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Copyright � 2007 by the Genetics Society of AmericaDOI: 10.1534/genetics.107.072066
Biochemical and Genetic Analyses Provide Insight Into the Structuraland Mechanistic Properties of Actin Filament Disassembly by the
Aip1p–Cofilin Complex in Saccharomyces cerevisiae
Michael G. Clark1 and David C. Amberg2
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210
Manuscript received February 13, 2007Accepted for publication April 27, 2007
ABSTRACT
Explication of the Aip1p/cofilin/actin filament complex may lead to a more detailed understanding ofthe mechanisms by which Aip1p and cofilin collaborate to rapidly disassemble filaments. We further char-acterized the actin–Aip1p interface through a random mutagenic screen of ACT1, identifying a novelAip1p interaction site on actin. This finding is consistent with our current ternary complex model andoffers insights into how Aip1p may disturb intersubunit contacts within an actin filament. In addition, site-directed mutagenesis aimed at interfering with salt bridge interactions at the predicted Aip1p–cofilininterface revealed hyperactive alleles of cof1 and aip1 that support the ternary complex model and suggestthat conformational changes in cofilin structure may be transmitted to actin filaments, causing increaseddestabilization. Furthermore, these data support an active role for Aip1p in promoting actin filamentturnover.
RAPID actin filament turnover is crucial for main-taining adaptable cytoskeletal networks. Ongoing
cellular dynamics and a finite supply of monomericactin demand that existing filaments are efficiently re-cycled through an intricate progression of disassembly,exchange of monomer-bound nucleotides, and read-dition of ATP-actin subunits to the barbed ends of elon-gating filaments. To expedite filament disassembly, anassortment of actin-binding proteins collaborate to ac-celerate the pointed-end off rate, block barbed-end sub-unit addition, sever filaments into smaller fragments, andsequester monomeric actin. Among the most prom-inent of these proteins is cofilin, which binds to F- andG-actin at a 1:1 ratio and has historically been implicatedin enhancement of pointed-end disassembly (Carlier
et al. 1997) and severing of actin filaments (MacIver
et al. 1991). However, recent in vitro findings refute theassertion of increased disassembly from pointed endsand limit severing activity to extreme substoichiometriclevels of cofilin to actin, proposing that cofilin stabilizesactin filaments when present at isostoichiometric levels(Andrianantoandro and Pollard 2006). Such analysesare valuable in delineating the potential roles of cofilinthrough a range of concentrations. However, cofilin’sin vivo activities proceed among an abundance of otheractin-interacting proteins that may function to regulate,
antagonize, or augment cofilin activities. The mostnotable enhancer of cofilin activity is actin-interactingprotein 1 (Aip1p), which always colocalizes with cofilinand has been shown to enhance filament severing bycofilin while also exhibiting a cofilin-dependent barbed-end regulatory effect that may involve filament capping(Okada et al. 1999, 2002, 2006; Rodal et al. 1999;Balcer et al. 2003; Mohri et al. 2004; Ono et al. 2004;Clark et al. 2006). Significantly, Aip1p’s activity hasbeen observed only at ratios of cofilin:actin .1:5 (Rodal
et al. 1999) and thus Aip1p is not predicted to be activewhen cofilin is at the levels believed to be optimal forsevering, but would be highly active at the stoichiome-tries at which cofilin alone is predicted to stabilize fila-ments. This has profound implications for the perceivedin vivo function of cofilin over a broad range of concen-trations. The experimentally estimated ratio of Aip1p:cofilin:actin in the cell is 1:1:5 or 1:1:10 (Rodal et al.1999). However, localized quantities of Aip1p and cofilinrelative to actin are expected to be much greater at sitessuch as cortical actin patches. Due to the large amountof Aip1p maintained in vivo, its activities must be takeninto consideration when establishing how cofilin behavesin the cell.
Aip1p’s role in the cell could include expansion ordestruction of actin networks, and possibly both, de-pending upon its precise mechanism of activity, itslocal concentration, and the presence of additionalactin-interacting proteins. For example, if Aip1p is in-volved only in enhancing severing, then we expect that itwould allow for the elaboration of actin networks bycreating more barbed ends. If it also plays a role in
1Present address: Department of Biology and Biotechnology, WorcesterPolytechnic Institute, Worcester, MA 01609.
2Corresponding author: Department of Biochemistry and MolecularBiology, SUNY Upstate Medical University, 750 E. Adams St., Syracuse,NY 13210. E-mail: [email protected]
Genetics 176: 1527–1539 ( July 2007)
capping barbed ends, then we expect it to break downactin networks by shortening filaments and allowingthem to disassemble from pointed ends. Similarly, ifAip1p-enhanced severing occurs in the presence ofcapping protein (Cap1p/Cap2p), filament disassemblywould occur. On the basis of preliminary findings, wehave previously proposed a novel model for actin fil-ament barbed-end regulation by Aip1p in which cofilin-dependent Aip1p severing increases the number ofcofilin-bound barbed ends, which could be prohibitivetoward binding by profilin-bound actin subunits (Clark
et al. 2006). In addition to its molecular function of pro-moting actin filament disassembly/turnover, variousphysiological roles have also been observed. These in-clude maintenance of cell morphology in Drosophila(Rogers et al. 2003), organization of body-muscle wallin Caenorhabditis elegans (Ono 2001), reliability of chro-mosome segregation, motility, cytokinesis, and endocy-tosis in Dictyostelium (Konzok et al. 1999; Gerisch et al.2004), leaf size and viability in Arabidopsis (Ketelaar
et al. 2004), assistance in disassembly of Listeria comettails (Brieher et al. 2006), as well as endocytic vesicleinternalization (Clark et al. 2006) and turnover of actincortical patches and actin cables (Okada et al. 2006) inSaccharomyces cerevisiae.
The binding interface between cofilin and actin hasbeen well characterized through mutational analyses in-corporating genetic, cell biological, and biochemical ap-proaches, as well as by visualization using cryo-electronmicroscopy and molecular modeling (Amberg et al.1995; Rodal et al. 1999; Lappalainen and Drubin 1997;McGough et al. 1997; Bobkov et al. 2002; Galkin et al.2003). Recent studies of the Aip1p–actin interactionhave detailed two distinct sites on Aip1p involved inF-actin binding, one on each b-propellor domain (Mohri
et al. 2004; Clark et al. 2006; Okada et al. 2006). While theactin-binding site on Aip1p’s N-terminal b-propellor do-main has been confirmed in S. cerevisiae and C. elegans,the C-terminal actin-binding domain has been detectedonly in yeast, and none of the identified residues forthis site are conserved. Thus, it is not clear if it ismaintained in other species. Findings indicate that theC-terminal binding site is the weaker of the two, al-though both are necessary for proper Aip1p activity invivo and in vitro (Clark et al. 2006; Okada et al. 2006).Analysis of ACT1 charged-to-alanine mutant alleles onthe basis of yeast two-hybrid data has outlined a likelyAip1p-binding site on actin, although it is not clear ifboth of Aip1p’s propellers can bind to this site orif the site is exclusively bound by one of Aip1p’s actin-binding domains (Rodal et al. 1999). Assuming thelatter, it is likely that actin contains a second Aip1p-binding site.
Recently the direct physical interaction betweencofilin and Aip1p has also been characterized (Clark
et al. 2006). This complex was first postulated on thebasis of yeast two-hybrid data demonstrating that the
Aip1p–actin interaction is dependent upon the abilityof actin to bind cofilin and that Aip1p and cofilin’s actin-binding sites appear to overlap (Rodal et al. 1999). Anunbiased mutagenesis strategy of Aip1p in conjunctionwith computational molecular docking with cofilin pre-dicted a cofilin-binding footprint on the concave sur-face of Aip1p, stretching across the cleft with essentialpoints of contact on each of the two b-propellordomains (Clark et al. 2006; Figure 1). Although the pre-cision of the molecular modeling approach revealed ahigh level of complementarity at the proposed inter-face, including eight predicted salt bridge interactions,it is imperative that this model be supplemented by mu-tational studies to confirm the predicted orientation ofcofilin within Aip1p’s binding cleft.
To more thoroughly define the Aip1p–actin physicalinteraction, we have undertaken a genetic dissection ofACT1 along with subsequent synthetic genetic andbiochemical analyses to map a previously unidentifiedAip1p-binding site on actin. In addition, we have pro-vided biochemical data that support the Aip1p–cofilinmolecular model in regard to the specific orientation ofcofilin within the concave surface of Aip1p. Surprisingly,through this effort we have discovered and characterizedthree hyperactive cofilin mutants and two hyperactiveaip1p mutants. These unique and previously unavailableresources have already contributed new insight regard-ing the role played by Aip1p in actin filament disassemblyand will prove to be useful reagents in future studies.Cumulatively, these findings represent an important stepforward in understanding the structural and mechanisticproperties of the Aip1p–cofilin–actin ternary complex.
MATERIALS AND METHODS
Yeast two-hybrid analysis of random act1 mutants: All yeaststrains used in this article are listed in Table 1. Plasmid pDAb7(also known as pRB1516), encoding a fusion of the GAL4DNA-binding domain (DBD) to ACT1 in vector pAS1-CYH2(Amberg et al. 1995), was randomly mutated by treatment withhydroxylamine (Amberg et al. 2005) and lithium acetate(LiOAc) transformed (Rose et al. 1989) into yeast strainY190 (Durfee et al. 1993). Successful transformants wereselected on casamino acid–tryptophan medium and then ana-lyzed for their abilities to interact with Aip1p and cofilin byyeast two-hybrid analysis (Fields and Song 1989). Briefly, Y190cells carrying mutant act1-DBD fusion plasmids were spottedon SC–trp and allowed to grow overnight. These were thenreplica plated to two duplicate YPD plates, which were eachoverlayed with a lawn of yeast strain Y187 transformed withplasmid pAip6 encoding a fusion of AIP1 to the GAL4 DNAactivation domain (Amberg et al. 1995) or plasmid pJT20encoding a fusion of COF1 to the GAL4 DNA activationdomain. The cells were allowed to mate for 1 day after whichdiploids were selected on SC media lacking tryptophan andleucine. After 2 days, diploids were replica plated to SDmedium plus 10 mg/ml adenine and 25, 50, or 100 mm 3,5-aminotriazole. Each transformant was screened for loss of theAip1p interaction while maintaing the cofilin interaction.Mutants showing such a defect were rescued, confirmed by
1528 M. G. Clark and D. C. Amberg
retesting, amplified in Escherichia coli by standard methods(Rose et al. 1989), and submitted for sequencing. Images ofact1p mutants were generated using Insight II Version 2000(Molecular Simulations, San Diego). Coordinates for Act1pwere retrieved from the Collaboratory for Structural Bioinfor-matics Protein Data Bank (PDB file 1ATN).
aip1-GST and cof1-GST mutant plasmid construction:Vector pAR3 (Rodal et al. 1999) was the template for site-directed mutagenesis of AIP1 by overlap extension fusion PCRusing external primers MCo-179 (59-GCGCGGGATCCATGTCATCTATCTCTTTG-39) and MCo-180 (59-GCGCGAAGCTTTCACTCGAGGACAACATT-39), which contain a 59 BamHIor HindIII site, respectively. Internal primers were specific tothe mutant allele. Final PCR products were digested withrestriction enzymes BamHI and HindIII and cloned into thesame sites of pAR3. All mutants were confirmed by sequencing.
Vector pBH360 was created by cloning a cofilin PCR pro-duct into the BamHI and EcoRI sites of pGEX-2T (GE Health-care). The primers used were BHp40 (59-CTCGGATCCAGATCTGGTGTTGCTGTTGCTGATGAATCC-39) and BHp48(59-GTGAGAATTCTTAATGAGAACCAGCGCCTCTGC-39),which contain a 59 BamHI or EcoRI site, respectively. Thisvector was the template for site-directed mutagenesis of COF1by overlap extension fusion PCR using the external primerslisted above and internal primers specific to the mutant allele.
Final PCR products were digested with restriction enzymesBamHI and EcoRI and cloned into the same sites of pBH360.All mutants were confirmed by sequencing.
Genomic integration of mutant alleles: To generate haploidstrains carrying act1 mutant alleles, vector pRB1456 (ACT1:HIS3) was the template for site-directed mutagenesis by overlapextension fusion PCR using external primers DAo-Act1-50 (59-GATCCTTTCCTTCCCAATCTC-39) and DAo-Act1-53 (59-CCCAGAAACAAAGGGTATGAG-39) and internal primers specific tothe mutant allele. Final PCR products were LiOAc trans-formed into diploid yeast strain DAY111x112, allowed to re-cover overnight in YPD, and plated on SC–his to select successfulintegrants. Heterozygous mutant diploids were then sporu-lated and dissected, and haploids containing the act1:HIS3cassette were isolated. Sequencing of PCR products from ge-nomic DNA templates confirmed that all mutants were in-tegrated and that no extra mutations were present.
To generate strains carrying mutated aip1 genes, a strategyusing triple integration of three DNA fragments was imple-mented. Fragment 1 was a PCR product that included�100 bpof genomic sequence upstream of AIP1 and the first 66 bp ofAIP1 (primers LGo-Aip1-1, 59-AATACTAGCTATTGCTTTCCG-39, and MCo-184, 59-AAAGTTATCCTGTGTCGAAGGCTGAGG-39) from template pMC60 (Clark et al. 2006). Fragment 2was a PCR product that included the final 90 bp of AIP1 followed
TABLE 1
S. cerevisiae strains
Name Genotype Source
FY23x86 MATa/a ura3-52/ura3-52 leu2D1/leu2D1 trp1D63/TRP1 HIS3/his3D200 Rodal et al. (1999)Y187 MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3,112 GAL—lacZ Bai and Elledge (1996)Y190 MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3,112 URA3T
GAL—lacZ LYS2TGAL—HIS3cyhr
Bai and Elledge (1996)
DAY111x112 MATa/a ura3-52/ura3-52 leu2D1/leu2D1 trp1D63/trp1D63 his3D200/his3D200LGY2x3 MATa/a aip1DTURA3/aip1DTURA3 ura3-52/ura3-52 leu2D1/leu2D1
trp1D63/TRP1 HIS3/his3D200Rodal et al. (1999)
MCY50 MATa cof1-4:LEU2 his3D200MCY51 MATa cof1-4:LEU2 his3D200MCY52 MATa cof1-19:LEU2 his3D200MCY53 MATa cof1-19:LEU2 his3D200MCY92 MATa act1-105:HIS3 ura3-52 leu2D1 trp1D63 his3D200MCY93 MATa act1-105:HIS3 ura3-52 leu2D1 trp1D63 his3D200MCY94 MATa act1-113:HIS3 ura3-52 leu2D1 trp1D63 his3D200MCY95 MATa act1-113:HIS3 ura3-52 leu2D1 trp1D63 his3D200MCY106 MATa act1-210:HIS3 ura3-52 leu2D1 trp1D63 his3D200MCY107 MATa act1-210:HIS3 ura3-52 leu2D1 trp1D63 his3D200MCY114 MATa act1-212:HIS3 ura3-52 leu2D1 trp1D63 his3D200MCY115 MATa act1-212:HIS3 ura3-52 leu2D1 trp1D63 his3D200MCY118 MATa act1-213:HIS3 ura3-52 leu2D1 trp1D63 his3D200MCY119 MATa act1-213:HIS3 ura3-52 leu2D1 trp1D63 his3D200BBY420 MATa act1-101:HIS3MCY125 MATa aip1-151:G418 r ura3-52 leu2D1 trp1D63 his3D200MCY126 MATa aip1-151:G418 r ura3-52 leu2D1 trp1D63MCY127 MATa aip1-150:G418 r ura3-52 leu2D1 trp1D63 his3D200MCY128 MATa aip1-150:G418 r ura3-52 leu2D1 trp1D63 his3D200MCY129 MATa cof1-157:LEU2 ura3-52 leu2D1 trp1D63 his3D200MCY130 MATa cof1-157:LEU2 ura3-52 leu2D1 his3D200MCY133 MATa cof1-159:LEU2 ura3-52 leu2D1 trp1D63 his3D200MCY134 MATa cof1-159:LEU2 ura3-52 leu2D1 his3D200BBY97 MATa tpm1D:URA3 ura3-52 leu2D1 Bettinger et al. (2007)DAY158 MATa act1-159:HIS3 ura3-52 leu2D1 his3D200
Actin Disassembly by Aip1p–Cofilin 1529
by an inserted G418r marker gene and �100 bp of genomic se-quence downstream of AIP1 (primers MCo-235, 59-CACAAGAGAGGTGTTAACAACCTTTTA-39, and LGo-Aip1-4, 59-TTCTCATGTTCAACTTCGGAA-39) from template pMC60. Fragment3 was a BamHI/HindIII fragment from the appropriate aip1-GST vector. The three fragments were cotransformed into anaip1DTURA3 haploid strain such that genomic integration ofthe mutant aip1 gene required prior recombination with frag-ments 1 and 2 near its start and stop codons, respectively.Successful transformants were selected on YPD1G418 mediaand the loss of ability to grow on media lacking uracil. In-tegration location, presence of the proper mutation, and ab-sence of additional mutations were confirmed by sequencingPCR products generated from using the external primers LGo-Aip1-1 and MCo-Aip1-130 (59-AGTCTTTTCCTTACCCAT-39,inside G418r gene) on genomic DNA templates.
Cofilin mutant integration was achieved in a similar fashion.Fragment 1 contained �100 bp of genomic sequence up-stream of COF1, continued to base pair 195 of COF1, andincluding the intron (primers MCo-289, 59-AGCAACTAGCAAAAA-39, and MCo-214, 59-GTAAAGAGCGTCGTTTTCTGGCAATTT-39) from template pPL8 (Lappalainen et al. 1997).Fragment 2 contained the final 63 bp of COF1 followed by anintegrated LEU2 marker and�100 bp of downstream genomicsequence (primers MCo-241, 59-TTGGAAGATGTCAGCAGAGGCGCTGGT-39, and MCo-292, 59-CTTCCAAAGCGTGAG-39)from template pPL8. Fragment 3 was a BamHI/EcoRI frag-ment from the appropriate cof1-GST vector. Fragments weretransformed into a cof1DTG418r heterozygous diploid and suc-cessful transformants were selected on media lacking leucineand for the inability to grow on media containing G418. Integra-tion location, presence of the proper mutation, and absenceof additional mutations were confirmed by sequencing PCRproducts generated from using external primers BHp40 andBHp48 (both described above) on genomic DNA templates.
Synthetic sick/lethal testing of act1 mutants: For act1 3 cof1-4 crosses, act1 mutant strains (MATa act1-x:HIS3) were crossedto MCY51 and diploid selection was achieved by pickingzygotes. Diploid strains were transformed with vector pJT59(2m Aip1p overexpression vector), sporulated in sporulationmedia lacking uracil to maintain the vector, and dissected.Single- and double-mutant haploids containing the AIP1 over-expression vector pJT59 (2.2-kb genomic BstBI fragmentcontaining AIP1 cloned into the BstBI site of YEplac195) weregrown to saturation in casamino acid–ura media or YPD.Cultures were then diluted and 103 serial dilutions from 5000to 5 cells/ml were spotted on appropriate media (2 ml/spot).Cas–ura cultures were spotted onto cas–ura plates to showgrowth in the presence of aip1 overexpression. YPD cultureswere spotted on 5-FOA plates to show growth in the absence ofaip1 overexpression (endogenous Aip1p was still present).Growth proceeded for 3 days at 30�. act1 3 sac6D crosses weredone the same way, using the sac6D null allele from theEUROSCARF nonessential gene deletion collection in S. cere-visiae (Research Genetics, Birmingham, AL).
For act1 3 aip1D and act1 3 cof1-19 crosses, act1 strains werecrossed to LGY3 and MCY53, respectively. Following sporula-tion and dissection, single and double mutants were grown tosaturation, diluted as described above, and spotted onto YPDmedia. Growth proceeded for 3 days at 37�.
Protein purification: Yeast actin was purified by a modifiedDNaseI affinity purification procedure (Goode 2002). Briefly,10 liters of yeast strain FY23x86 were grown to saturation, re-suspended to a final volume of 120 ml in G-buffer (1ATP,DTT, PMSF, and Calbiochem protease inhibitor cocktail), andpassed twice through a French press at 1200 pounds-force persquare inch gauge (PSIG). The lysate was clarified in a BeckmanJA-20 rotor at 12,000 rpm for 30 min at 4� and then in a
Beckman Ti50.2 rotor at 50,000 rpm for 50 min at 4�. Thesupernatant was loaded in equal volumes onto two DNase I-sepharose columns, each with a 5-ml bed volume (DNAse I,Roche; Sepharose 4B, Sigma, St. Louis), at a flow rate of �1–2ml/min. Columns were washed with 25 ml G-buffer 1 10%deionized formamide, 25 ml G-buffer 1 0.2 m NH4Cl, and 25ml G-buffer. The actin was eluted with 25 ml G-buffer 1 50%deionized formamide and dialyzed overnight in 1 liter G-buffer(0.05 mm ATP). Samples were concentrated using an AmiconUltra spin column (Millipore, Bedford, MA) and then furtherpurified by HPLC ½Bio-Rad (Hercules, CA) Biologic DuoFlow�on a UNO-Q1 column (washed at 1 ml/min with G-buffer andthen eluted with a linear KCl gradient of 100–400 mm in G-buffer). Peak fractions were polymerized in F-buffer for 30 minat room temperature, and then the KCl concentration wasincreased to 0.6 m and the samples were incubated for 1 hr.Polymermized actin was pelleted by ultracentrifugation in aBeckman TLA100.2 rotor at 90,000 rpm. Pellets were resus-pended in G-buffer to 1–2 mg/ml, incubated on ice for 4 hr,dialyzed overnight in 1 liter G-buffer, snap frozen, and stored at�80�. Concentrations were determined by Bradford assay.
Yeast cofilin was expressed in E. coli DH5a cells as aglutathione-S-tranferase (GST) fusion protein under the con-trol of the Plac promoter. GST–cofilin was purified as pre-viously described (Lappalainen and Drubin 1997), but withthe following alterations. Cells were grown to stationary phasein 1 liter of LB1ampicillin, and then cofilin expression wasinduced by treatment with 200 mm IPTG for 3 hr. PBS wassubstituted with TBS (50 mm Tris, pH 7.5, 100 mm NaCl) in allrelevant steps. Following induction, cells were passed twicethrough a French press at 1200 PSIG, and then were spun ina Beckman centrifuge using a JA-20 rotor at 12,000 rpm for30 min. The supernatant was extracted, added to 2 ml of a50% resin slurry, and rocked for 30–60 min at 4�. After washingwith 4 3 10 ml cold TBS, 5 units of thrombin (Novagen) wasadded to the beads (3 ml TBS, 10.4 ml 0.5 m CaCl2) and in-cubated at room temperature for 4 hr. Following cleavage,the supernatant was collected and the resin was washed with2 3 2 ml TBS; washes were added to the supernatant. Thissample was dialyzed overnight against 50 mm Tris, pH 7.5 at4�. Samples were further purified by HPLC (Bio-Rad BiologicDuoFlow) on a UNO-Q1 column and washed at 1 ml/min with50 mm Tris, pH 7.5, and then eluted with a linear KCl gradient(100–400 mm) in 50 mm Tris, pH 7.5. Peak fractions weredialyzed overnight against 10 mm Tris, pH 7.5, and 50 mm NaClfor 5 hr at 4�, concentrated (Microcon YM-10 columns),confirmed by visualization on a polyacrylamide gel, and storedat �80�.
Yeast Aip1p was expressed in yeast strain FY86 as a GSTfusion protein under the control of a galactose-induciblepromoter ½pEG(KT)�. Cells were inoculated into 1 liter SC–ura1 3% glycerol 1 1% EtOH 1 0.1% glucose and grown for 20–24 hr at 30�. Galactose was added to a 2% final concentrationand induction continued for 10 hr. GST–Aip1p purificationproceeded as described for GST–cofilin, with the followingalterations. TBS (150 mm, Tris pH 7.5, 100 mm NaCl) was usedin all relevant steps. Prior to binding to glutathione–agaroseresin, cell lysate was pelleted in a Beckman centrifuge using aJA-20 rotor at 12,000 rpm for 30 min, and then the supernatantwas spun again in a Beckman ultracentrifuge using a 70 Tirotor at 50,000 rpm for 50 min at 4�.
Actin filament pelleting assay: To evaluate actin filamentsedimentation in the presence of Aip1p and cofilin, 4.0 mm
actin was polymerized at room temperature in F-buffer (1 mm
Tris, pH 7.5, 0.7 mm ATP, 0.2 mm CaCl2, 2 mm MgCl2, 285 mm
KCl, 0.001 mm EGTA, 0.2 mm DTT) for 1 hr. A total of 12.5 mlpolymerized actin was aliquoted into polycarbonate ultracen-trifuge tubes (Beckman Instruments, Fullerton, CA) and 12.5 ml
1530 M. G. Clark and D. C. Amberg
of a premixed Aip1p, cofilin, F-buffer, and water mixturewas added to each, yielding final concentrations of 2 mm actinand 13 F-buffer. The reactions were gently vortexed andincubated at room temperature for 20 min and centrifuged at90,000 rpm for 20 min at 25� in a TLA100 rotor (BeckmanInstruments) to pellet the actin filaments. Equal proportionsof the pellets and supernatants fractions were run on 13%SDS–PAGE gels and proteins were visualized by SYPRO Rubystaining (Invitrogen).
Yeast immunofluorescence: Immunofluorescence was per-formed by standard protocols using a methanol/acetone fix-ation (Amberg et al. 2005). Affinity-purified anti-Aip1p antibody(primary) was used at a dilution of 1:100 (Rodal et al. 1999).FITC-conjugated goat anti-rabbit IgG (Cappel; ICN Biochem-icals) was used at 1:1000.
Rhodamine–phalloidin staining: Staining of the actin cyto-skeleton was performed using a standard protocol (Amberg
et al. 2005). Briefly, a yeast cell culture grown to 2 3 107 wassubjected to incubation with EM-grade formaldehyde at a finalconcentration of 4%, washed with PBS, and treated withrhodamine-labeled phalloidin (1:10 dilution of 6.6 mm inmethanol). After washing again with PBS, cells were sus-pended in mounting solution and viewed by fluorescencemicroscopy.
Latrunculin A plate assay: Yeast cultures were grown tostationary phase, and then 10 ml of culture was mixed into 2 mlof 23 YPD. A total of 2 ml of 1% agar (55�) was then added andthe mixture was vortexed and evenly poured onto the surfaceof thin YPD agar plate. Once the top layer of agar solidified,four discs of Whatman’s paper were placed on the agar surfaceand 10 ml of the following was pipetted onto each respectivedisk: ddH2O, 0.5 mm latrunculin A (LatA), 1.0 mm LatA, and2.0 mm LatA. The plates were then incubated for 2 days at 30�and scored.
Molecular structures: Coordinates of actin, Aip1p, andcofilin were obtained from the Protein Data Bank (PDB files1YAG, 1PI6, and 1CFY, respectively).
RESULTS
Identification of additional Aip1p interaction siteson actin by differential interaction screening: TheAip1p- and cofilin-binding footprints on actin’s surfacehave been previously described on the basis of yeast two-hybrid analyses, as illustrated in Figure 1A (Rodal et al.1999). The cofilin-binding site depicted is also in agree-ment with cryo-EM and molecular modeling data (Galkin
et al. 2003). By combining these data with our recentlydescribed model of the Aip1p–cofilin complex, we wereable to extrapolate how Aip1p is likely to bind cofilin-decorated actin filaments. From these observations,it appears that Aip1p’s N-terminal actin interaction sitebinds in the vicinity of the previously characterizedAip1p-binding site on subdomain 4 (SD4) of actin. How-ever, Aip1p’s C-terminal actin interaction site is notlocated near SD4, but rather appears most likely tointeract with SD1 of actin. Therefore, we sought to pro-vide experimental support for this novel Aip1p-bindingsite on actin through an unbiased mutagenesis screen.
The ACT1 gene was randomly mutated and a yeasttwo-hybrid screen was implemented to identify mutantisolates that demonstrated a differential interaction
phenotype in which the actin mutant could interact wellwith cofilin, but not with Aip1p. Of five isolates meetingthis standard, two contained an A7T point mutation, andthe remaining three contained one each of the follow-ing point mutations: D11N, D157N, and P332H (Figure2). The residues altered by the act1-212 (D157N) andact1-213 (P332H) alleles fall within the previously iden-tified cofilin-binding footprint (Rodal et al. 1999), sug-gesting that the Aip1p-specific phenotype results froman alteration in cofilin binding that does not prevent thecofilin two-hybrid interaction, but alters it in such a waythat Aip1p binding is prohibited (Figure 1A). Interestingly,
Figure 1.—Location of relevant mutants on the crystalstructures of actin, Aip1p, and cofilin. (A) An actin monomeris oriented such that the DNAse-binding loop is at the topright region of the molecule. Residues in red are charge-to-alanine mutants that are specific to the cofilin interaction(Rodal et al. 1999). Residues in blue are charged to alaninemutants that are specific to the Aip1p interaction (Rodal
et al. 1999). Residues in purple are charge-to-alanine mutantsthat do not interact well with certain aip1p mutants. Residuesin green are randomly generated mutants that are specificto the Aip1p interaction. (B) The Aip1p (green)–cofilin(orange) model (Clark et al. 2006) is shown such that theAip1p N-terminal propeller is on the left. Colored residuesare predicted to be involved in salt bridge interactions be-tween Aip1p (blue or purple) and cofilin (red or pink). Saltbridges in blue/red were tested in this study. Salt bridges inpurple/pink were not tested.
Actin Disassembly by Aip1p–Cofilin 1531
the amino acid changes conferred by the act1-210 (A7T)and act1-211 (D11N) alleles are on the opposite side ofthe cofilin-binding site from where the previously iden-tified Aip1p-binding site is located, suggestive of a novelAip1p-binding site on actin (Figure 1A).
To gain more insight into other potential sites ofAip1p interaction on the actin surface, several actin- andcofilin-specific mutant aip1 alleles previously identifiedby our group, aip1-56 and aip1-59 (Clark et al. 2006),were tested by two-hybrid analysis against the Wertmancollection of cluster-charged-to-alanine Wertman alleles(Wertman et al. 1992). Our intention was that by weak-ening the affinity of Aip1p for the cofilin–actin com-plex, additional points of contact between Aip1p andactin could be identified. Of the 28 Wertman mutantsnot previously associated with the Aip1p or cofilin inter-actions, act1-101p (D363A, E364A), act1-105p (E311A,R312A), and act1-113p (R210A, D211A) emerged asdefective for the Aip1p interaction (Figure 2). The
residues altered by act1-105 are located between theknown Aip1p and the cofilin-binding footprints, whilethose altered by act1-113 lie within the known cofilin-binding footprint (Figure 1A). Interestingly, the resi-dues mutated by act1-101, like those changed by act1-210and act1-211, are on the opposite side of the cofilin-binding site from the known Aip1p-binding footprint,supporting the belief that a second Aip1p interactionsite exists on actin (Figure 1A).
Synthetic genetic phenotypes support a secondAip1p interaction site on actin: To strengthen ourargument for a novel Aip1p-binding site on actin, weimplemented a series of genetic crosses to determine ifour new actin mutants behave in a manner consistentwith what we would expect from an actin mutant that isexclusively defective for Aip1p binding. These data aresummarized in Table 2. Each act1 allele was integratedinto the yeast genome, with the exception of the act1-211 allele, which we were unable to recover, possibly in-dicating a dominant lethal phenotype. To specificallyinvestigate actin mutants that had impairments in Aip1pbinding and not major structural defects and/or inabil-ities to interact with other important actin-binding pro-teins, only mutant strains that had near-normal-growthphenotypes were considered for genetic experimenta-tion (as an aip1D strain grows normally). This elimi-nated the act1-101 and act1-105 alleles, as well as thealleles that make up the previously identified Aip1p-binding footprint: act1-109 and act1-111 (Wertman et al.1992). Also, act1-212 had a considerable growth defect(Table 2), possibly due to its proximity to the ATP-binding pocket of actin. The remaining act1 mutants(act1-113, act1-210, and act1-213) were analyzed for theability to phenocopy an aip1D allele, which is syntheticlethal with cof1-4 (Rodal et al. 1999). Simultaneously,the ability of AIP1 overexpression to suppress any ob-served synthetic lethality was tested. Mutant act1 andcof1-4 haploid strains were mated and the resultingdouble heterozygous diploids were transformed with aURA3-marked AIP1 overexpression 2m vector. Transfor-mants were then sporulated and dissected, and double-mutant haploids containing the overexpression vectorwere isolated. These strains were tested by serial dilutiongrowth assays on media lacking uracil to maintain thevector and on media containing 5-FOA to select for lossof the vector (Figure 3A). act1-113 is not synthetic lethalwith cof1-4, which is inconsistent with a failure to inter-act with Aip1p in vivo. Both act1-210 and act1-213 aresynthetic lethal with cof1-4, and this synthetic lethalityis able to be suppressed by AIP1 overexpression, con-sistent with in vivo loss of Aip1p binding in the actinmutants.
To show that AIP1 overexpression can specificallysuppress the act1 alleles as opposed to just suppressingcof1-4, we utilized the known severe synthetic slow-growth interaction between AIP1 and SAC6 (Rodal
et al. 1999; Clark et al. 2006). The sac6D allele alone also
Figure 2.—Two-hybrid analysis of act1 mutants. Yeast two-hybrid interactions of actin mutants against cofilin, Aip1p,and aip1p mutants were measured on the basis of the activa-tion of expression of the HIS3 reporter (as detected bygrowth on minimal media containing 25, 50, and 100 mm
3-aminotriazol concentrations).
1532 M. G. Clark and D. C. Amberg
has a moderate-growth phenotype that is not suppressedby overexpression of AIP1 (Figure 3B). As observed whentesting with the cof1-4 allele, act1-210 phenocopies theaip1D allele by demonstrating a severe growth defectwhen combined with the sac6D allele. However, act1-213has only a slight growth defect, which is not consistentwith an aip1D phenotype. cof1-4 is also severely sick withsac6D. AIP1 overexpression did suppress the growth de-fect of act1-210 sac6D and cof1-4 sac6D double mutants,allowing their growth phenotypes to improve, but neverto levels better than that of the sac6D allele alone. Thus,suppression is specific to the act1-210 allele and is con-sistent with its inability to interact with Aip1p by two-hybrid interaction. Notably, the act1-210 sac6D doublemutant is sicker than would be expected, even in thepresence of AIP1 overexpression. It is likely that this mu-tation also disrupts the interaction between actin and anunidentified actin-binding protein, which we believe isspecific to SAC6 on the basis of findings described below.
We next sought to genetically differentiate the actinalleles on the basis of the locations of charged residueswithin (act1-213p) or outside (act1-210p) of the cofilin-binding footprint. To this end, we tested for syntheticgrowth defects in act1/aip1D double-mutant haploids(Figure 3C). Since an aip1D allele causes no growth de-fects alone, we expected that any actin mutant specifi-cally deficient for Aip1p binding would be redundantwith the aip1D allele. Therefore, act1 aip1D double mu-tants should grow normally. However, if an actin mutant’sfailure to interact with Aip1p results from propagationthrough a cofilin-related defect, as we expected to bethe case for the a mutant within the cofilin-binding site,we predicted that additional functional defects woulddemonstrate a synthetic growth defect with the aip1D
allele, as is true for several cofilin mutants (Rodal et al.1999). Consistent with this reasoning, we found that whenact1 aip1D strains were tested at 37� for slow-growthphenotypes, act1-213 was very slow while act1-210 wascomparable to wild type (Figure 3C). Using similar logic,we then tested for synthetic growth phenotypes in act1cof1-19 double mutants (Figure 3D). cof1-19 is defectivefor its Aip1p interaction and thus should grow normallyif actin is unable to bind Aip1p (Rodal et al. 1999;Clark et al. 2006). Testing at 37� showed that act1-213causes severe defects when combined with cof1-19, whileact1-210 was similar to wild type. These findings supportour proposition that the residues mutated in act1-213are within the cofilin interaction domain and are dis-ruptive to normal cofilin binding, while the residues mu-tated in act1-210 are outside of the cofilin interactiondomain and do not interfere with cofilin binding.
In vitro analyses of act1-210p and act1-213p wereconducted by actin pelleting assays and revealed nofunctional defects (data not shown). While unexpected,these observations are not without precedent as Aip1pmutants defective for the two-hybrid interaction withcofilin or actin (Clark et al. 2006) also did not show a
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Actin Disassembly by Aip1p–Cofilin 1533
defect in the actin pelleting assay. We attribute thesefindings to the extensive binding interface of Aip1p withcofilin-bound actin filaments and have found thatin vivo analyses are most effective in detecting mutantphenotypes involving disruption of this complex.
Mutagenesis strategy for confirmation of the Aip1p–cofilin model: We recently provided genetic and bio-chemical data demonstrating that a cofilin-binding sitestretches across Aip1p’s concave surface, making essen-tial points of contact on each of the two b-propeller do-mains (Clark et al. 2006). In addition, we presented amolecular model based on a computational dockingsimulation that precisely details how these two proteinscould fit together, including eight stabilizing salt bridgeinteractions at the binding interface (Figure 1B). Al-though the model is supported by genetic and biochem-ical evidence, additional mutational data are necessary tocorroborate the precise orientation of cofilin within theconcave surface of Aip1p. Unfortunately, the transientand actin filament-dependent nature of this interactiondoes not allow for analysis of a stably bound complex.
Charge reversal mutagenesis reveals hyperactivecofilin and Aip1p mutants: During a mutagenic analysisof amino acid residues predicted to participate in saltbridge interactions between Aip1p and cofilin, we un-expectedly isolated three hyperactive cofilin mutantsthat are Aip1p independent. Listed from least to great-est for enhancement of actin filament disassembly, thesemutants are cof1-157p, cof1-159p, and cof1-158p, whichencode for the amino acid changes R135D, R82D, andK80E, respectively (Figure 4A and Table 3). These res-idues cluster relatively close to one another on the samesurface at the narrow end of the cofilin molecule (Figure1B), indicative that changes within this region lead totheir enhanced activity. Increased filament disassemblywas not detected at an actin:cofilin ratio of 5:1 or higher.Although the nature of this hyperactivity is not clearlydefined, the high relative concentration of cofilin re-quired for observation of increased disassembly indi-cates that the hyperactivity is not due to sequestering ofcofilin-bound actin subunits, as this would have causedactin’s pellet:supernatant ratios to mirror the actin:cofilinratio of each sample, which was not the case. Furthermore,inability to detect hyperactivity by these mutants at
Figure 3.—Synthetic genetic interactions of act1 mutant al-leles. (A) act1 mutants were tested for the ability to pheno-copy an aip1D allele by recapitulation of synthetic growthdefects with the cof1-4 allele, with and without overexpressionof AIP1. (B) act1 mutants were crossed with a sac6D allele todemonstrate synthetic growth defects and to show that sup-pression by AIP1 overexpression is specific to the act1 alleles.(C) To determine if the act1 mutants were redundant with anaip1D allele, each was combined with the aip1D allele. Redun-dancy was indicated by lack of an additive growth defect ascompared to each mutant alone. (D) Also to test redundancybetween the act1 mutants and aip1D, each was combined withcof1-19, which is known to not have a synthetic growth defectwith the aip1D allele.
1534 M. G. Clark and D. C. Amberg
actin:cofilin stoichiometries ,2:1 suggests that thehyperactivity results when filaments are more denselybound by cofilin. When F-actin, wild-type cofilin, andhyperactive cofilin were combined at a ratio of 20:20:1,no gain in activity was observed compared to wild-typecofilin alone, indicating that these cofilin mutants donot mimic the phenomenon by which low levels of
Figure 4.—Biochemical analyses of cofilin and aip1pcharge swap mutants reveal gain-of-function alleles. Polymer-ized actin filaments (2 mm) were incubated with or withoutcofilin and/or Aip1p. Filament disassembly was detected byhigh-speed centrifugation (360,000 3 g) followed by SDS–PAGE and SYPRO Ruby staining to compare the relativeamounts of actin in the pellet (F-actin) vs. actin in the super-natant (G-actin). (A) Cofilin mutants cof1-157p, cof1-158p,and cof1-159p were tested through a range of concentrationsto observe the stoichiometric requirements of the mutant ac-tivity, revealing that significant gains in function require acofilin:actin ratio .5:1. (B) Aip1p mutants aip1-150p andaip1-151p, when compared to Aip1p wild type, show an in-creased ability to disassemble cofilin-decorated actin fila-ments. The ratio of actin:cofilin:Aip1p is 10:10:1.
TABLE 3
Summary of biochemical analyses of aip1 and cof1 mutants
Allele Mutations Actin filament disassembly
Aip1p R18-Cof1 D106AIP1 allele
aip1-132 R18D NormalCOF1 alleles
cof1-122 D106A Weak loss of functioncof1-124 D106R Moderate loss of function
Aip1p D85-Cof1 K82AIP1 allele
aip1-152 D85K No expressionaip1-182 D85R Poor expression
COF1 allelescof1-112 K82A Gain of function (less than K82D)cof1-159 K82D Gain of function
Aip1p E111-Cof1 R80AIP1 allele
aip1-151 E111R Gain of functionCOF1 alleles
cof1-110 R80A Gain of function (less than R80E)cof1-158 R80E Gain of function
Aip1p K410-Cof1 D91AIP1 alleles
aip1-135 K410A Normalaip1-137 K410D Normal
COF1 allelescof1-114 D91A Normalcof1-117 D91K Normal
Aip1p K533-Cof1 D123/E126AIP1 allele
aip1-177 K533D Weak loss of functionCOF1 alleles
cof1-126 D123A Loss of function with Aip1pcof1-129 E126A Loss of function with Aip1pcof1-178 D123K/E126K No expression
Aip1p D585-Cof1 R135AIP1 allele
aip1-150 D585R Gain of functionCOF1 allele
cof1-157 R135D Gain of function
Combinatory mutantsAIP1 alleles
aip1-145 R18D/K410D Normalaip1-154 D85K/D585R No expressionaip1-162 D85K/K410D No expressionaip1-163 R18D/D585R D585R-associated gain of functionaip1-164 E111R/K410D E111R-associated gain of functionaip1-166 R18D/E111R E111R-associated gain of functionaip1-169 R18D/E111R/
D585RLoss of function
COF1 allelescof1-130 R80A/D123A Poor expressioncof1-133 R80A/E126A Poor F-actin bindingcof1-135 K82A/D123A Poor F-actin bindingcof1-136 K82A/E126A Poor F-actin bindingcof1-138 D91K/D123A No expressioncof1-140 D91K/D126A Poor F-actin bindingcof1-142 D106R/D123A Loss of functioncof1-144 D106R/D126A Loss of functioncof1-160 R80E/R135D R135D-associated gain of functioncof1-175 D123A/R135D R135D-associated gain of functioncof1-170 R80E/D123A Poor F-actin bindingcof1-171 R80E/E126A Poor F-actin bindingcof1-172 K82D/D123A Poor expressioncof1-173 K82D/E126A Poor F-actin binding
Actin Disassembly by Aip1p–Cofilin 1535
Aip1p lead to rapid disassembly of cofilin-decoratedactinfilaments(Rodal et al. 1999)(datanot shown).There-fore, these data suggest that the activities of hyperactivecofilins are specific to the way in which they stably bindto actin filaments. It is not clear if this causes increaseddepolymerization or enhanced filament severing.
Surprisingly, when we tested the activity of aip1pmutants that contain the reciprocal charge reversal foreach respective hyperactive cofilin mutant (Figure 1B),each was found to have a cofilin-dependent enhancementof actin filament disassembly (we were unable to expressthe D85K ora D85R mutant)(Figure4B). While the inten-sified activities associated with these are not as robust asthose observed for the cofilin mutants, it is intriguing tonote that the level of increased disassembly associatedwith each Aip1p mutant corresponds to the relative gainsof its respective salt bridge partner on cofilin. These ob-servations suggest that the hyperactive aip1p mutants actby inducing a change in cofilin, causing it to transitionfrom normal to hyperactive. This hypothesis is consistentwith the Aip1p–cofilin docked structure in that the saltbridge residues involved in enhanced disassembly line upwith one another as the model predicts.
Genetic analyses of cof1 and aip1 hyperactivemutants: To characterize how the cof1p and aip1phyperactive mutants behave in vivo, cof1-157TLEU2,cof1-159TLEU2, aip1-150TG418r, and aip1-151TG418r
cassettes were each recombined into the normal COF1or AIP1 genomic loci in S. cerevisiae and were subjectedto an array of analyses (summarized in Table 4). We wereunable to recover genomic integrants of cof1-158, sug-gesting that the allele may be lethal in vivo. Normal ex-pression of all integrated mutants was confirmed byWestern blotting (data not shown). Of the integratedalleles tested, only cof1-159 caused cell growth or mor-phology defects at 30�. Also, actin cytoskeletal stainingby rhodamine–phalloidin revealed that cof1-159 has a
disorganized actin cytoskeleton and excessive filamen-tous actin structures (Figure 5A). Interestingly, thisphenotype indicates a loss of function rather than again of function for cof1-159 in vivo. To uncover defectsin cof1-157, aip1-150, and aip1-151, growth on mediacontaining Lat A was assayed and a series of geneticcrosses were implemented (summarized in Table 4).Both aip1 and cof1-157 alleles demonstrated slight andmoderate levels of resistance, respectively, to latrunculinA (data not shown), indicating increased actin filamentstabilization rather than weakened filaments, consistentwith the cytoskeletal defects of cof1-159. When each wascombined with a tropomyosin null allele (tpm1D), whichhas a slow-growth defect resulting from destabilizedactin cables, no additive growth impairments wereobserved. In addition, cof1-157 and aip1 mutants werecombined with the act1-159 allele, which has a hyper-stabilized actin cytsokeleton, and the act1-212 allele,which is a mutant introduced earlier in this work thatseems to have a weakened actin cytoskeleton potentiallydue to a mutation located in the ATP-binding cleft. cof1-157 is synthetic lethal with act1-159 but not with act1-212,again indicating a decreased ability to destabilize actinfilaments. Surprisingly, aip1-150 was also synthetic lethalwith act1-159 but had no defect with act1-212, while aip1-151 was synthetic sick with act1-212 but not with act1-159.The differential phenotypes of these two aip1 mutantswere unexpected due to their similar in vitro pheno-types, but were reconfirmed when each was combinedwith the cof1-4 allele, as aip1-150 was synthetic lethal andaip1-151 grew normally. Localization of these aip1p mu-tants by immunofluorescence revealed that aip1-151plocalizes normally to cortical actin patches, while aip1-150p does not localize, explaining why it phenocopiesan aip1D allele when crossed to cof1-4 and act1-159(Figure 5B). The phenotypic defects of aip1-150 suggestthat it does not properly bind actin and/or cofilin
TABLE 4
Phenotypic analyses of aip1 and cof1 gain-of-function mutants
Synthetic growth phenotypes (37�)
Allele MutationsWildtype cof1-4 aip1D act1-159 act1-212 tpm1D
Actinorganization
Aip1plocalization
Lat Aresistance
Pelletingassay
Wild type 1111 1111 1111 11 111 11 Normal Patches Normal Normalcof1-157 R135D 1111 NA 1111 � 111 11 Normal ND Moderate
increaseEnhanced
activitycof1-159 K82D 11 ND ND ND ND ND Excessive
F-actinND ND Enhanced
activityaip1-150 D585R 1111 � NA � 111 11 Normal Cytosol Slight
increaseEnhanced
activityaip1-151 E111R 1111 1111 NA 11 1 11 Normal Patches Slight
increaseEnhanced
activityaip1D Deletion 1111 � NA � 111 ND Thickened
cablesNA Slight
decreaseNA
NA, not applicable; ND, no data; 1111, wild-type growth; 111, moderate growth; 11, slow growth; 1, very slow growth; �,no growth.
1536 M. G. Clark and D. C. Amberg
in vivo. Previous reports have noted that Aip1p misloc-alizes in certain strains containing actin or cofilinmutants that do not properly bind Aip1p (Rodal et al.1999), which may explain why aip1-150p is found tomislocalize. Cumulatively, these phenotypes show that thefunctional gains observed by these mutants in vitro do notcarry over in vivo, perhaps due to altered binding kinetics,misregulation, or defective interactions with alternateligands (such as Srv2p for cofilin) within the cell.
DISCUSSION
Actin’s Aip1p interaction sites are consistent with thepredicted Aip1p–cofilin molecular model: Visual com-
parisons of the known cofilin and Aip1p-binding siteson actin to molecular models of the Aip1p–cofilin andcofilin–F-actin models lead us to believe that a secondAip1p-binding site must exist on actin. This was con-firmed by an unbiased mutagenesis screen of actin thatsucceeded in isolating actin mutants that interact withcofilin but not with Aip1p in the yeast two-hybrid system.Genetic crosses revealed that one of these mutants (act1-210) precisely phenocopies an aip1D allele and can besuppressed by overexpression of AIP1. This mutation(A7T) falls within SD1 of actin and is superficiallylocated such that it is likely to specifically disrupt anAIP1 interaction site. It is not surprising that the newlyidentified site was previously overlooked, as the originaltwo-hybrid screen used to footprint the first Aip1p-binding site on actin (Rodal et al. 1999) implementedcluster-charged-to-alanine alleles that do not cover thisregion of actin well. Given that our unbiased mutagen-esis strategy identified a binding site in the same regionthat we anticipated on the basis of comparisons betweenAip1p–cofilin and cofilin–actin molecular models, thesefindings also represent strong support for our previouslypredicted Aip1p–cofilin model.
Cofilin-induced actin filament destabilization is asso-ciated with a rotational change in the filament twist anddisruption of the contacts between SD1 and SD2 oflongitudinally adjacent actin subunits (McGough et al.1997; Galkin et al. 2003). It is possible that furtherweakening of filaments is propagated through theAip1p–actin SD1 interaction, potentially due to anadditional torque exerted on the filament, excess forceon cofilin that disturbs intersubunit contacts, or a con-formational change that further separates SD1 fromSD2. In vivo and in vitro observations agree that both ofAip1p’s actin-binding sites are necessary for optimalF-actin binding and disassembly (Mohri et al. 2004;Clark et al. 2006; Okada et al. 2006). This favors a modelin which both of Aip1p’s propellers need to bind theirrespective actin-binding sites for function. On the basisof our crude reconstruction of the Aip1p–cofilin–actinfilament model, it is not obvious how both of Aip1p’sactin interaction sites could simultaneously bind totheir respective contact sites on actin. However, we donot expect that this static model can serve to fullydescribe the dynamic complex, as we predict significantconformational alterations during the dramatic depoly-merization of cofilin-decorated actin filaments that oc-curs upon binding by Aip1p.
Hyperactive mutants offer unique insight into thepotential mechanism of actin filament disassembly: Anattempt to provide more definitive evidence for theAip1p–cofilin model resulted in the inadvertent discov-ery of three hyperactive cofilin mutants and two hyper-active Aip1p mutants. Our original intention was tocreate repulsive charge reversal mutations at the site ofeach predicted salt bridge on one molecule and thenalleviate the repulsion by making the compensatory
Figure 5.—Actin cytoskeletal staining and Aip1p localiza-tion in aip1 and cof1 mutant strains. Each panel presents amontage of cell images showing a representative sample oftotal cells viewed. (A) Rhodamine–phalloidin staining of theactin cytoskeleton shows that only cof1-159 has actin organiza-tional defects, including cytoskeletal depolarization and exces-sive accumulation of filamentous actin at cortical actin patchesand cytosolic actin cables. (B) Immunofluorescence revealsthat aip1-150 mislocalizes from cortical actin patches to thecytosol while aip1-151 appears normal, accounting for ob-served phenotypic differences between the mutant strains.
Actin Disassembly by Aip1p–Cofilin 1537
mutation on the opposite molecule. However, the robustAip1p–cofilin interaction in the presence of F-actin re-quired us to create combinatorial Aip1p or cofilin mutantsto see sufficient loss of biochemical activity in this study.Unfortunately, the use of multi-mutant proteins resultedin losses of function that could not be overcome bycomplementary charge reversals. This was particularly pro-blematic for the cofilin alleles, which were often rendereddefective for binding to actin filaments (Table 3).
Discovery of hyperactive mutations within cofilin andAip1p have provided unique and previously unavailablereagents with which to study actin filament disassemblyby the Aip1p–cofilin complex. Three cofilin mutants,cof1-157p (R135D), cof1-158p (R80E), and cof1-159p(K82D), each generate enhanced disassembly activitiesof varying intensity. Interestingly, these residues havepreviously been targeted by Lappalainen et al. (1997).In that study, a cluster-charged-to-alanine approachcreated the alleles cof1-16 (R80A, K82A) and cof1-22(E134A, R135A, and R138A), both of which resulted in aloss of cofilin binding to filamentous but not monomericactin in vitro (Lappalainen et al. 1997). Notably, these mu-tations do not reside at the putative interface with F-actin,but rather at the Aip1p interface, as we have demon-strated. Thus, the loss of F-actin binding by these alleles isindicative of widespread conformational alterations withinthe cofilin molcule. A similar observation was madepreviously, when the array of cluster-charged-to-alaninealleles of cofilin were used to footprint the actin- andAip1p-binding domains on cofilin by two-hybrid analysis(Rodal et al. 1999). Sites of mutation that caused the samedefects were observed on opposite sides of the molecule.
We suspect that each hyperactive cofilin mutation re-sults in an intramolecular conformational change thatallows cofilin to induce a more destabilizing torsionalstress on the filament, leading to increased severing.The nature of such a conformational shift is not known,although we predict that these alleles produce similarstructural alterations that may be discernible by molec-ular modeling or NMR.
The isolated hyperactive aip1p mutants, aip1-150p(D585R) and aip1-151p (E111R), contain mutations ofresidues that are the salt bridge partners with hyperac-tive cof-157p and -158p, respectively. We were unable topurify the salt bridge mutant paired with cof1-159p:aip1-152p (D85K) or aip1-182 (D85R). Our group haspreviously mutated these same residues as part ofan Aip1p cluster-charged-to-alanine scan, in whichtwo-hybrid analysis revealed normal Aip1p–cofilin andAip1p–actin interactions (Clark et al. 2006). Therefore,we suspect that when these hyperactive aip1p mutantsbind to cofilin, the charge reversals create a localized re-pulsion between the two molecules that is not sufficientto prevent the interaction but does exert a force againstthe R80 or R135 residue of cofilin strong enough to in-duce a conformational change that mimics the hyper-active cofilin alleles. The idea that the increased activity
is transmitted from aip1p through cofilin is supportedby three observations. First, the residues mutated ineach hyperactive aip1p directly line up with and areexpected to exert a repulsive force against the residuesresponsible for the functional gains in the hyperactivecof1p mutants. Second, the level of enhancement de-tected for each aip1p mutant correlates to the relativegain of function observed in its respective hyperactivecofilin salt bridge partner. Third, the residues mutatedin each hyperactive aip1p are on opposite propellers,making it highly unlikely that the observed functionalgains result from similar conformational changes in theoverall structure of Aip1p.
The observed alignment of mutated residues betweenhyperactive alleles of these two molecules provides fur-ther support that the orientation of cofilin within ourAip1p–cofilin molecular model is correct. In addition,these alleles may offer an interesting perspective to themechanism of actin filament disassembly by Aip1p andcofilin. Some published reports favor passive barbed-end capping of actin filaments as Aip1p’s primary ac-tivity in filament disassembly (Okada et al. 2002; Balcer
et al. 2003; Okada et al. 2006). However, doubt has beencast upon the existence and/or in vivo importance ofthis function (Ono et al. 2004; Brieher et al. 2006;Clark et al. 2006). For these reasons, we prefer a modelin which Aip1p actively contributes to filament disas-sembly by enhancing severing by cofilin. The proposi-tion that our hyperactive aip1p mutants act directlythrough the cofilin molecule by inducing a conforma-tional change is consistent with an active role for Aip1p.This does not rule out capping as a function of Aip1p,but we have previously proposed that the perceivedbarbed-end regulation by Aip1p in vivo could involve itspropensity for producing cofilin-bound barbed ends,which may serve as a selective gate for prohibiting bindingby profilin-bound actin subunits (Clark et al. 2006).Furthermore, recent findings have shown that the barbed-end elongation rate of actin filaments saturated withSchizosaccharomyces pombe cofilin is decreased by approx-imately twofold, although human cofilin did not dem-onstrate this effect (Andrianantoandro and Pollard
2006). These experiments were done with actin andcofilins from mixed species; thus additional experimen-tation is needed to understand the extent of barbed-endregulation by cofilin from like sources and what thein vivo significance of this activity may be.
The inability to observe in vivo gain-of-function phe-notypes for our integrated mutants (cof1-157, cof1-159,aip1-150, and aip1-151) is perplexing, likely reflecting thevast complexity of cofilin and Aip1p activity in the cellcompared to when isolated in a test tube. Cellular reg-ulation of cofilin and Aip1p has not been well character-ized and may be responsible for preventing overactivecofilin or Aip1p from participating in uncontrolled fila-ment disassembly. It is believed that tropomyosin blockscofilin from binding actin cables and that PIP2 can bind
1538 M. G. Clark and D. C. Amberg
cofilin to prevent its actin interaction (Yonezawa et al.1990; Ono and Ono 2002; Okada et al. 2006). However,no Aip1p-specific modes of regulation have been identi-fied. Given the rapid disassembly of actin filaments invitro by Aip1p and cofilin, and the fact that Aip1p andcofilin are always found to colocalize at filamentous actinstructures, it seems intuitive that some level of Aip1p-specific regulation occurs to allow filaments to exist.
The capabilities of cofilin and Aip1p to promote actinturnover in vitro have been well demonstrated. It is clearthat the intensity of this activity, and possibly even themechanism, can vary greatly, depending upon the relativeconcentration of each protein as well as the presenceof regulatory and/or other actin-interacting proteins.Therefore, to further understand the cooperative na-ture of these proteins in vivo, it will be important to con-sider the local environment in which filament dynamicsprogress and to further elucidate the modes of regula-tion that moderate this process.
We thank David Sept for helpful input and discussions regarding theAip1p–cofilin complex mutations, as well as members of the AmbergLaboratory for constructive contributions and manuscript review. Thisresearch was supported by National Institutes of Health grant GM-56189.
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Communicating editor: B. J. Andrews
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