, p. 677–695 Vol. 8, No. 51535-9778/09/$08.00�0 doi:10.1128/EC.00386-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

MINIREVIEWS

Lessons from Fungal F-Box Proteins�

Wilfried Jonkers and Martijn Rep*Plant Pathology, Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 318,

1098 SM Amsterdam, The Netherlands

The F-box domain, so called after a conserved domain foundin human cyclin F (5), was described in 1996 (6) after firstbeing denoted a conserved N-terminal domain found in a sub-set of proteins (110). The F-box hypothesis was introducedshortly after (162, 185) and holds that F-box-containing pro-teins (henceforth F-box proteins) act as scavengers in the cell,collecting “junk” proteins to deliver to a “waste processor,”called the SCF complex, to which they dock through theirF-box domain. In the SCF complex, the junk proteins aremarked with ubiquitin for “incineration” in the proteasome.F-box proteins do not act indiscriminately but recruit specific,often modified proteins to the SCF complex and in this wayregulate the level of certain proteins in a cell. F-box proteinsare found in all eukaryotes and display a large variety of func-tions. In fungi they are, for example, involved in control of thecell division cycle, glucose sensing, mitochondrial connectivity,and control of the circadian clock.

F-box proteins are commonly identified by the presence of astretch of primary sequence that matches the consensus for anF-box domain (Fig. 1). However, it can be questioned whetherjust the occurrence of an F-box domain in a protein sequenceis sufficient to assume compliance with the F-box hypothesis.The F-box hypothesis is based on the assumption that an F boxmediates assembly into an SCF complex through binding to theSkp1 subunit (Fig. 2). The SCF complex consists of Skp1 (sup-pressor of kinetochore protein mutant) (34), Cul1 (Cullin)(135), Rbx1 (ring-box protein) (86), and an F-box protein andcatalyzes (like other E3 ligases), in cooperation with the E1and E2 enzymes, the transfer of the small protein ubiquitin tothe target protein (108, 162). Among fungi, the methods ofregulation of the SCF complexes and hence the methods ofregulation of the F-box proteins in these complexes appearto differ. SCF complexes are likely activated and regulatedthrough a recycling mechanism (35), which involves three maincontributors: the neddylator protein DCN1, responsible for thetransfer of Nedd8 to Cul1 (112, 133, 155, 159, 174, 211); thedeneddylator CSN (COP9 signalosome) (136, 137, 146, 179,200) (reviewed in references 159, 201, and 208); and theCAND1 protein (127, 215), which binds to deneddylated Cul1and competes out the Skp1–F-box complex from the core ofthe SCF complex. A new round of neddylation removes

CAND1 and thereby creates binding space for a new Skp1–F-box complex. In budding yeast (Saccharomyces cerevisiae), de-letion mutants for Nedd8 and CSN5, the CSN subunit respon-sible for the deneddylation reaction, are both viable (36, 115,124). This means that, although the components of the SCFrecycling mechanism are present, this process is not requiredfor survival. A second difference in budding yeast in compar-ison to other fungi is that is does not have the CAND1 protein,adding to the notion that in budding yeast recycling actsdifferently. In fission yeast (Schizosaccharomyces pombe),CAND1 is present, and Nedd8 is required for survival (155),but the CSN5 subunit is not (145, 216). Apparently, in fissionyeast, the neddylation reaction is required for proper SCFfunction, but deneddylation is not, suggesting that in fissionyeast an alternative deneddylation may be present. In Neuros-pora crassa, a deletion mutant for subunit 2 of CSN, �csn-2, isviable but lacks a normal circadian rhythm and conidiation(64), while in Aspergillus nidulans, four CSN mutants, includingone for subunit 5 (�csne), all lack fruiting body development(25, 26). Together, these data suggest that, in filamentousfungi, proper recycling of the SCF is strictly required only forcertain developmental processes, in accordance with the re-quirements of CSN in development in more-complex, multi-cellular organisms (reviewed in reference 179).

Some F-box proteins appear to function without binding toSkp1, suggesting that not all F-box proteins take part in an SCFcomplex. This also means that not all proteins interacting withan F-box protein will be ubiquitinated and proteasomally de-graded. In another deviation from the F-box hypothesis, someF-box protein/Skp1 complexes do not seem to be involved inubiquitination. Furthermore, even when an F-box domain me-diates assembly into an SCF complex, the result may be self-ubiquitination rather than fulfillment of a scavenger function.

Since 1996, several review articles covering the emergingtheme of ubiquitin-mediated protein degradation and thewidespread occurrence of F-box proteins have been published(38, 68, 71, 72, 92, 118, 198, 204, 205). Here, we discuss fungalF-box proteins, including their targets (if identified), and whenpossible, classify these F-box proteins according to degree ofcompliance with the F-box hypothesis. Most literature on fun-gal F-box proteins covers those found in budding yeast and, toa lesser extent, fission yeast, but important findings have alsobeen reported for filamentous ascomycetes. In Table 1, fungalF-box proteins described in the literature are listed accordingto their main cellular function. The distantly related buddingand fission yeasts share 10 (likely) orthologous F-box proteins.Budding yeast contains an additional 11 F-box proteins and

* Corresponding author. Mailing address: Plant Pathology,Swammerdam Institute for Life Sciences, University of Amsterdam,Kruislaan 318, 1098 SM Amsterdam, The Netherlands. Phone: 31-20-5257764. Fax: 31-20-5257934. E-mail: [email protected].

� Published ahead of print on 13 March 2009.

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fission yeast 7 (89). Cdc4, Grr1, and Met30 from budding yeastand their counterparts in other fungi are the most studiedfungal F-box proteins and are conserved throughout the fungalkingdom. In total, 31 F-box proteins are discussed, exclusivelyfrom ascomycetes: the “model” fungi S. cerevisiae, S. pombe,Kluyveromyces lactis, A. nidulans, Hypocrea jecorina, and N.crassa and the pathogenic fungi Candida albicans, Fusariumgraminearum, Fusarium oxysporum, and Magnaporthe grisea.

F-BOX PROTEINS COMPLYING WITH THEF-BOX HYPOTHESIS

To date, the best-described fungal F-box proteins complywith the F-box hypothesis; the targets of these F-box proteinsare commonly first phosphorylated before being recognizedand ubiquitinated by the SCF complex and finally degraded bythe proteasome. In a study (113) in which interactors of Skp1and the SCF complex were identified in budding yeast, 13F-box proteins were found to bind Skp1, and these 13 could allbe copurified with an SCF complex. These are Cdc4, Ctf13,Dia2, Grr1, Hrt3, Mdm30, Rcy1, Ufo1, and five uncharacter-ized F-box proteins. In another study investigating the bindingpartners of Skp1 and Cul1 (181), Met30 and Saf1 were alsofound to bind Skp1, and two more uncharacterized F-box pro-teins were found to bind Skp1 and/or Cul1. In the study men-tioned above (113), autoubiquitination of F-box proteins wasalso investigated using two different E2 enzymes, Cdc34 andUbc4. Twelve out of the 13 F-box proteins showed self-ubiq-uitination; only Grr1 was found not to be ubiquitinated, andDia2 and Mdm30 showed very little ubiquitination. Also, it wasdemonstrated that these F-box proteins were differentiallyubiquitinated by the two different E2 enzymes and that differ-

ent numbers of ubiquitin molecules were attached to the F-boxproteins. In other reports, ubiquitination and degradation ofCdc4, Met30, and Grr1 were demonstrated (55, 217). It is stillan open question whether F-box proteins are ubiquitinatedand degraded together with their targets in each degradationround. Another possibility is that they are recycled after re-cruiting their targets and ubiquitinated and degraded onlywhen unbound to a target protein.

In a study with fission yeast, 11 F-box proteins investigated(Pop1/2, Pof1, Pof3, Pof5, Pof7, Pof8, Pof9, Pof10, Pof12,Pof13, and Fbh1/Pof15) (120) could all bind to Skp1. Theinteractions were further studied with a temperature-sensitivemutant of Skp1 with point mutations in the Skp1–F-box inter-action core. Only the binding to Pof1, Pof3, and Pof10 wasweaker with this mutant than with the wild-type Skp1. Theeffect of this weakened binding on the function of the individ-ual F-box protein is not known, and targets of most of thesefission F-box proteins remain to be identified.

C-TERMINAL PROTEIN-PROTEININTERACTION DOMAINS

Most of the fungal F-box proteins that comply with theF-box hypothesis have a recognizable C-terminal protein-pro-tein interaction domain (Table 1). Four F-box proteins carry aWD40 domain: Cdc4 (and its orthologs), Fwd1, Ufo1, andMet30 (and its orthologs). WD40 is a domain of about 40amino acids often terminating with the two amino acids Trpand Asp (WD) and forms a beta-propeller structure (150).Grr1 and its orthologs carry an LRR (leucine-rich repeat)domain (85), a repeat of about 25 amino acids forming anonglobular, crescent-shaped structure. Saf1 carries an RCC1(regulator of chromosome condensation 1) repeat (168), an-other domain forming a beta-propeller structure involved inprotein-protein interactions. Of the F-box proteins that complywith the F-box hypothesis, only Mdm30 does not contain anyknown protein-protein interaction motif, and it is unknownhow it interacts with its targets Fzo1, Mdm34, and Gal4c. Thepresence of a recognizable protein-protein interaction domainmight be an indication that an F-box protein complies with theF-box hypothesis. One of the uncharacterized budding yeastF-box proteins (Ylr352w) that binds Skp1 and Cul1 also con-tains an LRR domain, suggesting that this F-box protein mightalso comply with the F-box hypothesis.

For Cdc4, Dia2, Grr1, Met30, Mdm30, Saf1, and Ufo1 of bud-ding yeast, one or more targets are known, and most of thesetargets are degraded via the SCF complex. This suggests that atleast these seven F-box proteins completely fulfill the F-box hy-pothesis. For Cdc4, Dia2, Grr1, and Met30, homologs in other

FIG. 1. F-box consensus sequence. The motif is about 45 amino acids long and based on the HMM logo for the F-box motif (178). Highlyconserved amino acids are underlined, and the two most conserved amino residues, the leucine and proline at positions 6 and 7, respectively, areindicated in red. At each position, amino acids are ordered from top to bottom by decreasing occurrence in F-box domains.

FIG. 2. SCF complex and ubiquitination of target proteins. TheSCF complex functions within the ubiquitination reaction throughcombined action with the E1 and E2 enzymes. F-box proteins bind toSkp1 via their F-box domain and to targets via their C-terminal do-main, thereby presenting the target for ubiquitination. Ub, ubiquitin.

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fungal species have been found and characterized, in some casestogether with their targets. The degree of conservation of thefunctions of these F-box proteins between the different speciescan now be assessed by comparing the different phenotypes ofdeletion mutants and the conservation of targets.

Cdc4: AN F-BOX PROTEIN CONTROLLING THE CELLDIVISION CYCLE, MORPHOGENESIS, NUTRIENT

SENSING, AND CALCIUM SIGNALING

In S. cerevisiae, Cdc4 (cell division cycle 4) regulates multipleprocesses in the cell by recruiting various proteins for degra-dation (Fig. 3), and especially in the cell cycle process, Cdc4plays an important role by recruiting different cell cycle inhib-itors, a transcription factor, a cyclin, and a replication factor

for degradation. The CDC4 gene was first identified from ayeast mutant unable to initiate DNA replication during tran-sition from the G1 to the S phase (213).

Cdc4 AND CELL DIVISION CYCLE

Regulation of the transition from one cell cycle phase to thenext involves proteins that inhibit or promote progression.Proteins from both categories have to be degraded at somepoint, either to ensure progression or to prevent prematureinitiation of a new phase. Cdc4 is required for the degradationof Sic1 and Far1, proteins that inhibit cell cycle progression,and for the degradation of Cdc6, a protein that promotesprogression (97). Sic1 (substrate/subunit inhibitor of cyclin-dependent protein kinase 1) is phosphorylated by its inhibition

TABLE 1. Fungal F-box proteins described in the literature and discussed in this review

Cellular function(s) F-boxprotein Fungal species Additional motif(s) Target(s) Skp1 binding SCF

assembly

Cell division cycle, morphologicalswitch, and nutrient andcalcium sensing

Cdc4 S. cerevisiae WD40 Sic1, Swi5, Far1, Cdc6, Cbl6?,b

Tec1, Gcn4, Hac1, Rcn1,Ctf13

Yes Yes

Pop1 S. pombe WD40 Rum1, Cdc18, Cig2 Yes YesPop2 S. pombe WD40 Rum1, Cdc18, Cig2 Yes YesCdc4 C. albicans WD40 Sol1, Far1 ? ?

DNA replication Dia2 S. cerevisiae TPR � LRR Tec1 Yes YesPof3 S. pombe TPR � LRR ? Yes

Cell division cycle, glucoseuptake, growth on nonglucosecarbon sources, and retrograde

Grr1 S. cerevisiae LRR Cln1/2, Gic2, Ime2, Hof1, Std1,Mth1, Gis4, Pfk27, Tye7,Gala/b, Msk1

Yes Yes

signaling Grr1 K. lactis LRR Sms1 ? ?Grr1 C. albicans LRR Ccn1, Cln1, Hof1 ? ?GrrA A. nidulans LRR ? ? ?Fbp1 F. graminearum LRR ? Yes ?Pth1 M. grisea LRR ? ? ?

Methylmercury resistance Hrt3 S. cerevisiae ? Yes YesMitochondrial function Mdm30 S. cerevisiae Fzo1, Gal4c, Mdm34 Yes YesQuiescence Saf1 S. cerevisiae RCC1 Aah1, Ura7? Yes YesGenome stability Ufo1 S. cerevisiae WD40 � UIM Ho, Rad30 Yes YesCircadian clock Fwd1 N. crassa WD40 Frq Yes Yes

Sulfur metabolism Met30 S. cerevisiae WD40 Met4 Yes YesPof1 S. pombe WD40 Zip1 Yes ?Scon2 N. crassa WD40 Cys3 Yes ?SconB A. nidulans WD40 MetR Yes ?Lim1 H. jecorina WD40 ? ? ?

DNA repair Fbh1 S. pombe Helicase ? Yes ?Root infection Frp1 F. oxysporum ? Yes ?Peroxide resistance Pof14 S. pombe Erg9 (inhibited, not degraded) Yes ?Kinetochore assembly Ctf13 S. cerevisiae ? Yes No

Membrane trafficking Rcy1 S. cerevisiae SEC10 � CAAXa Snc1, Kex2 (both recycled, notdegraded)

Yes Yes

Pof6 S. pombe SEC10 � CAAX Sip1 (not recycled or degraded) Yes NoDNA repair Ela1 S. cerevisiae Elongin A �

coiled coilRpb1 No No

Mitochondrial function Mfb1 S. cerevisiae ? ? ?Exit from mitosis Amn1 S. cerevisiae LRR Not in insect

cells?

a SEC10, a domain of approximately 650 residues long found in proteins of the eukaryotic exocyst complex, which specifically affects the synthesis and delivery ofsecretory and basolateral plasma membrane proteins (126); CAAX motif, a C-terminal prenylation motif (where C is the prenylated cysteine, A is usually aliphatic,and X may be many different residues) (3).

b ?, unknown or uncertain.

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target, Cdc28, and another kinase, Pho85 (153, 209). Phos-phorylated Sic1 is recognized by Cdc4 and marked for degra-dation by polyubiquitination (49, 197). Recently, the transcrip-tion factor Swi5, which activates transcription of SIC1 (93), wasalso found to be degraded through interaction with Cdc4. Deg-radation of the transcription factor Swi5 via Cdc4 during theearly G1 phase allows efficient removal of Sic1 in the late G1

phase (93). This means that Cdc4 is responsible for Sic1 re-moval by degrading both the protein itself and the activator ofits transcription. Through Sic1 degradation, Cdc4 also regu-lates expression of OCH1, a gene encoding alpha-1,6-manno-syltransferase, suggesting that Cdc4 is involved in regulation ofcell wall composition during the cell cycle (40). Far1 (factorarrest 1) is also phosphorylated by the Cdc28 kinase complexand then recognized by Cdc4 (67). Degradation of Far1 isnucleus specific, suggesting that Cdc4 may act specifically inthe nucleus (20). Cdc6 (cell division cycle 6) is a DNA repli-cation initiation factor that is degraded via Cdc4 in the lateG1/early S phase as well as in the G2/M phase. Phosphorylationof Cdc6 to ensure recognition by Cdc4 at both time pointsrequires the Cdc28 kinase. Cdc6 degradations at these twotime points differ in the degradation rates and in the cyclinsthat take part in the Cdc28 kinase complex (42). The firstdifference is probably due to the fact that degradation dependson two different interaction domains in Cdc4 (163). It has alsobeen suggested that Cdc4 has a role in the degradation of Clb6(79), a cyclin that triggers, together with Clb5, the progressionfrom G1 into S phase. Clb6 is rapidly degraded at the end ofthe S phase and stabilized in cdc4 mutants. Moreover, itssequence harbors Cdc4 degron motifs. Direct interaction, how-

ever, has not yet been demonstrated. Cdc4 also targets anotherF-box protein involved in kinetochore assembly and function,Ctf13 (see Ctf13: a Kinetochore Assembly F-Box Protein). S.pombe contains two homologs of CDC4, called POP1 andPOP2 (polyploidy 1 and 2) (102). POP2 was also discovered inanother study, where it was called SUD1 (stops unwanted dip-loidization 1) (80). Pop1 and Pop2 are structurally related butfunction independently from each other. The phenotypes ofboth deletion mutants are comparable in that both displaypolyploidization, but neither protein cannot fully take over thefunction of the other since overexpression of POP1 or POP2could not suppress the defects caused by loss of the other gene(101). The polyploidization phenotype is caused by the accu-mulation of the cyclin-dependent kinase (CDK) inhibitorRum1 (80, 102) (homolog of budding yeast Sic1) and S-phaseregulator Cdc18 (80, 207) (homolog of budding yeast Cdc6).These two proteins are normally degraded during definedstages of the cell cycle, but in the pop1 and pop2 mutants andthe pop1 pop2 double mutant, the levels of these proteins arehigh compared to those in the wild type. The accumulation andpolyubiquitination of Rum1 and Cdc18 in a proteasome-defi-cient mutant support the notion that Pop1 and Pop2 recruitthese two proteins for degradation. Also, a direct interactionbetween Pop1 and Cdc18 was found using coimmunoprecipita-tion. Pop1 and Pop2 can form homodimers and heterodimers,resulting in three alternative SCF complexes, SCFPop1/Pop1,SCFPop1/Pop2, and SCFPop2/Pop2, but the different molecularfunctions of these three complexes remain unclear (101). TheS-phase cyclin Cig2 (homolog of budding yeast cyclin Cln2) isalso stabilized in pop1 and pop2 deletion mutants, suggesting a

FIG. 3. Overview of Cdc4 targets in S. cerevisiae. Negatively regulating targets are depicted in red, positively regulating targets in green, Ctf13in white, and transcription factors in blue. See the text for descriptions of the different targets.



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role for both proteins in Cig2 degradation (210). Coimmuno-precipitation revealed that Pop1 and Cig2 interact in the cellindependently from Pop2 and that this interaction requiresphosphorylation of Cig2 and at least the central 93 amino acidresidues (residues 181 to 273). In fungi, Pop1 and Pop2 arecurrently the only examples of two homologous F-box proteinsfunctioning in the same degradation pathway. The advantageof having two F-box proteins for the same function might bethat degradation of certain proteins can be fine-tuned andregulated at an extra level. The methods of degradation ofCig2 are remarkably different between budding and fissionyeasts, since Cln2 is degraded by Grr1 in budding yeast (seeGrr1 and the Cell Cycle) but its fission yeast homolog Cig2 isdegraded by Pop1 (the role of Pop2 in degradation of Cig2 isstill unclear).

Cdc4 AND PSEUDOHYPHAL GROWTH

Cdc4 is also involved in degradation of transcription factorsthat regulate pseudohyphal growth. The transcription factorTec1 (transposon enhancement control 1) is phosphorylated bymitogen-activated protein kinase Fus3 and then recognized byCdc4, promoting its degradation (32). Tec1 is responsible forthe onset of filamentous growth in S. cerevisiae. This morpho-logical switch is made when nutrient availability is low, but theswitch needs to revert after pheromone sensing to allow mat-ing. Whether Cdc4 is solely responsible for degradation ofTec1 is disputable, because it has been shown that anotherF-box protein, Dia2, is also able to induce degradation of Tec1after pheromone sensing (9). In the human pathogen C. albi-cans, Cdc4 plays a role in the switch from hyphal to yeastlikegrowth, as demonstrated by deletion of CDC4, which results inconstitutive hyphal growth (4). This dimorphic switch is impor-tant for pathogenicity, as the hyphal form contributes to theability to penetrate the body and cause candidemia. In contrastto that of budding yeast, the CDC4 deletion mutant of C.albicans is viable and does not display an arrest in the G1

phase. Possibly, Cdc4 in C. albicans has fewer targets than itsbudding yeast counterpart or accumulation of the same targetsin a C. albicans �cdc4 mutant does not (fully) inhibit growth.Similarity between the Cdc4 proteins of the two yeasts is 18%for the first 300 amino acids and 48% from residues 360 to 743,which includes the F-box domain and the WD40 motif. Thetwo proteins indeed appear to have different functions, sinceCDC4 from C. albicans cannot complement the cdc4 strain ofbudding yeast (182). The constitutive hyphal growth phenotypeof the C. albicans cdc4 strain is not due to the accumulation ofC. albicans Far1, the homolog of the Cdc4 target Far1 inbudding yeast. Sol1, the closest homolog of Sic1 in C. albicans,is degraded via Cdc4, but high levels of Sol1 are not respon-sible for the constitutive hyphal growth of the cdc4 mutant.Another possible target of Cdc4 involved in filamentousgrowth is Tec1, but no elevated levels were observed in C.albicans cdc4 mutants (4). This means that the target of Cdc4in C. albicans whose removal is required for the dimorphicswitch has not yet been identified and could be different froma Cdc4 target in budding yeast.

Cdc4 AND GROWTH RESPONSES AFTERNUTRIENT SENSING

The transcription factors Hac1 (homologous to Atf/Creb1)and Gcn4 (general control nonderepressible 4) are both in-volved in the activation of unfolded-protein-responsive geneswhose products assist in the folding of proteins in the endo-plasmic reticulum (ER) lumen upon ER stress. Hac1, a basicleucine zipper transcription factor, is degraded in the nucleusvia Cdc4 when ER stress is removed (158). Gcn4 is also re-quired for the activation of transcription of amino acid andpurin biosynthesis genes during starvation. After the switchfrom poor to rich medium, Gcn4 is degraded via Cdc4, likelyafter being phosphorylated by Pho85 (139). The degree ofconservation of this Cdc4 function is unclear because the re-spective targets have not been studied in this respect for otherfungi.

Cdc4 AND CALCIUM SENSING

Cdc4 acts in calcium homeostasis by targeting Rcn1 fordestruction upon calcium availability (94). Rcn1 (regulator ofcalcineurin 1) inhibits calcineurin, a phosphatase that medi-ates cellular responses after stress and Ca2� uptake (41).Calcineurin mediates its own inhibition by a negative-feedbackloop: it stimulates the expression of RCN1 and stabilizes Rcn1by dephosphorylation. Phosphorylation of Rcn1 makes it rec-ognizable for Cdc4 and marks it for degradation, allowingcalcineurin to break out of its negative-feedback loop andincrease its activity.

From the details described above, it is clear that betweenyeasts, Cdc4 is conserved in some functions, like the degrada-tion of the CDK inhibitors Sic1 and Far1. Whether the func-tion of Cdc4 in regulation of pseudohyphal growth is conservedbetween budding yeast and C. albicans is uncertain, since thetarget protein in this pathway in C. albicans has not yet beenfound. The involvement of Cdc4 in nutrient sensing and cal-cium signaling in filamentous fungi is unlikely, since deletion ofCDC4 in these fungi has not been reported to lead to defectsin these processes. A main difference between budding yeastand the other fungi is that in budding yeast the search fortargets has been more intensive, for example, by use of yeasttwo-hybrid screens (93). Application of such screens to otherfungi would be helpful to more fully evaluate the conservationof targets between the different fungi.

Although no genetic studies of the Cdc4 homolog in N.crassa have been reported, this homolog was found to be tar-geted by a plant-defensive peptide. By use of a yeast two-hybrid screen, defensin 1 from Pisum sativum was shown tointeract specifically with Cdc4 (131). Defensins are plant pep-tides exhibiting an antifungal activity as part of the plant innateimmune system. Interaction with Cdc4 can explain how defen-sin 1 inhibits fungal growth, namely, by interference with thefungal cell cycle. By use of microscopy, it was observed thatdefensin 1, tagged with a fluorophore, was localized in thenuclei of N. crassa and Fusarium solani, suggesting that defen-sin 1 can enter the fungal cell and interfere in the nucleus withthe cell division cycle.



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Dia2: AN F-BOX PROTEIN INVOLVED INDNA REPLICATION

The F-box protein Dia2 (digs into agar 2) plays a role inDNA replication in S. cerevisiae and is thereby also involved incell growth and division. As mentioned earlier, Tec1, a tran-scription factor regulating filamentation genes, is degraded viaDia2 (9), probably in joint action with Cdc4 (32). These twoF-box proteins are also both capable of degrading ectopicallyexpressed human cyclin E (99), even though they bear differentprotein-protein interaction domains (LRR and WD40, respec-tively). Deletion of DIA2 in budding yeast causes a defect ininvasive and pseudohyphal growth, slower growth at low tem-peratures, early entry into the S phase, and accumulation ofDNA damage (98). These defects were also observed in DIA2�F-box mutants, suggesting that binding of Dia2 to Skp1 isnecessary for these functions. Dia2 binds both early- and late-firing origins and is thereby involved in resetting the origin. Itrecruits the SCF complex to the replication origins, suggestingthat a possible target becomes ubiquitinated there. Yra1, pre-viously described as a protein involved in mRNA export (189),is an interaction partner of Dia2 and is required for Dia2function at replication origins (191). Possibly, the SCFDia2

complex binds origins with the assistance of Yra1. In anotherstudy (18), deletion of DIA2 resulted in accumulation of DNAdamage after the collapse of replication forks. This suggeststhat a possible target of Dia2 may be found among proteinsthat interfere with replication fork stability in certain genomicregions. Also, genetic interactions of Dia2 were found withDNA replication, repair, and checkpoint pathways (18). A rolefor Dia2 in DNA repair was suggested as well by the require-ment of Dia2 for resistance to certain DNA-damaging com-pounds. These observations indicate that there are likely moretargets or functions of Dia2 than only targeting Tec1 for deg-radation.

Pof3 from S. pombe is the ortholog of Dia2 from buddingyeast. Deletion of POF3 results in multiple phenotypes: G2-phase delay (probably due to activation of the DNA damagecheckpoints), hypersensitivity to UV radiation, telomere dys-function, and chromosome instability and segregation defects(89). Targets of Pof3 are not yet known but may be foundamong proteins playing a role in chromatin structure and/orfunction. Fission yeast does not have a Tec1 ortholog, so tar-gets different from Tec1 must be responsible for the phenotypeof the deletion mutant. A protein that was found to interactwith Pof3 is Mcl1, ortholog of the budding yeast S-phase reg-ulator Ctf4 (134). Mcl1 is a protein essential for chromosomemaintenance and contains WD40 repeats and SepB boxes (100,206). A �mcl1 strain shows phenotypes similar to those of the�pof3 mutant. Normally, Mcl1 is not rapidly degraded in wild-type cells, and no ubiquitination of Mcl3 could be demon-strated, suggesting that Mcl1 is not a target of Pof3. This is alsoin accordance with the fact that the two deletion mutants sharethe same phenotype, something that would not be expected ifMcl1 were a target of Pof3.

Dia2 is conserved not only in fission yeast but also in fila-mentous fungi, suggesting a well-conserved function (BLASTsearches and our observations). It would be worthwhile toinvestigate whether and how Dia2 regulates DNA replicationin filamentous fungi.

Grr1: AN F-BOX PROTEIN INVOLVED IN GLUCOSE ANDAMINO ACID SENSING, CELL DIVISION CYCLE,

MEIOSIS, AND RETROGRADE SIGNALING

Grr1 (glucose repression resistant 1) in S. cerevisiae plays arole in a large number of cellular processes: retrograde signal-ing, pheromone sensitivity and cell cycle regulation, nutrition-ally controlled transcription, glucose sensing, and cytokinesis(122) (Fig. 4). Grr1 was initially found in budding yeastthrough a mutation causing resistance to glucose repression,with a deletion mutant additionally showing growth defects(7, 52).

Grr1 AND THE CELL CYCLE

That Grr1 is involved in cell cycle control was shown by theaccumulation of the cyclins Cln1 and Cln2 in a GRR1 deletionmutant (12, 95, 116, 177, 180, 186). Degradation of the cyclinsCln1 and Cln2 via Grr1 is required after completion of the G1

phase or when cells have to arrest in G1, for instance, afterpheromone sensing. Binding of Grr1 to Cln2 has been estab-lished in multiple ways, but binding to Cln1 has never beendetected, suggesting that Grr1 might target Cln1 indirectly.Another target of Grr1 is Gic2, a protein that accumulatesthroughout the G1 phase and reaches its peak just before budemergence. At that time, Cdc42, a Rho-related GTP-bindingprotein required for polarized growth of the cytoskeleton dur-ing bud emergence, is activated and binds Gic2. When the budhas emerged, polarized growth ceases and Gic2 is degraded toavoid morphological defects. Only Gic2 bound to Cdc42 canbe phosphorylated and eventually recognized by Grr1 (81).During cytokinesis, the process of cell separation, Grr1 isresponsible for the degradation of Hof1. Hof1 first forms aring around the bud neck of the mother cell and then formsanother ring in the daughter cell. Just after septum forma-tion and separation, Hof1 normally disappears (196). Grr1 isrecruited to the mother bud neck and binds to Hof1 afterthe activation of the mitotic exit network (19). This suggeststhat Grr1 not only is active in the nucleus and cytoplasm butalso can be recruited to specific cellular structures. The Grr1protein of C. albicans is 46% identical to Grr1 from buddingyeast, and C. albicans GRR1 can fully complement a yeast�grr1 strain. A �grr1 strain of C. albicans exhibits pseudohy-phal growth under yeastlike growth-inducing conditions anddoes not grow on glucose (123). The constitutive pseudohy-phal growth phenotype (27) of the �grr1 deletion straincould be explained by the stabilization of the two G1 cyclinsCcn1 and Cln3. That Grr1 mediates degradation of Ccn1and Cln3 was demonstrated by the fact that both cyclins arestabilized; additionally, Cln3 was found as a hyperphosphor-ylated protein in the �grr1 strain. Elevated levels of Hof1were also detected in the �grr1 strain. These data suggestthat Grr1 function in degradation of cyclins and Hof1, aswell as glucose uptake, is conserved between the two yeastspecies. The fact that the genes from C. albicans can func-tionally replace budding yeast GRR1 suggests that interac-tions of Grr1 with its targets are conserved between the twoyeasts.



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Grr1 AND MEIOSIS

Generally, the cell cycle is closely connected to the availabil-ity of nutrients. In low-glucose medium, diploid cells tend toundergo meiosis and sporulation rather than grow and divide.A role for Grr1 in preventing untimely meiosis and sporulationwas demonstrated in a study of the degradation of Ime2, aprotein kinase required for multiple steps throughout thesporulation process (166). In a �grr1 mutant, accumulation of(nonubiquitinated) Ime2 was found and meiosis still occurred,even under high-glucose conditions. In A. nidulans, GRRA, theortholog of budding yeast GRR1, was found in a subtractionhybridization screen aimed at identification of genes that arespecifically expressed during fruiting body development (107).GRRA is able to complement the �grr1 phenotype in yeastpartially or, when the gene is overexpressed, almost fully. Com-plemented phenotypes of the yeast deletion mutant include themorphological abnormalities and changes in gene expressionupon a carbon source shift (107). This demonstrated that GrrAfrom A. nidulans is probably able to bind endogenous targets inbudding yeast, suggesting that these interactions are still con-served within GrrA. However, the phenotype resulting fromdeletion of GRRA in A. nidulans is quite different from that ofthe yeast mutants. A. nidulans �grrA mutants showed impairedascosporogenesis, asexual conidiation, and sexual develop-ment, while displaying a normal vegetative growth. A similarphenotype was observed with CSN subunit mutants of A. nidu-lans (25, 26). This suggests that CSN may be involved in thefunctioning of GrrA in A. nidulans. From further cytologicalexamination, it was concluded that meiosis, giving rise to cro-

zierlike structures that contain diploid nuclei, does not takeplace in the grrA mutant. In striking contrast, meiosis doesoccur in the budding yeast grr1 mutant, even under meiosis-suppressing conditions. In light of this, it would be interestingto investigate whether and how the Ime2 ortholog in A. nidu-lans is involved in GrrA-controlled sexual development. In theplant-pathogenic fungus F. graminearum (Gibberella zeae), theortholog of budding yeast Grr1, denoted Fbp1, was found in arestriction enzyme-mediated insertion screen for nonpatho-genic mutants (60). The virulence of fbp1 mutants on barleyheads was severely reduced compared to that of the wild type,and growth on potato dextrose agar and carrot agar producedless mycelium. Furthermore, Fbp1 plays a role in sexual repro-duction. FBP1 deletion caused a loss of perithecium formationas females in self-crosses and smaller and fewer perithecia as amale in the outcross. The asci contained incomplete octads ofabnormal spores and did not segregate in a one-to-one man-ner. Deletion constructs lacking the F box, the LRR, or bothdomains were nonfunctional both in the interaction with F.graminearum Skp1 and yeast Skp1 and in the ability to com-plement the sexual reproduction deficiency. Clearly, also in F.graminearum, protein turnover is required for sexual reproduc-tion, but whether the ortholog of budding yeast Ime2 is in-volved is not known. Grr1 from budding yeast was unable tocomplement the knockout phenotype. Conversely, FBP1 fromF. graminearum was able to partially complement the yeast grr1mutant. This suggests that during evolution, Grr1 has retainedthe ability to bind at least some heterologous targets, despitetheir diversification. Grr1 is likely conserved as a pathogenicity

FIG. 4. Overview of the Grr1 targets in S. cerevisiae. Negatively regulating targets are depicted in red, positively regulating targets in green, andtranscription factors in blue. See the text for descriptions of the different targets. Ub, ubiquitin.



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factor in plant-pathogenic fungi. In a screen for pathogenicitygenes of M. grisea using insertional mutagenesis, one mutanthad a disruption in a GRR1 ortholog, PTH1 (pathogenicity 1),and a subsequent deletion of this gene resulted in reduceddisease symptoms toward barley (192). Why Grr1 is requiredfor full pathogenicity in this fungus is not known.

Grr1 IN GROWTH ON GLUCOSE AND NONGLUCOSECARBON SOURCES

In addition to regulating meiosis, Grr1 from S. cerevisiaeconducts other glucose availability-related functions. Whenhigh levels of glucose are sensed, Grr1 not only initiates thedegradation of Ime2 but also activates hexose permeases(HXT) that allow the rapid import of glucose. Activation ofHXT expression is achieved by the degradation of Std1 andMth1, which promote the repression of HXT genes by bindingto the repressor Rtg1. Upon glucose sensing, Std1 and Mth1are phosphorylated, recognized by Grr1, and degraded. Free,unbound Rtg1 can then be phosphorylated, promoting an in-tramolecular interaction in Rtg1 that prevents DNA binding(165), thereby releasing repression of the HXT genes (91).Although not yet fully understood, the process of Snf1 proteinkinase inactivation is also required for degradation of Std1 andMth1 (160). In K. lactis, Grr1 was characterized as an F-boxprotein required for glucose signaling, just as described forbudding yeast (70). Complementation of S. cerevisiae �grr1with K. lactis GRR1 showed full restoration of the growth andmorphological defects of the deletion strain, demonstratingthat GRR1 from K. lactis is a functional homolog of buddingyeast GRR1. It was also shown that K. lactis GRR1 controls thelevels of Sms1, the single ortholog of Mth1 and Std1, thebudding yeast Grr1 targets. The Sms1 level decreased dramat-ically after glucose addition, suggesting rapid degradation ofSms1 to allow expression of the hexose transporter genes.Other targets of Grr1 in K. lactis have not yet been found, butas the complementation of S. cerevisiae �grr1 with K. lactisGRR1 shows, Grr1 from K. lactis is probably able to bindtargets in budding yeast. These targets also include Mth1 andStd1, suggesting that these interactions are still conserved,even though in K. lactis only Sms1 is present.

In addition to activating genes required for glucose uptake,Grr1 from S. cerevisiae is required for the assimilation of al-ternative carbon sources (52). Grr1 mediates this process byrecruiting Gis4, a target that is ubiquitinated but not degraded(117). The ubiquitinated form of Gis4 binds and activatesphosphorylated forms of Snf1, which results in derepression ofseveral genes required for the assimilation of alternative car-bon sources. Gis4 is a rare example of a target that is notdegraded after ubiquitination but is instead activated. Thisshows that although Grr1 function generally complies with theF-box hypothesis, ubiquitination of Gis4 is an exception to thisrule. Whether and how Gis4 is phosphorylated before recog-nition by Grr1 and how it is rescued from degradation afteraddition of ubiquitin are not known.

Grr1 also regulates other metabolic processes in the cellthrough its involvement in the degradation of Tye7 (131a) andPfk27 (15). Tye7 is a transcription factor that activates severalglycolytic genes (152), and Pfk27 synthesizes the second mes-senger fructose-2,6-biphosphate (154). After glucose deple-

tion, the removal of these proteins via Grr1 probably facilitatesthe switch from glycolysis to gluconeogenesis. This shows thatGrr1 is active not only during glucose availability but alsoduring glucose depletion.

Furthermore, together with Mdm30, another F-box protein,Grr1 regulates the activation of the Gal4 transcription activa-tion complex. This complex regulates the transcription of genesinvolved in galactose assimilation. Degradation of the Gal4isoforms Gal4a and Gal4b via Grr1 is required when glucosebecomes available and galactose assimilation is shut down(147). This was demonstrated by deletion of GRR1, whichresults in stabilization of Gal4a/b and increased activation ofGal4 targets.

Grr1 AND AMINO ACID SENSING

Grr1 also plays a role in amino acid sensing by promotingthe expression of several amino acid permease genes uponamino acid availability (17, 77). The activation of amino acidpermease genes is mediated by the transcription factors Stp1and Stp2. These two proteins are cleaved after activation of thePtr3/Ssy5 amino acid sensing pathway and transported to thenucleus (130). Stp1 cleavage depends on Grr1, suggesting thatGrr1 targets a protein that normally inhibits cleavage. A can-didate might be Ssy5, which is involved in the amino acidpermease expression pathway and which is normally degradedupon amino acid availability. On the other hand, higher pro-tein levels of Stp2 were found in �grr1 cells than in wild-typeGRR1 cells (15).

Grr1 AND RETROGRADE SIGNALING

Mitochondrial retrograde signaling (RTG) is a pathway con-necting mitochondria to the nucleus, allowing cells to react tochanges in the functional state of mitochondria. The RTGpathway targets two transcription factors, Rtg1 and Rtg3.These two proteins form heterodimers and activate RTG-re-sponsive genes (128). Grr1 functions in this pathway by deg-radation of Msk1, a negative regulator that inhibits localizationof Rtg1 and Rtg3 to the nucleus (129). Grr1 targets Mks1 onlywhen it is unbound to either Rtg2 or Bmh1, a 14-3-3 protein.When the RTG pathway is off, Bmh1 protects Mks1 and allowsit to inhibit Rtg1 and Rtg3. When the pathway is on, Mks1instead binds to Rtg2 and is thereby inactivated. Degradationof free Mks1 via Grr1 ensures that the switch is quick andunder tight control.

Grr1 seems to be conserved among yeasts, considering thecell cycle and glucose uptake. Regarding meiosis, however, therole of Grr1 in budding yeast and the role of Grr1 in filamen-tous fungi seem opposite of each other. Another differencebetween Grr1 in yeasts and Grr1 in filamentous fungi is in-volvement in glucose uptake, since, for example, Mig1 andSnf1 repression in filamentous fungi is different from that inbudding yeast (28, 172). Regarding other functions, like aminoacid sensing and retrograde signaling, hardly anything is knownabout Grr1 involvement in other fungi, partly because of a lackof knowledge but probably also partly because these processesare differently regulated. Once again, fewer studies to findGrr1 targets have been carried out for other fungi than forbudding yeast (15).



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Hrt3: AN F-BOX PROTEIN ENHANCINGMETHYLMERCURY RESISTANCE

Hrt3 (high-level expression reduces Ty3 transposition) andYlr224w in S. cerevisiae both promote resistance to methylmer-cury, a highly toxic compound (75). Overproduction of thesetwo F-box proteins elevated resistance to the toxic compound,in contrast to 15 other F-box proteins studied. This resistancerequired the F-box domain of these two proteins and also theproteasome, suggesting that degradation of a target protein isinvolved. Targets of Hrt3 or Ylr224w that could explain theroles of these F-box proteins in methylmercury resistance havenot yet been identified. Interactions of Hrt3 other than withubiquitin conjugation proteins were with alcohol dehydroge-nase (Adh2) and Idh1, a subunit of mitochondrial NAD�-dependent isocitrate dehydrogenase, which catalyzes the oxi-dation of isocitrate to alpha-ketoglutarate in the tricarboxylicacid cycle (73, 109). The biological relevance of the interactionwith these two catabolism-related proteins is not clear, but theymight be involved in sensitivity to methylmercury. Other inter-actions with ribosomal proteins Rpl12A and Guf1 and a phos-phatase functioning in the G1/S-phase transition were found.

Hrt3 is conserved in the entire fungal kingdom (BLASTsearches and our observations), suggesting that it serves afundamental function in fungi. Still, its characterization is lim-ited; only the overexpressing phenotype was investigated for S.cerevisiae. Investigation of a (conditional) deletion mutant of S.cerevisiae or other fungi will be crucial to further explore thefunctions of Hrt3.

Mdm30: A MITOCHONDRION-ASSOCIATEDF-BOX PROTEIN

Mdm30 (mitochondrial distribution and morphology) andMfb1 (mitochondrion-associated F-box protein) in S. cerevisiaeboth control membrane fusion dynamics of mitochondria. Themembranes of mitochondria continuously undergo fusion andfission to maintain a dynamic morphology. A target of Mdm30is Fzo1 (mitofusin), a membrane-bound GTPase involved inmembrane fusion. Fzo1 is ubiquitinated and targeted to theproteasome in an Mdm30-controlled manner (33). Anothertarget of Mdm30 is Mdm34, a mitochondrial outer membraneprotein (157). Interaction between Mdm30 and Mdm34 is es-sential for growth on nonfermentable carbon sources and fornormal mitochondrial morphology. If and how ubiquitinationof Mdm34 contributes to these functions are not yet under-stood.

Additionally, Mdm30 (alternatively called Dsg1 [does some-thing to Gal4]) is required for destruction of Gal4c, the inhib-itory isoform of Gal4, and thereby plays a role in carbon as-similation (147) together with Grr1, which is required for thedegradation of Gal4a/b, the Gal4 active isoforms. A �dsg1strain shows elevated levels of Gal4c, which results in theinability to use galactose as a carbon source.

Mdm30 binds to Skp1 via its F-box domain, and these twoproteins together with other components of the SCF complexparticipate in Fzo1 degradation (33). This means that Mdm30,in contrast to earlier views (68, 71), can be part of an SCFcomplex and conforms to the F-box hypothesis. Mdm30 is notconserved in other fungi, but insight into how mitochondrial

morphology is regulated by F-box proteins in S. cerevisiae isvaluable, as in other fungi alternative F-box proteins or at leastubiquitination and protein turnover could also be involved inthis intriguing process.

Saf1: AN F-BOX PROTEIN INVOLVED IN ENTRYINTO QUIESCENCE

Saf1 (SCF-associated factor 1) is an F-box protein requiredfor the degradation of adenine deaminase 1 (Aah1) in S. cer-evisiae. A microarray study showed clear AAH1 downregula-tion during the shift from proliferation to quiescence (47, 48).Quiescence, also known as the stationary phase, is a state thatyeast cells enter when nutrients are limiting. A SAF1 deletionmutant showed no downregulation of AAH1 expression, andstabilized protein levels of Aah1 were detected upon entry intoquiescence. Degradation of Aah1 relies on Saf1, Skp1, and theproteasome and is dependent on the interaction between Saf1and Skp1 via the F-box domain of Saf1. Saf1 interacts in a yeasttwo-hybrid experiment with both Aah1 and Skp1. Loss or mu-tation of the F-box domain of Saf1 abolished the interactionwith Skp1 but not with Aah1, although the latter interactionwas slightly weakened. Mutation of the lysine at position 329 ofAah1 did not affect the interaction with Saf1 but increased thestability of Aah1, suggesting that this lysine might be the ubiq-uitination site. Other targets of Saf1 are not yet known, but acandidate might be Ura7, a protein that is present at reducedlevels in SAF1-overexpressing strains and is stabilized in saf1strains (36). Curiously, although the known target(s) of Saf1 isconserved among fungi, Saf1 itself is not. In fungi other thenbudding yeast, degradation of these Saf1-targeted proteinsmight not be required upon quiescence or the proteins mightbe turned over in a different manner.

Ufo1: AN F-BOX PROTEIN INVOLVED IN DNADAMAGE RESPONSE

In S. cerevisiae, Ufo1 (UV-F box-HO 1) targets the endonu-clease Ho for proteasomal degradation and functions in ge-nome stability and in response to DNA damage (88). AfterDNA damage, the MEC1/RAD9/CHK1 pathway phosphoryl-ates Ho, stimulating its recognition and degradation. Ufo1itself is also degraded via self-ubiquitination. This ubiquitina-tion reaction is mediated by the ubiquitin interaction motifs(UIMs) in the C terminus of Ufo1 that bind during assembly inthe SCF complex to Ddi1, a protein containing ubiquitinlike(UBL) and ubiquitin-associated (UBA) domains (78). Re-moval of the UIM domain in Ufo1 (Ufo1�uim) stabilizes theprotein and inhibits the degradation of other proteins normallydegraded by SCF complexes. It therefore seems that Ufo1�uimmay prevent assembly of other F-box proteins into an SCFcomplex.

Ufo1 also appears to regulate the degradation of Rad30,since that protein is stabilized in proteasome mutants and incells lacking Skp1 or Ufo1. Direct interaction between Ufo1and Rad30 has, however, not yet been demonstrated. Rad30 isa polymerase eta necessary for DNA replication near damagedDNA (184) and is removed again after replication because ofits high error frequency.

Recently, a study of the interactome of green fluorescent



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protein (GFP)-labeled Ufo1 identified new proteins takingpart in Ufo1 function (9a). The proteins interacting specificallywith GFP-Ufo1 and bearing PEST degrons—potential phos-phorylation sites that are often found in proteins targeted fordegradation (167)—are Rbp2, Spt5, Fas2, and Gip2. Rpb2 isan RNA polymerase II (Pol II) subunit (39), and Spt5 is aprotein that mediates both activation and inhibition of tran-scription elongation (125). Fas2 is a fatty acid synthetase com-ponent (142), and Gip2 is a putative regulatory subunit of theprotein phosphatase Glc7p, involved in glycogen metabolism(195). Whether these proteins are targets of Ufo1 is notknown. Ufo1 is not conserved in other fungi, suggesting thatthis type of regulation of the DNA damage response is re-stricted to (close relatives of) budding yeast.

Fwd1: AN F-BOX PROTEIN CONTROLLING THECIRCADIAN CLOCK

In N. crassa, Fwd1 (F-box protein containing a WD40 re-peat) was found to be involved in controlling the circadianclock via degradation of Frequency (Frq) (65, 66). Circadianclocks regulate a wide variety of physiological and molecularprocesses during oscillation between day and night. Besidesbeing regulated by Frq, the circadian clock in Neurospora isfurther regulated by light and controlled by the transcriptionfactors Wc-1 and Wc-2 (44). Frq inhibits its own transcriptionby inhibiting Wc-1 and Wc-2 (1, 2). When Frq is hyperphos-phorylated by CK1 and CKII (63), it is recognized by Fwd1 anddegraded. This releases Wc-1 and Wc-2 activity, leading to theproduction of new Frq.

The function of Fwd1 in the SCF complex is regulated by theCOP9 signalosome CSN. Disruption of a subunit of CSN im-paired the degradation of Frq, probably because reducedamounts of Fwd1 were present in the csn mutant: the half-lifeof Fwd1 is reduced from 6 to 9 h to 45 min, and other com-ponents of the SCF complex proved to be unstable. In a �csn-2mutant, SCF is constitutively neddylated, which enhances thedegradation rate of Fwd1. This degradation is probably inde-pendent of binding of Frq to Fwd1, resulting in reducedamounts of Fwd1 and impaired degradation of Frq1 (64). TheCSN-2 deletion mutant also exhibits slow growth and reducedproduction of aerial hyphae compared to levels for the wild-type CSN-2, suggesting that other F-box proteins might also beaffected. N. crassa is the main model organism for the investi-gation of circadian rhythms in fungi, and only for this fungushas Fwd1 been studied intensively. Nevertheless, this protein,as well as circadian rhythms, is present in other filamentousfungi (14, 114), as are homologs of N. crassa clock components,like Frq, WC-1, and WC-2 (132). However, in A. nidulans nohomolog of FRQ is present (57), even though an FWD1 ho-molog is present (our observations). This suggests that at leastin some fungi, Fwd1 has other targets.

Interestingly, in plants, involvement in rhythmic processeshas also been demonstrated for several F-box proteins, likeZEITLUPE, FKF1, and AFR (61, 151, 187), which play a rolein photocontrol of the circadian period, the circadian clock,and phytochrome A-mediated light signaling, respectively.

Met30: AN F-BOX PROTEIN INVOLVED INSULFUR METABOLISM

As described above, the F-box hypothesis states that thetargets of F-box protein are degraded after ubiquitination, butubiquitination of the Grr1 target Gis4 does not lead to degra-dation. Met30 is another example of an F-box protein whosetarget is not necessarily degraded. Met30 is an F-box proteinfrom S. cerevisiae that can recruit its target to the SCF fordegradation, but it can also activate its target by ubiquitinationwhen the target is assembled into a transcription activationcomplex.

A major Met30 target is Met4, a transcriptional activator ofthe sulfate assimilation pathway controlling MET and SAMgenes for uptake and biosynthesis of sulfur-containing com-pounds (194). Met4 is also required for cadmium tolerance byactivating the expression of genes involved in glutathione bio-synthesis (10, 21, 212).

The transcription of Met30 is regulated through a feedbackloop, as Met4 controls the activation of Met30 (171). The wayMet4 is regulated by Met30 has been under discussion forseveral years (23, 24, 83, 140, 141). A picture in which Met30regulates Met4 in multiple ways has emerged (30). First, Met30activates Met4 when low levels of methionine are available,resulting in the expression of MET and SAM genes. When,through MET and SAM activation, higher intracellular levels ofmethionine are obtained, the intracellular concentration ofcysteine also increases through the S-adenosyl-methionine andcysteine biosynthesis pathways. High levels of cysteine againlead to the inactivation of Met4 by Met30. The alternativeactivation and inactivation of Met4 by Met30 are explained ina two-step model. In the inactive state, dimerization of Met4causes low interaction with cofactors, leading to intermediateexpression of MET and SAM genes. Met30 relieves this dimer-ization through degradation of one of the dimerized Met4proteins, leaving the other Met4 subunit free to assemble intoan activation complex, thus triggering expression of the METand SAM genes (step 1). When higher levels of sulfur-contain-ing amino acids are present, Met30 binds to Met4 in the as-sembled promoter complex, leading to Met4 ubiquitination(step 2); this ubiquitinated promoter complex represses tran-scription of the MET and SAM genes. Eventually, Met4 isdegraded and the complex disassembles, making space for newcomplexes to form on the promoter when levels of sulfur-containing amino acids are low again.

In S. pombe, the Met30 homolog Pof1 is an essential proteinthat targets the Met4 homolog Zip1 (a basic leucine zipper)(62). Like in S. cerevisiae, Zip1 mediates cadmium tolerance byactivation of cadmium response genes. Regulation of Met4 byMet30 and regulation of Zip1 by Pof1 show similar patterns.However, one difference between the two systems is that Zip1is required for the biosynthesis of sulfur-containing amino ac-ids only under low levels of sulfur and is not required duringnormal growth conditions, as is Met4 (13). In N. crassa, theMet30 homolog Scon2 (sulfur controller) is also required forsulfur uptake and assimilation (110, 111). Cys3, the Met4 or-tholog of N. crassa, is degraded via Scon2 and regulates theentire set of sulfur uptake and assimilation genes. Interactionbetween Scon2 and Scon3 (N. crassa Skp1) was observed usinga yeast two-hybrid screen and coimmunoprecipitation and was



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dependent on the F-box motif in Scon2 (183). Cys3 activatesnot only sulfur utilization genes but also the transcription ofCYS3 itself and of the SCON2 gene. Therefore, when Scon2targets Cys3 for proteolysis, its own activation is also reduced,ensuring the possibility of rapidly activating Cys3 again. Mu-tational analysis of Scon2 showed that the F-box domain playsan important role in the regulation of Cys3. Eleven out of 14mutations in the F-box domain gave rise to a constitutivelyrepressed phenotype corresponding to the �cys3 phenotype.This is at first sight surprising, since mutation of the F box isexpected to impair Skp1 binding (although loss of binding toSkp1 was not verified) and thereby decrease the ability todegrade Cys3. This would in turn be expected to lead to aconstitutive activation phenotype of CYS3. It is possible thatScon2 is required not only for the degradation of Cys3 but alsofor its activation, as demonstrated for Met30 and Met4 inbudding yeast. The role of the Met30 homolog SconB (148) inA. nidulans has proven to be similar to that of Scon2 in N.crassa, including binding to Skp1, called SconC in A. nidulans(164). Still, differences have also been found: SCONB is nottranscriptionally activated by the Cys3 counterpart of A. nidu-lans, MetR (149), and although MetR and Cys3 both recognizethe same DNA sequences, full-length CYS3 cannot fully com-plement the �metR phenotype.

In addition to its role in sulfur metabolism, Met30 is essen-tial for cell cycle progression. It regulates multiple aspects ofthe cell cycle, including the expression of cyclins required forG1-phase progression and the accumulation of proteins in-volved in replication and progression through the M phase(161, 190). A target of Met30 was believed to be Swe1 (34), aWee1 family kinase that inhibits Cdc28 by phosphorylation,since high activity of Swe1 and nonubiquitinated forms of Swe1were found in �met30 cells. An in vivo interaction betweenMet30 and Swe1 was also demonstrated (34). A later studyconcluded, however, that Met30 is not responsible for degra-dation of Swe1 but that degradation is a result of the interac-tion between Swe1 and Hsl7 (84, 138). This interaction withHsl7 mediates the translocation of Swe1 out of the nucleus tothe mother bud neck, where its degradation takes place in anunknown manner. The involvement of Met30 in Swe1 degra-dation is therefore disputable but cannot be ruled out entirely.Regardless of the exact mechanisms, it is now clear that theactivation and degradation of proteins involved in the cell cycleare under the control of multiple F-box proteins (Cdc4, Grr1,and Met30). These F-box proteins either target cell cycle pro-teins directly or regulate their levels indirectly.

Finally, recently a Met30 homolog, Lim1, was found in H.jecorina through a yeast one-hybrid screen, and it was demon-strated that Lim1 can bind promoter sequences of the cello-biohydrolase gene CBH2 (58). Clarification of the role in tran-scription of Lim1 and the involvement of possible targetsawaits further investigation.

F-BOX PROTEINS WITHOUT IDENTIFIED TARGETS

F-box proteins from which targets are known to be ubiquiti-nated through binding to Skp1 and assembly into an SCFcomplex are described above. For several other fungal F-boxproteins, binding to Skp1 does not appear to be required forfunction or target inactivation. The functions of these proteins,

then, seem to fall outside the F-box hypothesis, as probably notargets are recruited to an SCF complex. The F-box domain insome of these proteins could interact with proteins other thanSkp1. Alternatively, interaction with Skp1 is required only forself-ubiquitination of the F-box protein to control proteinlevels.

Fbh1: A DNA REPAIR F-BOX PROTEIN

Fbh1 (F-box DNA helicase) is an F-box protein from S.pombe involved in the regulation of recombination levels andDNA repair (144, 156, 175). Like its human homolog, Fbh1contains a helicase domain to unwind DNA. Human Fbh1functions downstream of the recombinase enzyme Rhp51 (theortholog of S. cerevisiae Rad51). Although Fbh1 binds Skp1, itappears that the F box is not necessary for Fbh1 to promoteDNA repair, since two mutations in the F-box domain did notalter growth or genotoxin resistance (120). A mutation in thehelicase domain, however, did affect the DNA repair function.The human homolog assembles into an SCF complex, but itstargets, if any, are also still unknown. Possibly, Skp1 bindingmediates only self-ubiquitination of Fbh1. Remarkably, Fbh1is the only fungal protein that contains an F box combined witha helicase domain, and it is found only in fission yeast.

Frp1: AN F-BOX PROTEIN REQUIRED FORROOT INVASION

In F. oxysporum f. sp. lycopersici, a vascular wilt pathogen oftomato, Frp1 (F-box protein required for pathogenicity 1) wasfound using an insertional mutagenesis screen for pathogenic-ity genes (46). Frp1 is required for assimilation of various(nonsugar) carbon sources as well as induction of genes forcell wall-degrading enzymes, which would explain the deficientplant root colonization and penetration by the �frp1 mutant(W. Jonkers et al., in press). Frp1 binds to Skp1 in yeasttwo-hybrid and pull-down assays, but mutations in the F-boxdomain of Frp1 that impair binding to Skp1 do not affect thephenotype, suggesting that the main function of Frp1 does notdepend on ubiquitination of targets (W. Jonkers and M. Rep,unpublished results). Because FRP1 orthologs are present inother plant-pathogenic fungi, it will be interesting to studytheir role in pathogenicity in these fungi. The deletion of theFRP1 ortholog in F. graminearum has been reported previ-ously, but an initial characterization revealed no obvious dif-ferences from the wild type (60).

Pof14: AN F-BOX PROTEIN THAT INHIBITSERGOSTEROL SYNTHESIS

Pof14 is an F-box protein in S. pombe required for survivalupon hydrogen peroxide stress (193). In response to suchstress, Pof14 binds and inhibits Erg9, a squalene synthase in-volved in ergosterol synthesis. Ergosterol enhances the perme-ability of the membrane and thereby the uptake of hydrogenperoxide. Pof14 and Erg9 bind to each other in a membrane-bound complex, as was demonstrated by tagging both proteinswith fluorescent tags. Binding of Pof14 to Erg9 inhibits theactivity of Erg9, and overexpression of POF14 leads to de-creased levels of squalene synthase activity and ergosterol.



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Transcription of POF14 is induced after treatment with hydro-gen peroxide, and deletion of POF14 decreases viability afterhydrogen peroxide treatment (193). Decreased viability wasnot observed upon deletion of the F-box domain, suggestingthat binding of Pof14 to Skp1 is not required for peroxideresistance. However, binding of Pof14 to Skp1 may promotethe degradation of Pof14 itself. In wild-type cells, Pof14 has ahalf-life of 20 to 40 min, but in temperature-sensitive mutantsof Skp1, Pof14 is stable for at least 60 min (193). Whether thisstabilization is due to defective assembly of Skp1 and Pof14into an SCF complex for self-ubiquitination is not known.

NON-SCF F-BOX PROTEINS

Ctf13 and Rcy1 are two F-box proteins that were found tobind Skp1 but nevertheless function independently of an SCFcomplex and probably also do not have targets to be ubiquiti-nated; therefore, they are unlikely to be involved in proteininactivation. The binding of these proteins to Skp1 may beevolutionarily conserved but may have acquired an alternativefunction.

Ctf13: A KINETOCHORE ASSEMBLY F-BOX PROTEIN

In S. cerevisiae, Ctf13 (chromosome transmission fidelity 13) ispart of the CBF3 complex, which in turn is part of the centro-mere-bound scaffold, where the microtubule binding componentsof kinetochores assemble. The CBF3 complex consists of fourcomponents: Skp1, Ctf13, p64 (encoded by CEP3 and contain-ing a zinc finger centromere binding domain), and p110 (aprotein complex encoded by three genes, Cbf2, Ndc10, andCtf14) (105, 214). The binding of Ctf13 to Skp1 requires thephosphorylation of Ctf13 (87, 173) and the interaction withSgt1 and Hsp90 (8, 188) for the assembly and function of thekinetochore complex. When mutations that prevent binding ofCtf13 to Skp1 were introduced, severely impaired cell growthwas observed. Interestingly, Ctf13 is targeted by another F-boxprotein, Cdc4, for degradation, which is in accordance withCtf13 not being part of an SCF complex itself. Binding of Ctf13to p64 rescues Ctf13 from degradation. Probably only freeCtf13 is degraded via Cdc4, and Ctf13 degradation might berequired to tightly regulate kinetochore assembly.

Rcy1: AN F-BOX PROTEIN INVOLVED INVESICLE TRAFFICKING

Rcy1 (recycling 1) was found in a genetic screen for S.cerevisiae mutants defective in membrane trafficking throughthe endocytic pathway. Deletion of RCY1 results in an arrest ofthe endocytic pathway and leads to accumulation of enlargedcompartments close to areas of cell expansion (202). For Rcy1to function, it needs to bind Skp1, but other components of theSCF are not required for recycling or for degradation of Rcy1itself. Rcy1 contains two SEC10 domains and a CAAX box,implicated in mediating interaction with membranes and alsoneeded for recycling. Rcy1 is required for the recycling of thev-SNARE Snc1p, a membrane protein that fuses exocytic ves-icles with the plasma membrane (56). During vegetativegrowth, Snc1p is localized at the plasma membrane and con-tinually recycles through the Golgi body (121, 143). Rcy1 binds

via its C-terminal domain to two GTPases, proteins that reg-ulate vesicle transport during exo- and endocytosis and arerequired for Golgi body function in yeast (16). Rcy1 interactsspecifically with the active forms of the two GTPases, andtogether they colocalize to the Golgi body and endosomes.

A second recycled protein by Rcy1 is Kex2, a calcium-de-pendent serine protease involved in preprotein processing.Kex2p is a membrane-bound protein cycling between trans-Golgi vesicles and late endosomal compartments (54, 203).These studies suggest that the involvement of Rcy1 in vesicletransport is not related to protein degradation. Indeed, ubiq-uitination of the interacting proteins seems unlikely, since as-sembly into an SCF complex is not required for Rcy1 function.The F box of Rcy1 is required for binding to Skp1, but thebiochemical function of this small complex during vesicle traf-ficking remains unclear. Perhaps surprisingly, Rcy1 has beenfound in an SCF complex (113), but it remains unknownwhether this is a functional complex. The S. pombe homolog ofRcy1, Pof6, also forms a complex with Skp1 and does notfunction in an SCF complex (69). Pof6 is required for septumprocessing and sporulation. Deletion of POF6 results in theformation of a thick septum and the absence of viable spores,which differs from the deletion phenotype of Rcy1, which is notlethal. Recently, a specific Pof6 interactor, Sip1, was foundusing TAP (tandem affinity purification) purification andMudPIT (multidimensional protein identification technology)analysis (82). It was shown that Pof6 and Sip1 form a non-SCFcomplex with Skp1 and that both proteins require interactionwith Skp1 for stability. Sip1 is a widely conserved protein ineukaryotes and consists of HEAT (Huntington, elongation fac-tor 3, the regulatory A subunit or protein phosphatase 2fA andTor1) repeats required for interaction with other proteins.Like Pof6, Sip1 is essential and plays a role in endocytosis andcytokinesis. The budding yeast ortholog of Sip1, Laa1, has notyet been identified as an interactor of Rcy1 but might also bepart of the Rcy1-Skp1 complex, as it too mediates proteintransport between the trans-Golgi network and endosomes(50).

Clearly, the role of Rcy1 is conserved between budding yeastand fission yeast. In fact, Rcy1 seems to play a fundamentalrole in vesicle trafficking in fungi, since it is conserved through-out the fungal kingdom (BLAST searches and our observa-tions).

NON-Skp1 BINDING F-BOX PROTEINS

Some proteins with an F-box domain do not bind Skp1 butinstead bind another E3 ligase subunit. For other F-box pro-teins, binding to Skp1 could not be demonstrated or has notbeen investigated. The F-box domain in some of the latterproteins may also mediate assembly into different complexes.

Ela1: AN ELONGIN COMPLEX F-BOX PROTEIN

Ela1 (elongin A 1) and Elc1 (a Skp1 homolog) in S. cerevi-siae were identified as the homologs of mammalian elongincomplex components (106). Also, in yeast, Ela1 and Elc1 arepresent in the same complex. Probably, Ela1 does not act in anSCF complex, since it binds Elc1 instead of Skp1 and sinceElc1 does not bind Cul1. Ela1 and Elc1 likely bind to Cul3



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instead. Cul3 is a Cullin that is normally part of an E3 ligasecomplex called BC3B, consisting of Cul3, a BTB domain-con-taining protein, and Rbx1. Apparently, Ela1 exists in a complex(Ela1/Elc1/Cul3/Rbx) that is a combination of the human E3ligase Von Hippel-Lindau (VHL) and the BC3B complexes.This new combinatory complex was not reported earlier, and itshows the possibility that subunits from different complexescan interchange to form new complexes, potentially broaden-ing the arsenal of ubiquitin ligase superfamilies.

Ela1 and Cul3 were found to be required for cell survivalafter treatment with UV or the mutagen 4-nitroquinoline 1-ox-ide. Both proteins are also required for degradation and poly-ubiquitination of subunit Rpb1 of RNA Pol II. Pol II is nor-mally removed from damaged DNA to make room for thenuclear excision repair machinery to assemble at that site andrepair damaged DNA strands (169).

Mfb1: A MITOCHONDRION-ASSOCIATEDF-BOX PROTEIN

Mfb1 (mitochondrion-associated F-box protein) in S. cerevi-siae controls membrane fusion dynamics of mitochondria, likeMdm30, described above. Deletion of MFB1 results in abnor-mal mitochondrial morphologies, including short tubules, ag-gregates, and fragments in different combinations (103). Bind-ing to Tom71 localizes Mfb1 to mitochondria, and binding toTom70 ensures stable association with these organelles (104).The paralogous TPR (tetratricopeptide repeat) proteinsTom70 and Tom71 are both associated with mitochondrialprotein import (22, 176). Loss of MDM30 also results in shorttubules, aggregates, and fragments but in a different distribu-tion than in �mfb1 mutants (53). A double knockout of MFB1and MDM30 results in a decreased number of short tubules butmore aggregates and fragments. On rich dextrose and glycerolplates, an mfb1 mutant grows like the wild type, an mdm30mutant grows more slowly, and a double mutant displays asevere growth problem, probably due to mitochondrial DNAinstability (45). Possible targets of Mfb1 are proteins involvedin mitochondrial morphogenesis, but for none of the candidateproteins were larger amounts seen in the �mfb1 mutant than inthe wild-type MFB1. This observation and the lack of demon-stration that Mfb1 binds Skp1 suggest that Mfb1 may notfunction as part of an SCF complex.

Amn1: A MITOSIS EXIT STATE F-BOX PROTEIN

Amn1 (antagonist of mitotic exit network 1) from S. cerevi-siae is listed as one of the 21 budding yeast F-box proteins in anearlier review (205). The protein shares homology with an-other F-box/LRR protein, Pof2 of fission yeast, with little con-servation of the F-box domain, in part because of an inter-spersed region of 56 amino acids in the motif. Although agenetic interaction has been found, a physical interaction be-tween Amn1 and Skp1 could not be demonstrated (in insectcells), perhaps because of the interspersed region in the F-boxdomain. Amn1 itself might be targeted for SCF-mediated pro-teolysis, since stabilized forms of Amn1 were found in cul1 andskp1 mutant strains. AMN1 expression peaks at the M/G1

phase, and Amn1 is normally degraded when cells enter the Sphase, showing an accumulation pattern similar to that of the

Cdc4 target Sic1. Amn1 is required to turn off the mitotic exitpathway after it is completed, and it inhibits the function ofTem1, a small GTPase that activates the mitotic exit network,which causes spindle breakdown, degradation of mitotic cyc-lins, cytokinesis, and cell separation (11). It was shown thatAmn1 binds to Tem1 and inhibits its function by obstructingthe binding of Tem1 to Cdc15. This ensures that the cell canexit from mitosis and enter the G1 phase (199). Tem1 levels areelevated in a �amn1 mutant (153), suggesting that Amn1 mayregulate Tem1 levels. Since Amn1 apparently does not bindSkp1, this regulation may not involve ubiquitination.

OTHER FUNGAL F-BOX PROTEINS

Broad, genomics-based interaction and localization studieshave provided some information on F-box proteins that havenot been investigated individually (Table 2). Most of theseproteins bind Skp1 and can assemble into an SCF complex(113, 181), and some also interact with ribosomes (Ynl311c)(51) or other proteins, like Sgt1 (Ynl311c and Ydr306c) (43).Sgt1 binds to Skp1 and other SCF components (96) and acts asa “client adaptor,” linking the chaperone Hsp90 to SCF andCBF3 complexes containing Skp1 (29). For one F-box protein(Ymr258c), it was determined that it localizes to the cyto-plasm and nucleus using a GFP fusion (74), and for another(Ylr224w), it was demonstrated that it is readily monoubiq-uitinated in vitro by SCF-Ubc4 complexes (113).

Finally, two other F-box proteins, Cos111 and Pof10, cannotbe classified into one of the above-mentioned categories buthave been studied and can be related to specific cellular pro-cess. In S. cerevisiae, the F-box protein Cos111 was identifiedfrom a mutant that showed increased sensitivity to ciclopiroxolamine, an antifungal agent that chelates iron and other ionsand thereby inhibits metal-dependent enzymes (119). Thecos111 mutant is sensitive to ciclopirox olamine at 36°C and isalso sensitive to hydroxyurea. In addition, the cos111 deletion

TABLE 2. Additional F-box proteins in S. cerevisiae and S. pombewithout targets and ascribed cellular function or

Skp1/SCF binding data

F-boxprotein Yeast Additional

motifSkp1

bindingSCF

binding

Cos111 S. cerevisiae ?c ?Das1a S. cerevisiae Yes YesYdr306c S. cerevisiae RNI-likeb Yes YesPof5 S. pombe RNI-like Yes ?Ynl311c S. cerevisiae Yes YesYlr224c S. cerevisiae Yes YesYmr258c S. cerevisiae Yes ?Ydr131c S. cerevisiae Yes YesYlr352w S. cerevisiae LRR Yes YesPof10 S. pombe WD40 Yes ?Pof11 S. pombe ? ?Pof12 S. pombe Yes ?Pof13 S. pombe Yes ?Pof16 S. pombe ? ?

a Das1 (Dst1� 6-azauracil sensitivity) from S. cerevisiae is a putative SCFubiquitin ligase F-box protein of which a null mutant suppresses �dst1 sensitivityto 6-azauracil (http://db.yeastgenome.org/cgi-bin/locus.pl?locus�DAS1) (S.Chavez, personal communication).

b RNI-like, RNase inhibitor-like.c ?, unknown or uncertain.



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mutant showed sensitivity to caffeine, which might indicate adefect in the cyclic AMP signal transduction pathway. Deletionof COS111 does not lead to any growth defects under normalor stress conditions, like temperature shifts, high salt, or os-motic or oxidative stress, and no alterations in morphology,sporulation, or mating were observed (170). It is unknownwhether Cos111 binds Skp1 and regulates protein degradationvia an SCF complex.

Pof10 from S. pombe is an F-box/WD40 protein that bindsSkp1 via its F-box domain (76). Deletion of POF10 does notresult in an obvious phenotype, which is remarkable sincePOF10 is conserved between fission yeast and filamentousfungi (BLAST search and our observations). On the otherhand, overexpression of POF10 results in lethality, probablydue to sequestration of Skp1, thereby preventing the formationof other SCF complexes. Viability was restored by concomitantoverexpression of SKP1, presumably by making more Skp1available for formation of other SCF complexes. Binding toSkp1 may not lead to self-ubiquitination, because Pof10 ishighly stable in contrast to other F-box proteins. AlthoughPof10 bears a protein-protein interaction domain (a WD40motif), targets of Pof10 have not been identified.

CONCLUDING REMARKS

Fungal F-box proteins take part in highly diverse cellularprocesses, but most share the same molecular function: re-moval or inactivation of specific proteins. Loss of a fungalF-box protein often results in a pleiotropic phenotype, espe-cially when the F-box protein has multiple targets. For Cdc4,which has 10 known targets, a null mutation is lethal. Con-versely, when a gene deletion shows no or little effect, theF-box protein may target only one or a few proteins. Forexample, the original deletion mutant of COS111 did not showany phenotype, but it was later demonstrated that COS111 mayhave a function in tolerance to an antifungal agent. Targets ofF-box proteins can vary from transcription factors, enzymes,DNA repair proteins, structural proteins, and cyclins to inhib-itors and/or activators of various other processes. These targetscan operate at an intermediate level of a signaling pathway, forexample, Rcn1 and Sic1, which are degraded via Cdc4, andMsk1 and Ime2, which are degraded via Grr1. Other targetsfunction at the end of a pathway, examples of which are thetranscription factors Tec1 and Gcn1, degraded via Cdc4, andFrq, degraded via Fwd1.

Targets of F-box proteins are recognized mostly when phos-phorylated. Such phosphorylation can be performed by manydifferent protein kinases, like CDKs, mitogen-activated proteinkinases, Pho kinases, and casein kinases, depending on thepathway or process in which the target protein functions. Dif-ferent forms of phosphorylation can be required for recogni-tion. For instance, a requirement for hyperphosphorylation ofa target causes a threshold before a target is being degradedand ensures that multiple phosphorylation steps control deg-radation, like for Cdc4-mediated degradation of Sic1 andFwd1-mediated degradation of Frq. An exceptional case of anunphosphorylated target is Msk1, which is degraded by Grr1when it is unbound to either Rtg2 or Bmh1. Apparently, thesite on Msk1 recognized by Grr1 is masked by these interactingproteins.

In addition to these well-studied F-box proteins, severalother F-box proteins interact with proteins without targetingthem for disposal, examples being Ctf13, Rcy1, and Pof14. Forstill other F-box proteins, no targets or interacting proteinshave been found, and these are often referred to as “orphanF-box proteins.” It might be that the targets are yet to be foundor that no targets exist for these orphan F-box proteins. Espe-cially for those of which mutation of the F-box domain doesnot (greatly) affect function (like Frp1 and Fbh1), the F-boxdomains may serve as degradation motifs required solely forself-ubiquitination. Instead of targeting other proteins, theymay, for instance, function as DNA binding proteins or per-form an enzymatic reaction. Another variation on the F-boxhypothesis is seen for Ela1, an F-box protein that does recruittargets for degradation but assembles in a complex differentfrom the SCF complex.

The levels of free and SCF-bound F-box proteins in thefungal cell are regulated by recycling of Skp1–F-box complexeswithin the SCF core via Nedd8 and CAND1 and by self-ubiq-uitination. It seems that in unicellular fungi, like budding andfission yeasts, recycling is less important and F-box proteins areregulated mostly by autoubiquitination, as shown for severalbudding yeast F-box proteins. The difference in regulationcould be related to the small number of F-box proteins in thesetwo yeasts (21 and 16, respectively) relative to the number infilamentous fungi, which can be 100 or more (BLAST searchesand our observations).

F-box proteins are probably also regulated by recycling andautoubiquitination to remove “free” F-box proteins (i.e., un-bound to a target) from SCF complexes, so that these com-plexes become available for other F-box proteins. Such a sce-nario was supported by overexpression of POF10, probablyleading to constitutive occupation of Skp1, which results inlethality (76), and for the Ufo1�uim mutant lacking the UIMs.Normally, the UIMs are required for self-ubiquitination ofUfo1; a mutant lacking the UIMs cannot be ubiquitinatedanymore and therefore remains in the SCF complex (78). In avariation on self-ubiquitination, Ctf13 is targeted by anotherF-box protein, Cdc4, when unassembled into a CBF complex.

Besides being regulated on the protein level by ubiquitina-tion and recycling, F-box proteins can be regulated at thetranscriptional level. An example is Met30, which creates anegative-feedback loop by degrading the transcription factorMet4, inactivating its own transcription. Another potentialmechanism of regulation is localization. Some F-box proteinsfunction specifically at certain sites in the cell or at certainregions on chromosomal DNA. To be transported to thesesites, interacting partners can play an important role, as dem-onstrated for Mfb1 and Dia2/Pof3.

In fungi, regulation of the activity of F-box proteins them-selves is usually not an integral part of a signal transductionpathway, in contrast to some cases with plants, where it hasbeen demonstrated that F-box proteins could be activated bydirect binding to a small molecule. These F-box proteins act asreceptors, with direct hormone binding triggering their activa-tion (reviewed in reference 214a). Such a mechanism remainsa possibility also with fungi, for instance, for Met30, which isactivated when high levels of methionine, S-adenosyl-methio-nine, or cysteine are present. Binding studies of these sulfur-



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containing amino acids or derivatives to Met30 could confirmthis possibility.

Perhaps less sophisticated, many fungal F-box proteins ap-pear to function simply as garbage collectors, removing wasteproteins that have been marked for degradation. However,several variations on this theme have emerged. For instance,Met30 regulates the transcription factor Met4 in complex waysand does not simply follow the standard F-box hypothesis.Further in-depth investigations of F-box proteins and theirpotential targets or other functions may reveal more such vari-ations.

Most F-box proteins discussed in this review are from S.cerevisiae, providing a fairly comprehensive overview of thevariety of functions that F-box proteins perform in a eukaryoticcell. The additional results obtained with orthologs and otherF-box proteins from fission yeast and filamentous fungi give animpression of the degree of functional conservation of F-boxproteins between fungal species. For example, functional con-servation of Grr1 is, not unexpectedly, less when species aremore distantly related—in contrast to the GRR1 ortholog of C.albicans, the orthologs from two filamentous fungi could notfully complement the �grr1 mutant of yeast. Since Skp1 ishighly conserved between species, this is probably due to dif-ferences in target recognition. Evolution of an F-box protein isconstricted by the requirement to recognize diverse targets.When an F-box protein encounters orthologs of its naturaltarget in another fungus or a “novel” target (i.e., not present inits natural environment), recognition might be less efficient,despite overall sequence conservation in the target recognitiondomain of the F-box protein. Conservation of F-box proteinfunction between different fungal species can also be assessedby the conservation of targets and the pathways leading to thephosphorylation of these targets. Fission yeast and C. albicansharbor orthologs of targets of budding yeast Cdc4 and Grr1,but these have not yet been found in other fungal species(Table 1). For Met4, a target of Met30, homologs are presentin fission yeast and in the filamentous fungi A. nidulans and N.crassa. Apparently, the Met30-Met4 interaction system hasremained relatively stable during fungal evolution.

Searches of fungal genome sequences allow an estimation ofthe numbers of genes encoding F-box proteins in differentfungal species. A. nidulans, for example, contains about 50genes encoding F-box proteins, and in different Fusarium spe-cies 60 to 95 genes encoding F-box proteins are present(Jonkers and Rep, unpublished). In comparing these numbersto the smaller numbers in yeast (21 in budding yeast and 16 infission yeast), it becomes clear that the potential variation ofprocesses regulated by F-box proteins in filamentous fungi ismuch more extensive than that for yeasts. Examples of this areregulation of the circadian clock with N. crassa and plant in-fection with F. oxysporum.

With fungi, it is relatively easy to investigate F-box proteins,due to the availability of knockout strains and the accessibilityto molecular manipulations. Sophisticated screens for targetidentification and deletion studies of all genes encoding F-boxproteins present in a fungal genome, combined with detailedinvestigations of protein-protein interactions and posttransla-tional modifications, will promote a deeper and broader insightinto the diverse functions of F-box proteins in eukaryotic cells.

Among the general lessons already learned from investiga-

tion of the fungal F-box arsenal are that these proteins func-tion in a very broad array of cellular functions and can targetmany different proteins for degradation. Furthermore, clearlynot all F-box proteins comply with the F-box hypothesis, andthe regulation of at least some of these proteins is more com-plex than expected. Concerning the conserved F-box proteinsfound in budding yeast and filamentous fungi, we learned thatthey can have both conserved and diversified targets and thataccumulation of conserved targets in deletion mutants of F-boxproteins can sometimes result in different phenotypes. Fungiremain a rich source for the discovery and understanding of agreat variety of intricate cellular processes with which F-boxproteins are involved.

ACKNOWLEDGMENTS

We thank Caroline Michielse and Ben Cornelissen for critical read-ing of the manuscript and Patrick Lane for enhancing the artwork.

REFERENCES

1. Aronson, B. D., K. A. Johnson, and J. C. Dunlap. 1994. Circadian clocklocus frequency: protein encoded by a single open reading frame definesperiod length and temperature compensation. Proc. Natl. Acad. Sci. USA91:7683–7687.

2. Aronson, B. D., K. A. Johnson, J. J. Loros, and J. C. Dunlap. 1994. Negativefeedback defining a circadian clock: autoregulation of the clock gene fre-quency. Science 263:1578–1584.

3. Ashby, M. N. 1998. CaaX converting enzymes. Curr. Opin. Lipidol. 9:99–102.

4. Atir-Lande, A., T. Gildor, and D. Kornitzer. 2005. Role for the SCFCDC4

ubiquitin ligase in Candida albicans morphogenesis. Mol. Biol. Cell 16:2772–2785.

5. Bai, C., R. Richman, and S. J. Elledge. 1994. Human cyclin F. EMBO J.13:6087–6098.

6. Bai, C., P. Sen, K. Hofmann, L. Ma, M. Goebl, J. W. Harper, and S. J.Elledge. 1996. SKP1 connects cell cycle regulators to the ubiquitin pro-teolysis machinery through a novel motif, the F-box. Cell 86:263–274.

7. Bailey, R. B., and A. Woodword. 1984. Isolation and characterization of apleiotropic glucose repression resistant mutant of Saccharomyces cerevi-siae. Mol. Gen. Genet. 193:507–512.

8. Bansal, P. K., R. Abdulle, and K. Kitagawa. 2004. Sgt1 associates withHsp90: an initial step of assembly of the core kinetochore complex. Mol.Cell. Biol. 24:8069–8079.

9. Bao, M. Z., M. A. Schwartz, G. T. Cantin, J. R. Yates III, and H. D.Madhani. 2004. Pheromone-dependent destruction of the Tec1 transcrip-tion factor is required for MAP kinase signaling specificity in yeast. Cell119:991–1000.

9a. Baranes-Bachar, K., I. Khalaila, Y. Ivantsiv, A. Lavut, O. Voloshin, and D.Raveh. 2008. New interacting partners of the F-box protein Ufo1 of yeast.Yeast 25:733–743.

10. Barbey, R., P. Baudouin-Cornu, T. A. Lee, A. Rouillon, P. Zarzov, M. Tyers,and D. Thomas. 2005. Inducible dissociation of SCF(Met30) ubiquitin ligasemediates a rapid transcriptional response to cadmium. EMBO J. 24:521–532.

11. Bardin, A. J., R. Visintin, and A. Amon. 2000. A mechanism for couplingexit from mitosis to partitioning of the nucleus. Cell 102:21–31.

12. Barral, Y., S. Jentsch, and C. Mann. 1995. G1 cyclin turnover and nutrientuptake are controlled by a common pathway in yeast. Genes Dev. 9:399–409.

13. Baudouin-Cornu, P., and J. Labarre. 2006. Regulation of the cadmiumstress response through SCF-like ubiquitin ligases: comparison betweenSaccharomyces cerevisiae, Schizosaccharomyces pombe and mammaliancells. Biochimie 88:1673–1685.

14. Bell-Pedersen, D., N. Garceau, and J. Loros. 1996. Circadian rhythms infungi. J. Genet. 75:387–401.

15. Benanti, J. A., S. K. Cheung, M. C. Brady, and D. P. Toczyski. 2007. Aproteomic screen reveals SCFGrr1 targets that regulate the glycolytic-glu-coneogenic switch. Nat. Cell Biol. 9:1184–1191.

16. Benli, M., F. Doring, D. G. Robinson, X. Yang, and D. Gallwitz. 1996. TwoGTPase isoforms, Ypt31p and Ypt32p, are essential for Golgi function inyeast. EMBO J. 15:6460–6475.

17. Bernard, F., and B. Andre. 2001. Ubiquitin and the SCF(Grr1) ubiquitinligase complex are involved in the signalling pathway activated by externalamino acids in Saccharomyces cerevisiae. FEBS Lett. 496:81–85.

18. Blake, D., B. Luke, P. Kanellis, P. Jorgensen, T. Goh, S. Penfold, B. J.Breitkreutz, D. Durocher, M. Peter, and M. Tyers. 2006. The F-box protein



.org/D

ownloaded from

http://ec.asm.org/

Dia2 overcomes replication impedance to promote genome stability inSaccharomyces cerevisiae. Genetics 174:1709–1727.

19. Blondel, M., S. Bach, S. Bamps, J. Dobbelaere, P. Wiget, C. Longaretti, Y.Barral, L. Meijer, and M. Peter. 2005. Degradation of Hof1 by SCF(Grr1) isimportant for actomyosin contraction during cytokinesis in yeast. EMBO J.24:1440–1452.

20. Blondel, M., J. M. Galan, Y. Chi, C. Lafourcade, C. Longaretti, R. J.Deshaies, and M. Peter. 2000. Nuclear-specific degradation of Far1 is con-trolled by the localization of the F-box protein Cdc4. EMBO J. 19:6085–6097.

21. Brennan, R. J., and R. H. Schiestl. 1996. Cadmium is an inducer of oxida-tive stress in yeast. Mutat. Res. 356:171–178.

22. Brix, J., G. A. Ziegler, K. Dietmeier, J. Schneider-Mergener, G. E. Schulz,and N. Pfanner. 2000. The mitochondrial import receptor Tom70: identi-fication of a 25 kDa core domain with a specific binding site for preproteins.J. Mol. Biol. 303:479–488.

23. Brunson, L. E., C. Dixon, L. Kozubowski, and N. Mathias. 2004. Theamino-terminal portion of the F-box protein Met30p mediates its nuclearimport and assimilation into an SCF complex. J. Biol. Chem. 279:6674–6682.

24. Brunson, L. E., C. Dixon, A. LeFebvre, L. Sun, and N. Mathias. 2005.Identification of residues in the WD-40 repeat motif of the F-box proteinMet30p required for interaction with its substrate Met4p. Mol. Genet.Genomics 273:361–370.

25. Busch, S., S. E. Eckert, S. Krappmann, and G. H. Braus. 2003. The COP9signalosome is an essential regulator of development in the filamentousfungus Aspergillus nidulans. Mol. Microbiol. 49:717–730.

26. Busch, S., E. U. Schwier, K. Nahlik, O. Bayram, K. Helmstaedt, O. W.Draht, S. Krappmann, O. Valerius, W. N. Lipscomb, and G. H. Braus.2007. An eight-subunit COP9 signalosome with an intact JAMM motif isrequired for fungal fruit body formation. Proc. Natl. Acad. Sci. USA 104:8089–8094.

27. Butler, D. K., O. All, J. Goffena, T. Loveless, T. Wilson, and K. A. Toenjes.2006. The GRR1 gene of Candida albicans is involved in the negativecontrol of pseudohyphal morphogenesis. Fungal Genet. Biol. 43:573–582.

28. Carlson, M. 1999. Glucose repression in yeast. Curr. Opin. Microbiol.2:202–207.

29. Catlett, M. G., and K. B. Kaplan. 2006. Sgt1p is a unique co-chaperone thatacts as a client adaptor to link Hsp90 to Skp1p. J. Biol. Chem. 281:33739–33748.

30. Chandrasekaran, S., and D. Skowyra. 2008. The emerging regulatory po-tential of SCFMet30-mediated polyubiquitination and proteolysis of theMet4 transcriptional activator. Cell Div. 3:11.

31. Reference deleted.32. Chou, S., L. Huang, and H. Liu. 2004. Fus3-regulated Tec1 degradation

through SCFCdc4 determines MAPK signaling specificity during mating inyeast. Cell 119:981–990.

33. Cohen, M. M., G. P. Leboucher, N. Livnat-Levanon, M. H. Glickman, andA. M. Weissman. 2008. Ubiquitin-proteasome-dependent degradation of amitofusin, a critical regulator of mitochondrial fusion. Mol. Biol. Cell 19:2457–2464.

34. Connelly, C., and P. Hieter. 1996. Budding yeast SKP1 encodes an evolu-tionarily conserved kinetochore protein required for cell cycle progression.Cell 86:275–285.

35. Cope, G. A., and R. J. Deshaies. 2003. COP9 signalosome: a multifunctionalregulator of SCF and other cullin-based ubiquitin ligases. Cell 114:663–671.

36. Cope, G. A., G. S. B. Suh, L. Aravind, S. E. Schwarz, S. L. Zipursky, E. V.Koonin, and R. J. Deshaies. 2002. Role of predicted metalloprotease motifof Jab1/Csn5 in cleavage of Nedd8 from Cul1. Science 298:608–611.

37. Reference deleted.38. Craig, K. L., and M. Tyers. 1999. The F-box: a new motif for ubiquitin

dependent proteolysis in cell cycle regulation and signal transduction. Prog.Biophys. Mol. Biol. 72:299–328.

39. Cramer, P., D. A. Bushnell, J. Fu, A. L. Gnatt, B. Maier-Davis, N. E.Thompson, R. R. Burgess, A. M. Edwards, P. R. David, and R. D. Kornberg.2000. Architecture of RNA polymerase II and implications for the tran-scription mechanism. Science 288:640–649.

40. Cui, Z., J. Horecka, and Y. Jigami. 2002. Cdc4 is involved in the transcrip-tional control of OCH1, a gene encoding alpha-1,6-mannosyltransferase inSaccharomyces cerevisiae. Yeast 19:69–77.

41. Cyert, M. S. 2003. Calcineurin signaling in Saccharomyces cerevisiae: howyeast go crazy in response to stress. Biochem. Biophys. Res. Commun.311:1143–1150.

42. Drury, L. S., G. Perkins, and J. F. X. Diffley. 2000. The cyclin-dependentkinase Cdc28p regulates distinct modes of Cdc6p proteolysis during thebudding yeast cell cycle. Curr. Biol. 10:231–240.

43. Dubacq, C., R. Guerois, R. Courbeyrette, K. Kitagawa, and C. Mann. 2002.Sgt1p contributes to cyclic AMP pathway activity and physically interactswith the adenylyl cyclase Cyr1p/Cdc35p in budding yeast. Eukaryot. Cell1:568–582.

44. Dunlap, J. C., and J. J. Loros. 2006. How fungi keep time: circadian systemin Neurospora and other fungi. Curr. Opin. Microbiol. 9:579–587.

45. Durr, M., M. Escobar-Henriques, S. Merz, S. Geimer, T. Langer, and B.Westermann. 2006. Nonredundant roles of mitochondria-associated F-boxproteins Mfb1 and Mdm30 in maintenance of mitochondrial morphology inyeast. Mol. Biol. Cell 17:3745–3755.

46. Duyvesteijn, R. G. E., R. van Wijk, Y. Boer, M. Rep, B. J. Cornelissen, andM. A. Haring. 2005. Frp1 is a Fusarium oxysporum F-box protein requiredfor pathogenicity on tomato. Mol. Microbiol. 57:1051–1063.

47. Escusa, S., J. Camblong, J. M. Galan, B. Pinson, and B. Daignan-Fornier.2006. Proteasome- and SCF-dependent degradation of yeast adeninedeaminase upon transition from proliferation to quiescence requires a newF-box protein named Saf1p. Mol. Microbiol. 60:1014–1025.

48. Escusa, S., D. Laporte, A. Massoni, H. Boucherie, A. Dautant, and B.Daignan-Fornier. 2007. Skp1-Cullin-F-box-dependent degradation ofAah1p requires its interaction with the F-box protein Saf1p. J. Biol. Chem.282:20097–20103.

49. Feldman, R. M. R., C. C. Correll, K. B. Kaplan, and R. J. Deshaies. 1997.A complex of Cdc4p, Skp1p, and Cdc53p/Cullin catalyzes ubiquitination ofthe phosphorylated CDK inhibitor Sic1p. Cell 91:221–230.

50. Fernandez, G. E., and G. S. Payne. 2006. Laa1p, a conserved AP-1 acces-sory protein important for AP-1 localization in yeast. Mol. Biol. Cell 17:3304–3317.

51. Fleischer, T. C., C. M. Weaver, K. J. McAfee, J. L. Jennings, and A. J. Link.2006. Systematic identification and functional screens of uncharacterizedproteins associated with eukaryotic ribosomal complexes. Genes Dev. 20:1294–1307.

52. Flick, J. S., and M. Johnston. 1991. GRR1 of Saccharomyces cerevisiae isrequired for glucose repression and encodes a protein with leucine-richrepeats. Mol. Cell. Biol. 11:5101–5112.

53. Fritz, S., N. Weinbach, and B. Westermann. 2003. Mdm30 is an F-boxprotein required for maintenance of fusion-competent mitochondria inyeast. Mol. Biol. Cell 14:2303–2313.

54. Fuller, R. S., A. Brake, and J. Thorner. 1989. Yeast prohormone processingenzyme (KEX2 gene product) is a Ca2�-dependent serine protease. Proc.Natl. Acad. Sci. USA 86:1434–1438.

55. Galan, J.-M., and M. Peter. 1999. Ubiquitin-dependent degradation ofmultiple F-box proteins by an autocatalytic mechanism. Proc. Natl. Acad.Sci. USA 96:9124–9129.

56. Galan, J.-M., A. Wiederkehr, J. H. Seol, R. Haguenauer-Tsapis, R. J.Deshaies, H. Riezman, and M. Peter. 2001. Skp1p and the F-box proteinRcy1p form a non-SCF complex involved in recycling of the SNARE Snc1pin yeast. Mol. Cell. Biol. 21:3105–3117.

57. Greene, A. V., N. Keller, H. Haas, and D. Bell-Pedersen. 2003. A circadianoscillator in Aspergillus spp. regulates daily development and gene expres-sion. Eukaryot. Cell 2:231–237.

58. Gremel, G., M. Dorrer, and M. Schmoll. 2008. Sulphur metabolism andcellulase gene expression are connected processes in the filamentous fungusHypocrea jecorina (anamorph Trichoderma reesei). BMC Microbiol. 8:174.

59. Reference deleted.60. Han, Y. K., M. D. Kim, S. H. Lee, S. H. Yun, and Y. W. Lee. 2007. A novel

F-box protein involved in sexual development and pathogenesis in Gib-berella zeae. Mol. Microbiol. 63:768–779.

61. Harmon, F. G., and S. A. Kay. 2003. The F box protein AFR is a positiveregulator of phytochrome A-mediated light signaling. Curr. Biol. 13:2091–2096.

62. Harrison, C., S. Katayama, S. Dhut, D. Chen, N. Jones, J. Bahler, and T.Toda. 2005. SCF(Pof1)-ubiquitin and its target Zip1 transcription factormediate cadmium response in fission yeast. EMBO J. 24:599–610.

63. He, Q., J. Cha, Q. He, H.-C. Lee, Y. Yang, and Y. Liu. 2006. CKI and CKIImediate the FREQUENCY-dependent phosphorylation of the WHITECOLLAR complex to close the Neurospora circadian negative feedbackloop. Genes Dev. 20:2552–2565.

64. He, Q., P. Cheng, Q. He, and Y. Liu. 2005. The COP9 signalosome regulatesthe Neurospora circadian clock by controlling the stability of the SCFFWD-1 complex. Genes Dev. 19:1518–1531.

65. He, Q., P. Cheng, Y. Yang, H. Yu, and Y. Liu. 2003. FWD1-mediateddegradation of FREQUENCY in Neurospora establishes a conservedmechanism for circadian clock regulation. EMBO J. 22:4421–4430.

66. He, Q., and Y. Liu. 2005. Degradation of the Neurospora circadian clockprotein FREQUENCY through the ubiquitin-proteasome pathway. Bio-chem. Soc. Trans. 33:953–956.

67. Henchoz, S., Y. Chi, B. Catarin, I. Herskowitz, R. J. Deshaies, and M. Peter.1997. Phosphorylation- and ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor Far1p in budding yeast. Genes Dev. 11:3046–3060.

68. Hermand, D. 2006. F-box proteins: more than baits for the SCF? Cell Div.1:30.

69. Hermand, D., S. Bamps, L. Tafforeau, J. Vandenhaute, and T. P. Makela.2003. Skp1 and the F-box protein Pof6 are essential for cell separation infission yeast. J. Biol. Chem. 278:9671–9677.

70. Hnatova, M., M. Wesolowski-Louvel, G. Dieppois, J. Deffaud, and M.Lemaire. 2008. Characterization of KlGRR1 and SMS1 genes, two new



.org/D

ownloaded from

http://ec.asm.org/

elements of the glucose signaling pathway of Kluyveromyces lactis. Eukaryot.Cell 7:1299–1308.

71. Ho, M., C. Ou, Y. R. Chan, C. T. Chien, and H. Pi. 2008. The utility F-boxfor protein destruction. Cell. Mol. Life Sci. 65:1977–2000.

72. Ho, M. S., P. I. Tsai, and C. T. Chien. 2006. F-box proteins: the key toprotein degradation. J. Biomed. Sci. 13:181–191.

73. Ho, Y., A. Gruhler, A. Heilbut, G. D. Bader, L. Moore, S.-L. Adams, A.Millar, P. Taylor, K. Bennett, K. Boutilier, L. Yang, C. Wolting, I. Donald-son, S. Schandorff, J. Shewnarane, M. Vo, J. Taggart, M. Goudreault, B.Muskat, C. Alfarano, D. Dewar, Z. Lin, K. Michalickova, A. R. Willems, H.Sassi, P. A. Nielsen, K. J. Rasmussen, J. R. Andersen, L. E. Johansen, L. H.Hansen, H. Jespersen, A. Podtelejnikov, E. Nielsen, J. Crawford, V.Poulsen, B. D. Sorensen, J. Matthiesen, R. C. Hendrickson, F. Gleeson, T.Pawson, M. F. Moran, D. Durocher, M. Mann, C. W. V. Hogue, D. Figeys,and M. Tyers. 2002. Systematic identification of protein complexes in Sac-charomyces cerevisiae by mass spectrometry. Nature 415:180–183.

74. Huh, W.-K., J. V. Falvo, L. C. Gerke, A. S. Carroll, R. W. Howson, J. S.Weissman, and E. K. O’Shea. 2003. Global analysis of protein localizationin budding yeast. Nature 425:686–691.

75. Hwang, G. W., Y. Ishida, and A. Naganuma. 2006. Identification of F-boxproteins that are involved in resistance to methylmercury in Saccharomycescerevisiae. FEBS Lett. 580:6813–6818.

76. Ikebe, C., K. Kominami, T. Toda, and K. Nakayama. 2002. Isolation andcharacterization of a novel F-box protein Pof10 in fission yeast. Biochem.Biophys. Res. Commun. 290:1399–1407.

77. Iraqui, I., S. Vissers, F. Bernard, J.-O. de Craene, E. Boles, A. Urrestarazu,and B. Andre. 1999. Amino acid signaling in Saccharomyces cerevisiae: apermease-like sensor of external amino acids and F-box protein Grr1p arerequired for transcriptional induction of the AGP1 gene, which encodes abroad-specificity amino acid permease. Mol. Cell. Biol. 19:989–1001.

78. Ivantsiv, Y., L. Kaplun, R. Tzirkin-Goldin, N. Shabek, and D. Raveh. 2006.Unique role for the UbL-UbA protein Ddi1 in turnover of SCFUfo1 com-plexes. Mol. Cell. Biol. 26:1579–1588.

79. Jackson, L. P., S. I. Reed, and S. B. Haase. 2006. Distinct mechanismscontrol the stability of the related S-phase cyclins Clb5 and Clb6. Mol. Cell.Biol. 26:2456–2466.

80. Jallepalli, P. V., D. Tien, and T. J. Kelly. 1998. sud1� targets cyclin-dependent kinase-phosphorylated Cdc18 and Rum1 proteins for degrada-tion and stops unwanted diploidization in fission yeast. Proc. Natl. Acad.Sci. USA 95:8159–8164.

81. Jaquenoud, M., M. P. Gulli, K. Peter, and M. Peter. 1998. The Cdc42peffector Gic2p is targeted for ubiquitin-dependent degradation by the SCF-Grr1 complex. EMBO J. 17:5360–5373.

82. Jourdain, I., N. Spielewoy, J. Thompson, S. Dhut, J. R. Yates III, and T.Toda. 2009. Identification of a conserved F-box protein 6 interactor essen-tial for endocytosis cytokinesis in fission yeast. Biochem. J. doi:10.1042/BJ20081659.

83. Kaiser, P., K. Flick, C. Wittenberg, and S. I. Reed. 2000. Regulation oftranscription by ubiquitination without proteolysis: Cdc34/SCFMet30-me-diated inactivation of the transcription factor Met4. Cell 102:303–314.

84. Kaiser, P., R. A. L. Sia, E. G. S. Bardes, D. J. Lew, and S. I. Reed. 1998.Cdc34 and the F-box protein Met30 are required for degradation of theCdk-inhibitory kinase Swe1. Genes Dev. 12:2587–2597.

85. Kajava, A. V. 1998. Structural diversity of leucine-rich repeat proteins. J.Mol. Biol. 277:519–527.

86. Kamura, T., D. M. Koepp, M. N. Conrad, D. Skowyra, R. J. Moreland, O.Iliopoulos, W. S. Lane, W. G. Kaelin, Jr., S. J. Elledge, R. C. Conaway, J. W.Harper, and J. W. Conaway. 1999. Rbx1, a component of the VHL tumorsuppressor complex and SCF ubiquitin ligase. Science 284:657–661.

87. Kaplan, K. B., A. A. Hyman, and P. K. Sorger. 1997. Regulating the yeastkinetochore by ubiquitin-dependent degradation and Skp1p-mediatedphosphorylation. Cell 91:491–500.

88. Kaplun, L., Y. Ivantsiv, A. Bakhrat, R. Tzirkin, K. Baranes, N. Shabek, andD. Raveh. 2006. The F-box protein, Ufo1, maintains genome stability byrecruiting the yeast mating switch endonuclease, Ho, for rapid proteasomedegradation. Isr. Med. Assoc. J. 8:246–248.

89. Katayama, S., K. Kitamura, A. Lehmann, O. Nikaido, and T. Toda. 2002.Fission yeast F-box protein Pof3 is required for genome integrity andtelomere function. Mol. Biol. Cell 13:211–224.

90. Reference deleted.91. Kim, J. H., V. Brachet, H. Moriya, and M. Johnston. 2006. Integration of

transcriptional and posttranslational regulation in a glucose signal trans-duction pathway in Saccharomyces cerevisiae. Eukaryot. Cell 5:167–173.

92. Kipreos, E. T., and M. Pagano. 2000. The F-box protein family. GenomeBiol. 1:3002.3001–3002.3007.

93. Kishi, T., A. Ikeda, N. Koyama, J. Fukada, and R. Nagao. 2008. A refinedtwo-hybrid system reveals that SCFCdc4-dependent degradation of Swi5contributes to the regulatory mechanism of S-phase entry. Proc. Natl. Acad.Sci. USA 105:14497–14502.

94. Kishi, T., A. Ikeda, R. Nagao, and N. Koyama. 2007. The SCFCdc4 ubiq-uitin ligase regulates calcineurin signaling through degradation of phosphor-

ylated Rcn1, an inhibitor of calcineurin. Proc. Natl. Acad. Sci. USA 104:17418–17423.

95. Kishi, T., and F. Yamao. 1998. An essential function of Grr1 for thedegradation of Cln2 is to act as a binding core that links Cln2 to Skp1.J. Cell Sci. 111:3655–3661.

96. Kitagawa, K., D. Skowyra, S. J. Elledge, J. W. Harper, and P. Hieter. 1999.SGT1 encodes an essential component of the yeast kinetochore assemblypathway and a novel subunit of the SCF ubiquitin ligase complex. Mol. Cell4:21–33.

97. Koepp, D. M., J. W. Harper, and S. J. Elledge. 1999. How the cyclin becamea cyclin: regulated proteolysis in the cell cycle. Cell 97:431–434.

98. Koepp, D. M., A. C. Kile, S. Swaminathan, and V. Rodriguez-Rivera. 2006.The F-box protein Dia2 regulates DNA replication. Mol. Biol. Cell 17:1540–1548.

99. Koepp, D. M., L. K. Schaefer, X. Ye, K. Keyomarsi, C. Chu, J. W. Harper,and S. J. Elledge. 2001. Phosphorylation-dependent ubiquitination of cyclinE by the SCFFbw7 ubiquitin ligase. Science 294:173–177.

100. Kohler, A., M. S. Schmidt-Zachmann, and W. W. Franke. 1997. AND-1, anatural chimeric DNA-binding protein, combines an HMG-box with regu-latory WD-repeats. J. Cell Sci. 110:1051–1062.

101. Kominami, K., I. Ochotorena, and T. Toda. 1998. Two F-box/WD-repeatproteins Pop1 and Pop2 form hetero- and homo-complexes together withcullin-1 in the fission yeast SCF (Skp1-Cullin-1-F-box) ubiquitin ligase.Genes Cells 3:721–735.

102. Kominami, K., and T. Toda. 1997. Fission yeast WD-repeat protein pop1regulates genome ploidy through ubiquitin-proteasome-mediated degrada-tion of the CDK inhibitor Rum1 and the S-phase initiator Cdc18. GenesDev. 11:1548–1560.

103. Kondo-Okamoto, N., K. Ohkuni, K. Kitagawa, J. M. McCaffery, J. M.Shaw, and K. Okamoto. 2006. The novel F-box protein Mfb1p regulatesmitochondrial connectivity and exhibits asymmetric localization in yeast.Mol. Biol. Cell 17:3756–3767.

104. Kondo-Okamoto, N., J. M. Shaw, and K. Okamoto. 2008. Tetratricopeptiderepeat proteins Tom70 and Tom71 mediate yeast mitochondrial morpho-genesis. EMBO Rep. 9:63–69.

105. Kopski, K. M., and T. C. Huffaker. 1997. Suppressors of the ndc10-2mutation: a role for the ubiquitin system in Saccharomyces cerevisiae ki-netochore function. Genetics 147:409–420.

106. Koth, C. M., M. V. Botuyan, R. J. Moreland, D. B. Jansma, J. W. Conaway,R. C. Conaway, W. J. Chazin, J. D. Friesen, C. H. Arrowsmith, and A. M.Edwards. 2000. Elongin from Saccharomyces cerevisiae. J. Biol. Chem.275:11174–11180.

107. Krappmann, S., N. Jung, B. Medic, S. Busch, R. A. Prade, and G. H. Braus.2006. The Aspergillus nidulans F-box protein GrrA links SCF activity tomeiosis. Mol. Microbiol. 61:76–88.

108. Krek, W. 1998. Proteolysis and the G1-S transition: the SCF connection.Curr. Opin. Genet. Dev. 8:36–42.

109. Krogan, N. J., G. Cagney, H. Yu, G. Zhong, X. Guo, A. Ignatchenko, J. Li,S. Pu, N. Datta, A. P. Tikuisis, T. Punna, J. M. Peregrín-Alvarez, M. Shales,X. Zhang, M. Davey, M. D. Robinson, A. Paccanaro, J. E. Bray, A. Sheung,B. Beattie, D. P. Richards, V. Canadien, A. Lalev, F. Mena, P. Wong, A.Starostine, M. M. Canete, J. Vlasblom, S. Wu, C. Orsi, S. R. Collins, S.Chandran, R. Haw, J. J. Rilstone, K. Gandi, N. J. Thompson, G. Musso, P.St. Onge, S. Ghanny, M. H. Y. Lam, G. Butland, A. M. Altaf-Ul, S. Kanaya,A. Shilatifard, E. O’Shea, J. S. Weissman, C. J. Ingles, T. R. Hughes, J.Parkinson, M. Gerstein, S. J. Wodak, A. Emili, and J. F. Greenblatt. 2006.Global landscape of protein complexes in the yeast Saccharomyces cerevi-siae. Nature 440:637–643.

110. Kumar, A., and J. V. Paietta. 1995. The sulfur controller-2 negative regu-latory gene of Neurospora crassa encodes a protein with beta-transducinrepeats. Proc. Natl. Acad. Sci. USA 92:3343–3347.

111. Kumar, A., and J. V. Paietta. 1998. An additional role for the F-box motif:gene regulation within the Neurospora crassa sulfur control network. Proc.Natl. Acad. Sci. USA 95:2417–2422.

112. Kurz, T., N. Ozlu, F. Rudolf, S. M. O’Rourke, B. Luke, K. Hofmann, A. A.Hyman, B. Bowerman, and M. Peter. 2005. The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae.Nature 435:1257–1261.

113. Kus, B. M., C. E. Caldon, R. Andorn-Broza, and A. M. Edwards. 2004.Functional interaction of 13 yeast SCF complexes with a set of yeast E2enzymes in vitro. Proteins 54:455–467.

114. Lakin-Thomas, P. L., and S. Brody. 2004. Circadian rhythms in microor-ganisms: new complexities. Annu. Rev. Microbiol. 58:489–519.

115. Lammer, D., N. Mathias, J. M. Laplaza, W. Jiang, Y. Liu, J. Callis, M.Goebl, and M. Estelle. 1998. Modification of yeast Cdc53p by the ubiquitin-related protein Rub1p affects function of the SCFCdc4 complex. GenesDev. 12:914–926.

116. Lanker, S., M. H. Valdivieso, and C. Wittenberg. 1996. Rapid degradationof the G1 cyclin Cln2 induced by CDK-dependent phosphorylation. Science271:1597–1601.

117. La Rue, J., S. Tokarz, and S. Lanker. 2005. SCF(Grr1)-mediated ubiquiti-



.org/D

ownloaded from

http://ec.asm.org/

nation of Gis4 modulates glucose response in yeast. J. Mol. Biol. 349:685–698.

118. Lechner, E., P. Achard, A. Vansiri, T. Potuschak, and P. Genschik. 2006.F-box proteins everywhere. Curr. Opin. Plant Biol. 9:631–638.

119. Leem, S. H., J. E. Park, I. S. Kim, J. Y. Chae, A. Sugino, and Y. Sunwoo.2003. The possible mechanism of action of ciclopirox olamine in the yeastSaccharomyces cerevisiae. Mol. Cells 15:55–61.

120. Lehmann, A., S. Katayama, C. Harrison, S. Dhut, K. Kitamura, N. Mc-Donald, and T. Toda. 2004. Molecular interactions of fission yeast Skp1 andits role in the DNA damage checkpoint. Genes Cells 9:367–382.

121. Lewis, M. J., B. J. Nichols, C. Prescianotto-Baschong, H. Riezman, andH. R. B. Pelham. 2000. Specific retrieval of the exocytic SNARE Snc1p fromearly yeast endosomes. Mol. Biol. Cell 11:23–38.

122. Li, F. N., and M. Johnston. 1997. Grr1 of Saccharomyces cerevisiae isconnected to the ubiquitin proteolysis machinery through Skp1: couplingglucose sensing to gene expression and the cell cycle. EMBO J. 16:5629–5638.

123. Li, W. J., Y. M. Wang, X. D. Zheng, Q. M. Shi, T. T. Zhang, C. Bai, D. Li,J. L. Sang, and Y. Wang. 2006. The F-box protein Grr1 regulates thestability of Ccn1, Cln3 and Hof1 and cell morphogenesis in Candida albi-cans. Mol. Microbiol. 62:212–226.

124. Liakopoulos, D., G. Doenges, K. Matuschewski, and S. Jentsch. 1998. Anovel protein modification pathway related to the ubiquitin system. EMBOJ. 17:2208–2214.

125. Lindstrom, D. L., S. L. Squazzo, N. Muster, T. A. Burckin, K. C. Wachter,C. A. Emigh, J. A. McCleery, J. R. Yates, III, and G. A. Hartzog. 2003. Dualroles for Spt5 in pre-mRNA processing and transcription elongation re-vealed by identification of Spt5-associated proteins. Mol. Cell. Biol. 23:1368–1378.

126. Lipschutz, J. H., V. R. Lingappa, and K. E. Mostov. 2003. The exocystaffects protein synthesis by acting on the translocation machinery of theendoplasmic reticulum. J. Biol. Chem. 278:20954–20960.

127. Liu, J., M. Furukawa, T. Matsumoto, and Y. Xiong. 2002. NEDD8 modi-fication of CUL1 dissociates p120CAND1, an inhibitor of CUL1-SKP1binding and SCF ligases. Mol. Cell 10:1511–1518.

128. Liu, Z., and R. A. Butow. 2006. Mitochondrial retrograde signaling. Annu.Rev. Genet. 40:159–185.

129. Liu, Z., M. Spirek, J. Thornton, and R. A. Butow. 2005. A novel degron-mediated degradation of the RTG pathway regulator, Mks1p, by SCFGrr1.Mol. Biol. Cell 16:4893–4904.

130. Liu, Z., J. Thornton, M. Spírek, and R. A. Butow. 2008. Activation of theSPS amino acid-sensing pathway in Saccharomyces cerevisiae correlates withthe phosphorylation state of a sensor component, Ptr3. Mol. Cell. Biol.28:551–563.

131. Lobo, D. S., I. B. Pereira, L. Fragel-Madeira, L. N. Medeiros, L. M. Cabral,J. Faria, M. Bellio, R. C. Campos, R. Linden, and E. Kurtenbach. 2007.Antifungal Pisum sativum defensin 1 interacts with Neurospora crassa cy-clin F related to the cell cycle. Biochemistry 46:987–996.

131a.Lohning, C., and M. Ciriacy. 1994. The TYE7 gene of Saccharomycescerevisiae encodes a putative bHLH-LZ transcription factor required forTyl-mediated gene expression. Yeast 10:1329–1339.

132. Lombardi, L. M., and S. Brody. 2005. Circadian rhythms in Neurosporacrassa: clock gene homologues in fungi. Fungal Genet. Biol. 42:887–892.

133. Lyapina, S., G. Cope, A. Shevchenko, G. Serino, T. Tsuge, C. Zhou, D. A.Wolf, N. Wei, A. Shevchenko, and R. J. Deshaies. 2001. Promotion ofNEDD8-CUL1 conjugate cleavage by COP9 signalosome. Science 292:1382–1385.

134. Mamnun, Y. M., S. Katayama, and T. Toda. 2006. Fission yeast Mcl1interacts with SCF(Pof3) and is required for centromere formation. Bio-chem. Biophys. Res. Commun. 350:125–130.

135. Mathias, N., S. L. Johnson, M. Winey, A. E. Adams, L. Goetsch, J. R.Pringle, B. Byers, and M. G. Goebl. 1996. Cdc53p acts in concert withCdc4p and Cdc34p to control the G1-to-S-phase transition and identifies aconserved family of proteins. Mol. Cell. Biol. 16:6634–6643.

136. Maytal-Kivity, V., E. Pick, R. Piran, K. Hofmann, and M. H. Glickman.2003. The COP9 signalosome-like complex in S. cerevisiae and links toother PCI complexes. Int. J. Biochem. Cell Biol. 35:706–715.

137. Maytal-Kivity, V., R. Piran, E. Pick, K. Hofmann, and M. H. Glickman.2002. COP9 signalosome components play a role in the mating pheromoneresponse of S. cerevisiae. EMBO Rep. 3:1215–1221.

138. McMillan, J. N., C. L. Theesfeld, J. C. Harrison, E. S. Bardes, and D. J.Lew. 2002. Determinants of Swe1p degradation in Saccharomyces cerevi-siae. Mol. Biol. Cell 13:3560–3575.

139. Meimoun, A., T. Holtzman, Z. Weissman, H. J. McBride, D. J. Stillman,G. R. Fink, and D. Kornitzer. 2000. Degradation of the transcription factorGcn4 requires the kinase Pho85 and the SCF(CDC4) ubiquitin-ligase com-plex. Mol. Biol. Cell 11:915–927.

140. Menant, A., R. Barbey, and D. Thomas. 2006. Substrate-mediated remod-eling of methionine transport by multiple ubiquitin-dependent mechanismsin yeast cells. EMBO J. 25:4436–4447.

141. Menant, A., P. Baudouin-Cornu, C. Peyraud, M. Tyers, and D. Thomas.

2006. Determinants of the ubiquitin-mediated degradation of the Met4transcription factor. J. Biol. Chem. 281:11744–11754.

142. Mohamed, A. H., S. S. Chirala, N. H. Mody, W. Y. Huang, and S. J. Wakil.1988. Primary structure of the multifunctional alpha subunit protein ofyeast fatty acid synthase derived from FAS2 gene sequence. J. Biol. Chem.263:12315–12325.

143. Morishita, M., R. Mendonsa, J. Wright, and J. Engebrecht. 2007. Snc1pv-SNARE transport to the prospore membrane during yeast sporulation isdependent on endosomal retrieval pathways. Traffic 8:1231–1245.

144. Morishita, T., F. Furukawa, C. Sakaguchi, T. Toda, A. M. Carr, H. Iwasaki,and H. Shinagawa. 2005. Role of the Schizosaccharomyces pombe F-boxDNA helicase in processing recombination intermediates. Mol. Cell. Biol.25:8074–8083.

145. Mundt, K. E., C. Liu, and A. M. Carr. 2002. Deletion mutants in COP9/signalosome subunits in fission yeast Schizosaccharomyces pombe displaydistinct phenotypes. Mol. Biol. Cell 13:493–502.

146. Mundt, K. E., J. Porte, J. M. Murray, C. Brikos, P. U. Christensen, T.Caspari, I. M. Hagan, J. B. A. Millar, V. Simanis, K. Hofmann, and A. M.Carr. 1999. The COP9/signalosome complex is conserved in fission yeastand has a role in S phase. Curr. Biol. 9:1427–1433.

147. Muratani, M., C. Kung, K. M. Shokat, and W. P. Tansey. 2005. The F boxprotein Dsg1/Mdm30 is a transcriptional coactivator that stimulates Gal4turnover and cotranscriptional mRNA processing. Cell 120:887–899.

148. Natorff, R., M. Piotrowska, and A. Paszewski. 1998. The Aspergillus nidu-lans sulphur regulatory gene sconB encodes a protein with WD40 repeatsand an F-box. Mol. Gen. Genet. 257:255–263.

149. Natorff, R., M. Sienko, J. Brzywczy, and A. Paszewski. 2003. The Aspergillusnidulans metR gene encodes a bZIP protein which activates transcription ofsulphur metabolism genes. Mol. Microbiol. 49:1081–1094.

150. Neer, E. J., C. J. Schmidt, R. Nambudripad, and T. F. Smith. 1994. Theancient regulatory-protein family of WD-repeat proteins. Nature 371:297–300.

151. Nelson, D. C., J. Lasswell, L. E. Rogg, M. A. Cohen, and B. Bartel. 2000.FKF1, a clock-controlled gene that regulates the transition to flowering inArabidopsis. Cell 101:331–340.

152. Nishi, K., C. S. Park, A. E. Pepper, G. Eichinger, M. A. Innis, and M. J.Holland. 1995. The GCR1 requirement for yeast glycolytic gene expressionis suppressed by dominant mutations in the SGC1 gene, which encodes anovel basic-helix-loop-helix protein. Mol. Cell. Biol. 15:2646–2653.

153. Nishizawa, M., M. Kawasumi, M. Fujino, and A. Toh-e. 1998. Phosphory-lation of sic1, a cyclin-dependent kinase (Cdk) inhibitor, by Cdk includingPho85 kinase is required for its prompt degradation. Mol. Biol. Cell 9:2393–2405.

154. Okar, D. A., and A. J. Lange. 1999. Fructose-2,6-bisphosphate and controlof carbohydrate metabolism in eukaryotes. Biofactors 10:1–14.

155. Osaka, F., M. Saeki, S. Katayama, N. Aida, E. A. Toh, K. Kominami, T.Toda, T. Suzuki, T. Chiba, K. Tanaka, and S. Kato. 2000. Covalent modifierNEDD8 is essential for SCF ubiquitin-ligase in fission yeast. EMBO J.19:3475–3484.

156. Osman, F., J. Dixon, A. R. Barr, and M. C. Whitby. 2005. The F-box DNAhelicase Fbh1 prevents Rhp51-dependent recombination without mediatorproteins. Mol. Cell. Biol. 25:8084–8096.

157. Ota, K., K. Kito, S. Okada, and T. Ito. 2008. A proteomic screen reveals themitochondrial outer membrane protein Mdm34p as an essential target ofthe F-box protein Mdm30p. Genes Cells 13:1075–1085.

158. Pal, B., N. C. Chan, L. Helfenbaum, K. Tan, W. P. Tansey, and M.-J.Gething. 2007. SCFCdc4-mediated degradation of the Hac1p transcriptionfactor regulates the unfolded protein response in Saccharomyces cerevisiae.Mol. Biol. Cell 18:426–440.

159. Pan, Z.-Q., A. Kentsis, D. C. Dias, K. Yamoah, and K. Wu. 2004. Nedd8 oncullin: building an expressway to protein destruction. Oncogene 23:1985–1997.

160. Pasula, S., D. Jouandot II, and J.-H. Kim. 2007. Biochemical evidence forglucose-independent induction of HXT expression in Saccharomyces cer-evisiae. FEBS Lett. 581:3230–3234.

161. Patton, E. E., C. Peyraud, A. Rouillon, Y. Surdin-Kerjan, M. Tyers, and D.Thomas. 2000. SCF(Met30)-mediated control of the transcriptional activa-tor Met4 is required for the G(1)-S transition. EMBO J. 19:1613–1624.

162. Patton, E. E., A. R. Willems, and M. Tyers. 1998. Combinatorial control inubiquitin-dependent proteolysis: don’t Skp the F-box hypothesis. TrendsGenet. 14:236–243.

163. Perkins, G., L. S. Drury, and J. F. Diffley. 2001. Separate SCF(CDC4)recognition elements target Cdc6 for proteolysis in S phase and mitosis.EMBO J. 20:4836–4845.

164. Piotrowska, M., R. Natorff, and A. Paszewski. 2000. sconC, a gene involvedin the regulation of sulphur metabolism in Aspergillus nidulans, belongs tothe SKP1 gene family. Mol. Gen. Genet. 264:276–282.

165. Polish, J. A., J.-H. Kim, and M. Johnston. 2005. How the Rgt1 transcriptionfactor of Saccharomyces cerevisiae is regulated by glucose. Genetics 169:583–594.

166. Purnapatre, K., M. Gray, S. Piccirillo, and S. M. Honigberg. 2005. Glucose



.org/D

ownloaded from

http://ec.asm.org/

inhibits meiotic DNA replication through SCFGrr1p-dependent destruc-tion of Ime2p kinase. Mol. Cell. Biol. 25:440–450.

167. Rechsteiner, M., and S. W. Rogers. 1996. PEST sequences and regulationby proteolysis. Trends Biochem. Sci. 21:267–271.

168. Renault, L., N. Nassar, I. Vetter, J. Becker, C. Klebe, M. Roth, and A.Wittinghofer. 1998. The 1.7 A crystal structure of the regulator of chromo-some condensation (RCC1) reveals a seven-bladed propeller. Nature 392:97–101.

169. Ribar, B., L. Prakash, and S. Prakash. 2007. ELA1 and CUL3 are requiredalong with ELC1 for RNA polymerase II polyubiquitylation and degrada-tion in DNA-damaged yeast cells. Mol. Cell. Biol. 27:3211–3216.

170. Rodríguez-Navarro, S., F. Estruch, and J. E. Perez-Ortín. 1999. Functionalanalysis of 12 ORFs from Saccharomyces cerevisiae chromosome II. Yeast15:913–919.

171. Rouillon, A., R. Barbey, E. E. Patton, M. Tyers, and D. Thomas. 2000.Feedback-regulated degradation of the transcriptional activator Met4 istriggered by the SCF(Met30) complex. EMBO J. 19:282–294.

172. Ruijter, G. J., and J. Visser. 1997. Carbon repression in aspergilli. FEMSMicrobiol. Lett. 151:103–114.

173. Russell, I. D., A. S. Grancell, and P. K. Sorger. 1999. The unstable F-boxprotein p58-Ctf13 forms the structural core of the CBF3 kinetochore com-plex. J. Cell Biol. 145:933–950.

174. Saha, A., and R. J. Deshaies. 2008. Multimodal activation of the ubiquitinligase SCF by Nedd8 conjugation. Mol. Cell 32:21–31.

175. Sakaguchi, C., T. Morishita, H. Shinagawa, and T. Hishida. 2008. Essentialand distinct roles of the F-box and helicase domains of Fbh1 in DNAdamage repair. BMC Mol. Biol. 9:27.

176. Schlossmann, J., R. Lill, W. Neupert, and D. A. Court. 1996. Tom71, anovel homologue of the mitochondrial preprotein receptor Tom70. J. Biol.Chem. 271:17890–17895.

177. Schneider, B. L., E. E. Patton, S. Lanker, M. D. Mendenhall, C. Wittenberg,B. Futcher, and M. Tyers. 1998. Yeast G1 cyclins are unstable in G1 phase.Nature 395:86–89.

178. Schuster-Bockler, B., J. Schultz, and S. Rahmann. 2004. HMM logos forvisualization of protein families. BMC Bioinformatics 5:7.

179. Schwechheimer, C. 2004. The COP9 signalosome (CSN): an evolutionaryconserved proteolysis regulator in eukaryotic development. Biochim. Bio-phys. Acta 1695:45–54.

180. Schweitzer, K., R. Cocklin, L. Garrett, F. Desai, and M. Goebl. 2005. Theubiquitin ligase SCFGrr1 is necessary for pheromone sensitivity in Saccha-romyces cerevisiae. Yeast 22:553–564.

181. Seol, J. H., A. Shevchenko, and R. J. Deshaies. 2001. Skp1 forms multipleprotein complexes, including RAVE, a regulator of V-ATPase assembly.Nat. Cell Biol. 3:384–391.

182. Shieh, J. C., A. White, Y. C. Cheng, and J. Rosamond. 2005. Identificationand functional characterization of Candida albicans CDC4. J. Biomed. Sci.12:913–924.

183. Sizemore, S. T., and J. V. Paietta. 2002. Cloning and characterization ofscon-3�, a new member of the Neurospora crassa sulfur regulatory system.Eukaryot. Cell 1:875–883.

184. Skoneczna, A., J. McIntyre, M. Skoneczny, Z. Policinska, and E. Sledz-iewska-Gojska. 2007. Polymerase eta is a short-lived, proteasomally de-graded protein that is temporarily stabilized following UV irradiation inSaccharomyces cerevisiae. J. Mol. Biol. 366:1074–1086.

185. Skowyra, D., K. L. Craig, M. Tyers, S. J. Elledge, and J. W. Harper. 1997.F-box proteins are receptors that recruit phosphorylated substrates to theSCF ubiquitin-ligase complex. Cell 91:209–219.

186. Skowyra, D., D. M. Koepp, T. Kamura, M. N. Conrad, R. C. Conaway, J. W.Conaway, S. J. Elledge, and J. W. Harper. 1999. Reconstitution of G1 cyclinubiquitination with complexes containing SCFGrr1 and Rbx1. Science 284:662–665.

187. Somers, D. E., T. F. Schultz, M. Milnamow, and S. A. Kay. 2000. ZEITLUPE encodes a novel clock-associated PAS protein from Arabidopsis.Cell 101:319–329.

188. Stemmann, O., A. Neidig, T. Kocher, M. Wilm, and J. Lechner. 2002. Hsp90enables Ctf13p/Skp1p to nucleate the budding yeast kinetochore. Proc.Natl. Acad. Sci. USA 99:8585–8590.

189. Strasser, K., and E. Hurt. 2000. Yra1p, a conserved nuclear RNA-bindingprotein, interacts directly with Mex67p and is required for mRNA export.EMBO J. 19:410–420.

190. Su, N. Y., K. Flick, and P. Kaiser. 2005. The F-box protein Met30 isrequired for multiple steps in the budding yeast cell cycle. Mol. Cell. Biol.25:3875–3885.

191. Swaminathan, S., A. C. Kile, E. M. MacDonald, and D. M. Koepp. 2007.Yra1 is required for S phase entry and affects Dia2 binding to replicationorigins. Mol. Cell. Biol. 27:4674–4684.

192. Sweigard, J. A., A. M. Carroll, L. Farrall, F. G. Chumley, and B. Valent.1998. Magnaporthe grisea pathogenicity genes obtained through insertionalmutagenesis. Mol. Plant-Microbe Interact. 11:404–412.

193. Tafforeau, L., S. Le Blastier, S. Bamps, M. Dewez, J. Vandenhaute, and D.Hermand. 2006. Repression of ergosterol level during oxidative stress byfission yeast F-box protein Pof14 independently of SCF. EMBO J. 25:4547–4556.

194. Thomas, D., L. Kuras, R. Barbey, H. Cherest, P. L. Blaiseau, and Y.Surdin-Kerjan. 1995. Met30p, a yeast transcriptional inhibitor that re-sponds to S-adenosylmethionine, is an essential protein with WD40 repeats.Mol. Cell. Biol. 15:6526–6534.

195. Tu, J., W. Song, and M. Carlson. 1996. Protein phosphatase type 1 interactswith proteins required for meiosis and other cellular processes in Saccha-romyces cerevisiae. Mol. Cell. Biol. 16:4199–4206.

196. Vallen, E. A., J. Caviston, and E. Bi. 2000. Roles of Hof1p, Bni1p, Bnr1p,and myo1p in cytokinesis in Saccharomyces cerevisiae. Mol. Biol. Cell11:593–611.

197. Verma, R., R. M. Feldman, and R. J. Deshaies. 1997. SIC1 is ubiquitinatedin vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activ-ities. Mol. Biol. Cell 8:1427–1437.

198. Vierstra, R. D. 2003. The ubiquitin/26S proteasome pathway, the complexlast chapter in the life of many plant proteins. Trends Plant Sci. 8:135–142.

199. Wang, Y., T. Shirogane, D. Liu, J. W. Harper, and S. J. Elledge. 2003. Exitfrom exit: resetting the cell cycle through Amn1 inhibition of G proteinsignaling. Cell 112:697–709.

200. Wee, S., B. Hetfeld, W. Dubiel, and D. A. Wolf. 2002. Conservation of theCOP9/signalosome in budding yeast. BMC Genet. 3:15.

201. Wei, N., and X. W. Deng. 2003. The Cop9 signalosome. Annu. Rev. CellDev. Biol. 19:261–286.

202. Wiederkehr, A., S. Avaro, C. Prescianotto-Baschong, R. Haguenauer-Tsapis, and H. Riezman. 2000. The F-box protein Rcy1p is involved inendocytic membrane traffic and recycling out of an early endosome inSaccharomyces cerevisiae. J. Cell Biol. 149:397–410.

203. Wilcox, C. A., and R. S. Fuller. 1991. Posttranslational processing of theprohormone-cleaving Kex2 protease in the Saccharomyces cerevisiae secre-tory pathway. J. Cell Biol. 115:297–307.

204. Willems, A. R., T. Goh, L. Taylor, I. Chernushevich, A. Shevchenko, and M.Tyers. 1999. SCF ubiquitin protein ligases and phosphorylation-dependentproteolysis. Philos. Trans. R. Soc. Lond. B 354:1533–1550.

205. Willems, A. R., M. Schwab, and M. Tyers. 2004. A hitchhiker’s guide to thecullin ubiquitin ligases: SCF and its kin. Biochim. Biophys. Acta 1695:133–170.

206. Williams, D. R., and J. R. McIntosh. 2002. mcl1�, the Schizosaccharomycespombe homologue of CTF4, is important for chromosome replication, co-hesion, and segregation. Eukaryot. Cell 1:758–773.

207. Wolf, D. A., F. McKeon, and P. K. Jackson. 1999. F-box/WD-repeat pro-teins Pop1p and Sud1p/Pop2p form complexes that bind and direct theproteolysis of Cdc18p. Curr. Biol. 9:373–377.

208. Wu, J.-T., Y.-R. Chan, and C.-T. Chien. 2006. Protection of cullin-RING E3ligases by CSN-UBP12. Trends Cell Biol. 16:362–369.

209. Wysocki, R., A. Javaheri, K. Kristjansdottir, F. Sha, and S. J. Kron. 2006.CDK Pho85 targets CDK inhibitor Sic1 to relieve yeast G1 checkpointarrest after DNA damage. Nat. Struct. Mol. Biol. 13:908–914.

210. Yamano, H., K. Kominami, C. Harrison, K. Kitamura, S. Katayama, S.Dhut, T. Hunt, and T. Toda. 2004. Requirement of the SCFPop1/Pop2ubiquitin ligase for degradation of the fission yeast S phase cyclin Cig2.J. Biol. Chem. 279:18974–18980.

211. Yang, X., J. Zhou, L. Sun, Z. Wei, J. Gao, W. Gong, R.-M. Xu, Z. Rao, andY. Liu. 2007. Structural basis for the function of DCN-1 in protein neddy-lation. J. Biol. Chem. 282:24490–24494.

212. Yen, J. L., N.-Y. Su, and P. Kaiser. 2005. The yeast ubiquitin ligaseSCFMet30 regulates heavy metal response. Mol. Biol. Cell 16:1872–1882.

213. Yochem, J., and B. Byers. 1987. Structural comparison of the yeast celldivision cycle gene CDC4 and a related pseudogene. J. Mol. Biol. 195:233–245.

214. Yoon, H. J., and J. Carbon. 1995. Genetic and biochemical interactionsbetween an essential kinetochore protein, Cbf2p/Ndc10p, and the CDC34ubiquitin-conjugating enzyme. Mol. Cell. Biol. 15:4835–4842.

214a.Yu, H., J. Wu, N. Xu, and M. Peng. 2007. Roles of F-box proteins in planthormone responses. Acta Biochim. Biophys. Sin. 39:915–922.

215. Zheng, J., X. Yang, J. M. Harrell, S. Ryzhikov, E.-H. Shim, K. Lykke-Andersen, N. Wei, H. Sun, R. Kobayashi, and H. Zhang. 2002. CAND1binds to unneddylated CUL1 and regulates the formation of SCF ubiquitinE3 ligase complex. Mol. Cell 10:1519–1526.

216. Zhou, C., V. Seibert, R. Geyer, E. Rhee, S. Lyapina, G. Cope, R. Deshaies,and D. Wolf. 2001. The fission yeast COP9/signalosome is involved in cullinmodification by ubiquitin-related Ned8p. BMC Biochem. 2:7.

217. Zhou, P., and P. M. Howley. 1998. Ubiquitination and degradation of thesubstrate recognition subunits of SCF ubiquitin-protein ligases. Mol. Cell2:571–580.



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