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Yeast Yeast 2003; 20: 803–811. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1002 Research Article ALG2, the Hansenula polymorpha isocitrate lyase gene Enrico Berardi*, Annalisa Gambini and Anna Rita Bellu Laboratorio di Genetica Microbica, Dipartimento di Biotecnologie, Universit` a Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy *Correspondence to: Enrico Berardi, Laboratorio di Genetica Microbica, Dipartimento di Biotecnologie, Universit` a Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy. E-mail: [email protected] Received: 7 December 2002 Accepted: 9 March 2003 Abstract To set the basis for molecular and cellular studies of the glyoxylate cycle in methy- lotrophic yeasts, we isolated and characterized ALG2, the Hansenula polymorpha isocitrate lyase gene. Complementation work and sequence analysis revealed an ORF of 1458 nucleotides, encoding a 486 amino acid protein with a predicted molecu- lar mass of 54.9 kDa. This protein is shorter than the Saccharomyces cerevisiae and Candida tropicalis ICLs, lacks a PST1 signal and possesses a PTS2-like signal. The transcriptional regulation of ALG2 mRNA levels by carbon source is mainly achieved by glucose repression–derepression, whereas ethanol induction plays only a minor role. We present evidence indicating that, in H. polymorpha, neither isocitrate lyase activity nor the ALG2 gene product are necessary for C 1 -peroxisome degradation triggered by ethanol. Therefore, the involvement of glyoxylate in degradation, as described by Kulachkovsky et al. (1997) for Pichia methanolica, does not necessar- ily apply to all methylotrophic yeasts. The relevant nucleotide sequence has been deposited at GenBank (Accession No. AF373067.1). Copyright 2003 John Wiley & Sons, Ltd. Keywords: isocitrate lyase; glyoxylate cycle; two-carbon metabolism; Hansenula polymorpha ; peroxisome degradation Introduction In yeasts, as in other heterotrophic microorganisms, the glyoxylate cycle is essential for the utilization of C 2 molecules (ethanol, acetate) and of com- pounds utilized via a C 2 intermediate (e.g. fatty acids) as sole carbon source. This sub-pathway of gluconeogenesis refills the tricarboxylic acid cycle with C 4 compounds when large amounts of oxaloacetate are diverted towards gluconeogene- sis. A key step in this process is the formation of glyoxylate and succinate from D-isocitrate — a Mg 2+ -dependent cleavage catalysed by isocitrate lyase (ICL; threo-DS-isocitrate glyoxylate-lyase; EC 4.1.3.1). ICL, typically a homotetramer of ca. 64 kDa subunits, has been characterized biochem- ically from different sources, including Candida tropicalis (Uchida et al., 1986), Candida lipolytica (Matsuoka et al., 1984) and Saccharomyces cere- visiae (Lopez-Boado et al., 1988a). The cloning of various ICL genes has also been reported, such as those of C. tropicalis and S. cerevisiae (Atomi et al., 1990; Fern´ andez et al., 1992; Sch¨ oler and Sch¨ uller 1993), and several ICL sequences from the three domains of living organisms are now avail- able in the public databases. Current issues in ICL research include their evo- lutionary origins and relationships (Vanni et al., 1990; Schnarrenberger and Martin, 2002, and ref- erences therein), structure–function links (Britton et al., 2000, 2001; Serrano and Bonete, 2001) and their significance in microbial virulence (e.g. Lorenz and Fink, 2001). In the yeast field, the control of ICL synthesis and activity, as well as its cellular localization have attracted considerable attention. In S. cerevisiae, ICL synthesis is subject to transcriptional regulation by two interacting mechanisms — glucose repression and ethanol induction (Barnett and Kornberg, 1960; Witt et al., 1968; Gonzalez, 1977; Sch¨ oler and Sch¨ uller, 1993; Fernandez et al., 1993). Its activity is regulated by feedback inhibition (Varimo and Oura, Copyright 2003 John Wiley & Sons, Ltd.

ALG2, the Hansenula polymorpha isocitrate lyase gene

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YeastYeast 2003; 20: 803–811.Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1002

Research Article

ALG2, the Hansenula polymorpha isocitrate lyase geneEnrico Berardi*, Annalisa Gambini and Anna Rita BelluLaboratorio di Genetica Microbica, Dipartimento di Biotecnologie, Universita Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona,Italy

*Correspondence to:Enrico Berardi, Laboratorio diGenetica Microbica,Dipartimento di Biotecnologie,Universita Politecnica delleMarche, Via Brecce Bianche,60131 Ancona, Italy.E-mail: [email protected]

Received: 7 December 2002Accepted: 9 March 2003

AbstractTo set the basis for molecular and cellular studies of the glyoxylate cycle in methy-lotrophic yeasts, we isolated and characterized ALG2, the Hansenula polymorphaisocitrate lyase gene. Complementation work and sequence analysis revealed an ORFof 1458 nucleotides, encoding a 486 amino acid protein with a predicted molecu-lar mass of 54.9 kDa. This protein is shorter than the Saccharomyces cerevisiae andCandida tropicalis ICLs, lacks a PST1 signal and possesses a PTS2-like signal. Thetranscriptional regulation of ALG2 mRNA levels by carbon source is mainly achievedby glucose repression–derepression, whereas ethanol induction plays only a minorrole. We present evidence indicating that, in H. polymorpha, neither isocitrate lyaseactivity nor the ALG2 gene product are necessary for C1-peroxisome degradationtriggered by ethanol. Therefore, the involvement of glyoxylate in degradation, asdescribed by Kulachkovsky et al. (1997) for Pichia methanolica, does not necessar-ily apply to all methylotrophic yeasts. The relevant nucleotide sequence has beendeposited at GenBank (Accession No. AF373067.1). Copyright 2003 John Wiley &Sons, Ltd.

Keywords: isocitrate lyase; glyoxylate cycle; two-carbon metabolism; Hansenulapolymorpha; peroxisome degradation

Introduction

In yeasts, as in other heterotrophic microorganisms,the glyoxylate cycle is essential for the utilizationof C2 molecules (ethanol, acetate) and of com-pounds utilized via a C2 intermediate (e.g. fattyacids) as sole carbon source. This sub-pathwayof gluconeogenesis refills the tricarboxylic acidcycle with C4 compounds when large amounts ofoxaloacetate are diverted towards gluconeogene-sis. A key step in this process is the formationof glyoxylate and succinate from D-isocitrate — aMg2+-dependent cleavage catalysed by isocitratelyase (ICL; threo-DS-isocitrate glyoxylate-lyase;EC 4.1.3.1). ICL, typically a homotetramer of ca.64 kDa subunits, has been characterized biochem-ically from different sources, including Candidatropicalis (Uchida et al., 1986), Candida lipolytica(Matsuoka et al., 1984) and Saccharomyces cere-visiae (Lopez-Boado et al., 1988a). The cloningof various ICL genes has also been reported, suchas those of C. tropicalis and S. cerevisiae (Atomi

et al., 1990; Fernandez et al., 1992; Scholer andSchuller 1993), and several ICL sequences from thethree domains of living organisms are now avail-able in the public databases.

Current issues in ICL research include their evo-lutionary origins and relationships (Vanni et al.,1990; Schnarrenberger and Martin, 2002, and ref-erences therein), structure–function links (Brittonet al., 2000, 2001; Serrano and Bonete, 2001)and their significance in microbial virulence (e.g.Lorenz and Fink, 2001). In the yeast field, thecontrol of ICL synthesis and activity, as well asits cellular localization have attracted considerableattention.

In S. cerevisiae, ICL synthesis is subjectto transcriptional regulation by two interactingmechanisms — glucose repression and ethanolinduction (Barnett and Kornberg, 1960; Witt et al.,1968; Gonzalez, 1977; Scholer and Schuller,1993; Fernandez et al., 1993). Its activity isregulated by feedback inhibition (Varimo and Oura,

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804 E. Berardi, A. Gambini and A. R. Bellu

1975), enzyme phosphorylation (Lopez-Boadoet al., 1988b; Ordiz et al., 1996) and proteolyticcatabolite inactivation (Holzer, 1976; Lopez-Boadoet al., 1987; Ordiz et al., 1995).

ICL is often found, along with other enzymesof the glyoxylate cycle, in a specialized type ofmicrobody, referred to as the glyoxysome (e.g.van den Bosch et al., 1992). The ICL localiza-tion and glyoxysomal import pathway have beenespecially investigated in plants and yeasts (Olsenet al., 1993; Taylor et al., 1996 and referencestherein; for a review on microbody import, see Sub-ramani, 1998). Whereas strong evidence suggeststhat the C. tropicalis isocitrate lyase is glyoxyso-mal (Kawamato et al., 1977; Atomi et al., 1990;Kamada et al., 1992), S. cerevisiae ICL was foundto be solely cytosolic, even under growth condi-tions that induce microbody proliferation (McCam-mon et al., 1990; Taylor et al., 1996). These resultsindicate that the presence of ICL in glyoxysomesis not essential for the functioning of the glyoxy-late cycle, and are in line with the finding thatmicrobody (peroxisome)-deficient mutants are usu-ally capable of growth on C2 compounds as solecarbon source (e.g. Erdmann et al., 1989).

An additional issue regarding methylotrophicyeasts (e.g. Hansenula polymorpha, Pichia pas-toris, P. methanolica) concerns the possible roleplayed by ICL and glyoxylate in triggering C1-peroxisome degradation. These yeasts are capa-ble of utilizing C1-compounds, such as methanoland formaldehyde, as sole carbon source, viaa complex metabolism, part of which is per-oxisomal (reviewed in Berardi, 1997). Glucoseand other carbon sources, including ethanol,repress the expression of genes involved in

C1 metabolism. In this respect, C2 compoundsare even stronger repressors than glucose andother sugars (see Veenhuis et al., 1983), sug-gesting that C1 and C2 metabolism (and, per-haps, C1- and C2-peroxisomes) are strongly anti-thetic. In addition, when methanol grown cellsare shifted to one of these repressing substrates,peroxisomes are promptly eliminated by com-plex peroxisome degradation phenomena (reviewedin Bellu and Kiel, 2002). Recent findings sug-gest that in the methylotrophic yeast P. methano-lica, ethanol-triggered C1-peroxisome degradationrequires glyoxylate in the cells, on the grounds thatacetyl-CoA synthetase or isocitrate lyase mutantsexhibit defective autophagic peroxisome degrada-tion (Kulachkovsky et al., 1997).

To set the basis for molecular and cellularstudies of the glyoxylate cycle in methylotrophicyeasts, we isolated and characterized ALG2, the H.polymorpha (syn. Pichia angusta) isocitrate lyasegene. The regulation of its mRNA levels by carbonsource, and its lack of involvement in peroxisomedegradation are also reported.

Materials and methods

Strains and plasmidsAll H. polymorpha strains used in this studyare derivatives of the wild-type homothallic hap-loid NCYC-495 (Table 1). They were main-tained in YPD medium. The Escherichia colistrain MC1061 [hsdR mcrB araD139 (araABC-leu)7679 lacX74 galU galK rpsL thi] was used asthe DNA manipulation host. The replicative vectorEBOX37p6 (ARS, CaLEU2, Ampr, ori) was usedfor all subcloning experiments.

Table 1. Hansenula polymorpha strain list and their ICL activities in E1 medium

Strain Genotype Source ICL activity*

L1 leu1-1 ALG2 Gleeson et al., 1986 110.7 ± 7M6 met6-1 ALG2 Gleeson et al., 1986 91.3 ± 6149-2 met6-1 alg2-1 Frank 1995 NDS149-2 leu1-1 alg2-1 Frank 1995 NDS149-2-p leu1-1 alg2-1..pE1-6 This study 121.7 ± 10S149-2-pA leu1-1 alg2-1..pA This study 92.0 ± 4S149-2-pB leu1-1 alg2-1..pB This study 112.7 ± 5A2�-T1 leu1-1 alg2�:: KanMX This study NDA2�-T2 leu1-1 alg2�:: KanMX This study NDA2�-T1 × M6 leu1-1 alg2�:: KanMX/met6-1 alg2-1 This study NDA2�-T2 × M6 leu1-1 alg2�:: KanMX/met6-1 alg2-1 This study ND

∗ nmol/min/mg protein ± SD. ND, not detectable.

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Media and growth conditionsYPD medium contained 2% glucose, 1% yeastextract and 1% peptone. ME medium contained2% malt extract. The minimal media (HDM,LDM, E1 and M2) contained 0.5% ammoniumsulphate, 0.2% yeast nitrogen base without aminoacids or ammonium sulphate (Difco); when neces-sary, 0.006% leucine or 0.002% methionine wereadded. In addition, HDM contained 2% glucose,LDM contained 0.2% glucose, E1 contained 2%ethanol, M2 contained 1% methanol. All cul-tures were grown in 250 ml flasks with 100 mlmedium, using an orbital incubator at 37 ◦C and250 rpm. For Northern blot experiments, after 12 hprecultivation in YPD, 106 cells/ml were inocu-lated in HDM, LDM and E1 to late exponentialphase. For C2-triggered degradation experiments,106 cells/ml (precultivated in YPD) were inocu-lated in M2 medium to late exponential phase,harvested and then shifted in E1 medium (107

cells/ml). Methanol oxidase activities were deter-mined at fixed intervals.

Biochemical methods

Crude cell extracts were obtained as described(Vallini et al., 2000). Enzyme activities of isoci-trate lyase (ICL; EC 4.1.3.1) and methanol oxi-dase (AO; EC 1.1.3.13) were assayed by estab-lished procedures (Dixon and Kornberg, 1959;van der Klei et al., 1990). Protein concentra-tions were determined using a Bio-Rad kit (Cat-alogue No. 500-0006; Bio-Rad, USA) with bovineserum albumin as the standard. Sodium dode-cyl sulphate–polyacrylamide gel electrophoresis(SDS–PAGE) was carried out as described (Laem-mli, 1970). Western blotting was performed usingthe Trans-blot SD immunoblotting system (Bio-Rad, USA) and polyclonal antibodies raised against

H. polymorpha AO. Ethanol was determined usinga Sigma kit (Catalogue No. 332-A; Sigma-AldrichCo., USA).

Molecular and genetic techniques

Restriction enzyme digestion, cloning, plasmid iso-lation and PCRs were performed by standardmethods (Sambrook et al., 1989). DNA sequenc-ing reactions were performed by the BigDye

Terminator Cyc Sequencing kit (Perkin-Elmer),according to the manufacturer’s instructions. Auto-matic sequencing was done with an ABI Prism310 (Perkin-Elmer). H. polymorpha was trans-formed by the LiAc method of Berardi and Thomas(1990). Crosses and sporulations were performedon ME plates, as described by Gleeson and Sud-bery (1988).

Cloning and sequencing of ALG2

A genomic library constructed in plasmid pYT3(Tan et al., 1995) was used to transform the mutantstrain S149-2. A 6200 bp clone (pE1-6) was iden-tified, which complemented the mutant strain forgrowth on ethanol (Figure 1). Different subcloneswere generated by inserting different fragments inEBOX37p6 plasmid. The shortest subclone (pA,900 bp, Figure 1) that complemented the mutantstrain for growth on ethanol was identified andsequenced. Surprisingly, this clone only expressedan amino-terminal truncated form of Alg2p (�1–198). Since the truncation occurred at the EcoRIsite, the adjacent EcoRI–EcoRI fragment was thensequenced using subclone pB (1150 bp, Figure 1),revealing the missing amino-terminus and its 5′flanking region. The relevant nucleotide sequencehas been deposited at GenBank (Accession No.AF373067.1). Two H. polymorpha STSs have been

ALG2

EE SS H HE

pApB

ALG2

E

pE1-6

1 Kbp

Figure 1. Schematic representation and physical map of the chromosomal fragment (thick line) which complements the H.polymorpha alg2-1 mutation (thin line: pYT3 regions adjacent to the genomic insert). The ALG2 open reading frame of 1458bp is indicated by the arrow. The two subclones capable of complementation are represented by grey boxes (E, EcoRI; S,SacI; H, HindIII)

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806 E. Berardi, A. Gambini and A. R. Bellu

recently disclosed that are highly similar to ALG2,and encompass part of the gene (GenBank Acces-sion Nos AL433051 and AL432278). Althoughmore than one frame-shift appears to have occurredin these sequences, several identities with Alg2pcan be underlined. For instance, the inverted com-plement of AL433051 translates a stretch equalto M3 –R32 of ALG2 in frame 3, and a longstretch equal to F139 –L314 of the same gene inframe 2.

Construction of alg2� strains

The 5′- and 3′-flanking regions of the ALG2ORF were amplified by overlap extension PCR,creating a geneticin resistance cassette betweenthe flanking regions as described (Wach et al.,1994). This PCR product was used to transformstrain L1. Transformants were selected on YPDplates containing 300 µg/ml geneticin, and theexpected chromosomal alteration in the alg2 dele-tion strains (A2�-T1 and A2�-T2), which hadbecome unable to grow on ethanol medium, wasconfirmed by PCR.

ALG2 mRNA levels

RNA was extracted using the method of Rose et al.(1990) and was analysed by northern blotting, usingformaldehyde as a denaturing agent (Sambrooket al., 1989). RNA was transferred to a Hybond-N+ membrane (Amersham, UK) and hybridizedusing standard conditions with probes made by therandom primer method (Sambrook et al., 1989),using DNA fragments derived from the ALG2gene of H. polymorpha and from rDNA encodingthe 25S rRNA of S. cerevisiae. The latter probewas used as an internal loading control for theassessment of ALG2 mRNA levels.

Results

ALG2 encodes a new member of the isocitratelyase family

Complementation of the ethanol growth defect ofthe alg2-1 mutant by a genomic DNA libraryidentified a 62 kbp clone that restored wild-typeisocitrate lyase activity during ethanol or acetategrowth (Table 1). Subcloning and sequence analy-sis revealed an ORF of 1458 nucleotides encoding

a 486 amino acid protein with a predicted molec-ular mass of 54.9 kDa (Figure 1). Sequencing theamino-terminus of Alg2p will disclose whether theactual starting amino acid is Met1 or Met3, andwhether the first methionine is removed by pro-cessing, as in the case, for instance, of S. cerevisiaeIcl1p (Fernandez et al., 1992).

Comparison of this ORF with the databasesrevealed that the ALG2 product is a new mem-ber of the isocitrate lyase gene family (Figure 2).Comparison of the Alg2p amino acid sequence withisocitrate lyases from other sources shows 72%similarity with four other yeast ICLs; they includethe C. tropicalis (63% identity) and P. pastoris(58% identity) ones. The enzymes from S. cere-visiae and E. coli show 69% and 52% similarity,with 56% and 37% identity, respectively.

Southern analyses showed that this gene ispresent in one copy in the H. polymorpha genome(not shown). Haploid strains of H. polymorphadeleted for ALG2 were constructed as describedin Materials and methods. The alg2� strains wereviable in glucose and methanol, but unable to growon ethanol or acetate as the sole carbon source;they showed absence of isocitrate lyase activity(Table 1), and could be complemented by an ALG2containing plasmid. When crossed with an alg2-1 mutant (mutant 149-2, ethanol−; Table 1), thesestrains yielded diploids also unable to grow onC2 compounds and lacking isocitrate lyase activ-ity (Table 1). These data confirm the essential roleof isocitrate lyase for the utilization of ethanol andacetate in H. polymorpha.

Analysis of Alg2p reveals conserved as well asdistinctive features

In Alg2p, highly conserved regions are revealed,such as the hexapeptides KKCGHM (ICL sig-nature; Figure 2) and SPSFNW (active site sig-nature; Figure 2). The former contains a Cys(Cys213), homologous to S. cerevisiae Cys195 andto E. coli Cys217, the significance of which hasbeen discussed (Ko and McFadden, 1990; Rehmanand McFadden, 1997; Robertson and Nimmo,1995); the latter contains the sequence SPS, whichis likely to take part in the formation of theenzyme–substrate bond (Ko et al., 1992). Towardsthe amino-terminus (residues 32–40), this proteincontains a short stretch (RLEGHQESL) rather sim-ilar to the consensus sequence for PTS2, one of

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ALG2, the Hansenula polymorpha ICL gene 807

Figure 2. Sequence alignment of H. polymorpha, C. tropicalis and S. cerevisiae Alg2p orthologues. The amino acid sequenceswere aligned using the ClustalW programme. Identical residues (black for 3/3 and dark grey for 2/3) and similar residues(light grey) are shaded. Similarity rules: A = G; D = E = H = K = R; N = Q = S = T; L = I = V = M = F = Y = W;dashes represents gaps. The two signature hexapeptides mentioned in the text (Hexa#1 and Hexa#2), as well as the point(>) corresponding to the EcoRI site in clone pA are indicated

the microbody (peroxisomal) targeting signals (seeSubramani, 1998). PTS1, the other type of per-oxisomal targeting signal (see Subramani, 1998)is not present in Alg2p. Subcloning experimentshave shown that nearly one half of Alg2p (AA1–198) is dispensable for isocitrate lyase functionand growth on C2 substrates (see Materials andmethods; Figure 2). We speculate that an adjacentsequence, PMR1, used as origin of replication inthe subcloning vector, might drive the transcrip-tion of this truncated version of ALG2. Not onlydoes the deleted protein still contain the active sitesignature already mentioned, but towards its amino-terminus another PTS2-like stretch is present (RLI-AIRTSA). Nonetheless, these premises leave opena number of significant questions, such as thatregarding the first possible amino acid of the trun-cated protein. If, for instance, this were Met216,then the truncated protein would lack a portion ofthe ICL signature, which would make it more dif-ficult to explain why the truncated protein remainsfunctional. Obviously, further work will be neces-sary to tackle these questions.

In S. cerevisiae, Thr53 is involved in a regula-tory mechanism necessary for short-term reversibleinactivation of ICL, probably mediated by its

cAMP-dependent phosphorylation (Ordiz et al.,1996). Interestingly, this residue, conserved inC. tropicalis, is not present in H. polymorpha.Other, as yet unidentified, residues are likely tobe the target of distinct protein kinases mediatingthe irreversible long-term inactivation of S. cere-visiae ICL. Another possible S. cerevisiae cAMP-dependent phosphorylation site is conserved inAlg2p (Thr339, KKFT; see Fernandez et al., 1992),together with other potential phosphorylation sites,which may suggest possible regulation by phos-phorylation of this protein. Finally, the decapep-tide sequence KTKRNYSARD of S. cerevisiae ICL(residues 37–46), important for glucose-inducedproteolytic inactivation (Ordiz et al., 1995) andrather conserved in C. tropicalis, is not found inH. polymorpha Alg2p.

ALG2 mRNA levels are regulated by carbonsource

In S. cerevisiae and other yeasts, the synthesis ofisocitrate lyase is induced by ethanol or acetateand repressed by glucose (Herrero et al., 1985).In H. polymorpha, significant ICL protein levelsand activities are found in ethanol or acetate, but

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not in glucose (Zwart et al., 1983; and our unpub-lished results), suggesting that the expression ofALG2 is controlled by tight regulatory mecha-nisms.

Preliminary RNA blot analysis demonstrated thatonly one kind of mRNA (1.5 kb) supposed tobe transcribed from this gene was present in thecells. The regulation of ALG2 mRNA levels bycarbon source was examined at late exponentialphase in an ALG2 strain (L1) grown in min-imal medium with high glucose content (2%),low glucose content (0.2%), ethanol or glucoseplus ethanol. Since preliminary experiments hadshown that ALG2 transcripts are also present inthe alg2-1 mutants, strain S149-2 (alg2-1) wasincluded in northern blot experiments. With thisstrain, ethanol induction was obtained by grow-ing it in 2% glucose, harvesting the cells andtransferring them in 2% ethanol medium for 12 h.As shown in Figure 3, the ALG2 regulation inthe two strains is quite similar. Growth in high-glucose medium show very low levels of transcript,whereas low-glucose as well as ethanol mediumshow strong ALG2 mRNA signals. Since, thelow-glucose medium did not show any detectableethanol during growth (as revealed by samplingevery hour until the end), we suggest that theobserved mRNA levels in this medium is notdue to the presence in the flask of ethanolexcreted by the cell population during growth.Finally, growth in high-glucose + ethanol mediumshow very low levels of transcript, comparableto those obtained in high-glucose medium (notshown).

ALG2

rDNA

1 2 3 4 5 6

Figure 3. Northern blot showing an analysis of theregulation of ALG2 transcription by carbon source in H.polymorpha L1 (ALG2) and s149-2 (alg2-1). Lane 1, ALG2 in2% glucose; lane 2, ALG2 in 0.2% glucose; lane 3, alg2-1 in0.2% glucose; lane 4, alg2-1 in 2% glucose; lane 5, ALG2 in1% ethanol; lane 6, alg2-1 in 1% ethanol (loading control:DNA for ribosomal 25S)

Therefore, our results indicate that glucoserepression, rather than ethanol induction, could bethe main controlling mechanism of isocitrate lyaseexpression in H. polymorpha.

ALG2 is not involved in peroxisome degradation

It has been suggested that, in the methylotrophicyeast Pichia methanolica, isocitrate lyase activ-ity (as well as acetyl-CoA synthetase activity) isessential for C1-peroxisome degradation triggeredby ethanol (Kulachkovsky et al., 1997; see Intro-duction). In order to examine whether ALG2 andthe isocitrate lyase activity are essential for per-oxisome degradation in H. polymorpha, we didtime-course experiments of methanol oxidase (AO)activity (a C1-peroxisomal marker activity) aftermethanol–ethanol shifts in a control strain as wellas in two independent alg2� disruptants and in analg2-1 mutant. As shown in Figure 4A, the kinet-ics of AO activity disappearance are similar in thefour strains, indicating that in H. polymorpha nei-ther the isocitrate lyase activity nor its gene productare necessary for peroxisome degradation triggeredby ethanol. Western analysis confirms that alg2�disruptant carries out normal AO degradation afterthe shift (Figure 4B).

Discussion

We have identified the H. polymorpha isocitratelyase gene by functional complementation of analg2-1 mutant strain previously isolated in ourlaboratory. Although, the amino acid similaritybetween some of the previously characterized yeastICL proteins and Alg2p is in some cases extremelyhigh, this protein reveals distinctive features, ifcompared to other yeast ICLs, and particularly tothose of S. cerevisiae and C. tropicalis.

Alg2p lacks, at the carboxy-terminus, threeamino acid stretches generally present in all otheryeast ICLs (see the three gaps towards the carboxy-terminus of Alg2p, Figure 2). With respect to S.cerevisiae and C. tropicalis, more than 60 residuesare missing, including the last 44. It is noteworthythat many of these sequences contain a PTS1 signalwhich, in some cases, has been shown to target ICLto the glyoxysome (e.g. AKV-COOH in C. tropi-calis). This fact implies that Alg2p does not makeuse of a PTS1 mechanism for glyoxysome import,

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0 1 4 6

D alg2

2 0 1 4 62

ALG2

0

20

40

60

80

100

0 5 10 15 20 25

AO

act

ivity

(%

)

A

B

Time (h)

Figure 4. (A) Evolution of methanol oxidase activity in methanol-grown cells of H. polymorpha after shift in ethanolmedium. -�-, L1 (control); -�-, alg2-1; -©-, alg2�–T1; -♦-, alg2�–T2. (B) Western blot analysis of AO degradation inwild-type and alg2�–T1. Samples were taken at t = 0, 1, 2, 4 and 6 h after the shift (equal amounts of protein were loadedin each lane)

which is in line with the lack, in this protein, ofany reasonable PTS1 consensus. In this respect,Alg2p resembles the N. crassa and A. nidulansICLs, which are only one or two amino acids longerthan the E. coli ICL. It has been suggested that alsothese ICL species do not use a PTS1 mechanismfor import (Taylor et al., 1996). Since subcellularlocalisation studies suggest that the H. polymor-pha ICL is glyoxysomal (Zwart et al., 1983), ALG2isolation opens up the possibility of tackling itstargeting and import mechanism. Interestingly, aPTS2-like signal is present in this isocitrate lyase,although this sequence differs for one amino acidfrom the canonical PTS2 consensus (serine insteadof glutamine or histidine in penultimate position).Whether or not this sequence is required for importclearly needs further investigation.

Alg2p lacks homologues to some of the S.cerevisiae sequences known to be implicated inshort- and long-term glucose inactivation of ICL(see Results). These include threonine 53 and thedecapeptide KTKRNYSARD. Thus, if Alg2p issubject to post-translational regulation, other sitesmust be involved, as already discussed. Again, thisimportant issue will have to await further work.

The results presented in this paper provide clearevidence for strong regulation of ALG2 mRNA

levels by carbon source. In contrast to S. cere-visiae, transcriptional regulation of ALG2 is mainlyachieved by repression–derepression mechanisms,ethanol induction playing only a minor role. Thereason for this ‘gratuitous’ induction of ALG2transcription (and possibly that of genes encod-ing other glyoxylate enzymes) is unknown, and itsunderstanding will require more detailed metabolicstudies. However, since extracts of low glucose-grown cells show low ICL activities despite theirhigh mRNA levels (our unpublished results), wespeculate that, in these cultural conditions, Alg2pis somehow inactive. Therefore, the presence ofethanol and the absence of glucose may trigger theactivation of Alg2p, thus achieving a rapid cellu-lar launch of the two-carbon metabolism. Promoteranalysis, as well as the identification of trans-actingelements and of post-translational regulatory mech-anisms, will help elucidate the network acting uponthe ICL activity in H. polymorpha.

Finally, the finding that in H. polymorpha nei-ther the isocitrate lyase activity nor the ALG2gene product are necessary for C1-peroxisomedegradation triggered by ethanol, indicates that themechanism suggested by Sibirny and co-workersfor P. methanolica (Kulachkovsky et al., 1997)does not necessarily apply to all methylotrophic

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yeasts. Therefore, the nature of the effector andof the mechanisms evolved by H. polymorpha todegrade C1-peroxisomes in response to ethanolremains to be determined.

Acknowledgements

We thank Jan Kiel and Marten Veenhuis for useful discus-sion and for providing the AO antiserum. We are gratefulto Veronica Vallini for assistance throughout the project.

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