JOURNAL OF BIOIXOICAL 269, No. 1, Issue of 7, pp. 556-566 ...Tm JOURNAL OF BIOIXOICAL CHEMISTRY 0 1994 by The American Soeiety for Biochemistry and Molecular Biology, Inc. Vol. 269,

Tm JOURNAL OF BIOIXOICAL CHEMISTRY 0 1994 by The American Soeiety for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 1, Issue of January 7, pp. 556-566, 1994 Printed in U S A .

PAY4, a Gene Required for Peroxisome Assembly in the Yeast Yarrowia ZipoZytica, Encodes a Novel Member of a Family of Putative ATPases*

(Received for publication, July 13, 1993)

William M. Nuttley, Anthony M. Brade, Gary A. Eitzen, Marten VeenhuisS, John D. Aitchisong, Rachel K. SzilardR John R. Glove* and Richard A. Rachubinskill From the Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 325, Canada and the $Laboratory for Electron Microscopy, University of Groningen, 9750 AA Haren, The Netherlands

PAY genes are required for peroxisome assembly in the yeast Yarrowia lipolytica. Here we characterize one mutant, pay4, and describe the cloning and sequencing of the PAY4 gene. The pay4 mutant shows no identifiable peroxisomes by biochemical and morphological criteria. The complementing PAY4 gene encodes a polypeptide, Pay4p, 1025 amino acids in length and having a pre- dicted molecular mass of 112,258 Da. The predicted Pay4p sequence contains two putative ATP-binding do- mains and shows structural relationships to other po- tential ATP-binding proteins involved in biological pro- cesses as diverse as peroxisome biogenesis, vesicle- mediated protein transport, cell cycle control, and transcriptional regulation. These proteins all share a highly conserved stretch of approximately 175 amino acids that contains a consensus sequence for ATP bind- ing. Pay4p shows sequence conservation with Paslp and PasSp, putative ATPases required for peroxisomal as- sembly in the yeasts Saccharomyces cerevisiae and Pichia pasforis, respectively. Pay4p, Paslp, and PasSp are presumably related members of a family of putative ATPases involved in peroxisome biogenesis. Pay4p is synthesized in low amounts in K lipolytica cells grown in glucose, and there is a rapid and pronounced increase in the levels of Pay4p upon transfer of the cells to a medium containing oleic acid as the sole carbon source.

Eukaryotic cells have evolved a complex set of organelles, with each organelle possessing a specific complement of en- zymes required for its particular metabolic role. This compart- mentalization of biochemical functions permits a level of meta- bolic control unavailable to prokaryotes. However, it presents the eukaryotic cell with the problem of targeting proteins from their sites of synthesis in the cytoplasm to their specific organ- ellar locations. Organelles like the mitochondrion, chloroplast, and endoplasmic reticulum have been extensively investigated, and much is now known about their biogenesis and how pro-

Research Council of Canada. The costs of publication of this article were * This work was funded by the Natural Sciences and Engineering

defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s1 reported in this paper has been submitted to the GenBankTMIEMBL Data Bank with accession number($ L23858.

5 Current address: Laboratory for Cell Biology, The Rockefeller Uni- versity, New York, N Y , 10021. ll Recipient of Studentship from the Medical Research Council of

Canada. 11 Medical Research Council of Canada Scientist. To whom correspond-

ence should be addressed McMaster University, Dept. of Biochemistry, 1200 Main St. West, Hamilton, ON L8N 325, Canada. Tel.: 416-525- 9140 (ext. 2316); Fax 416-522-9033.

teins are targeted to each. In contrast, peroxisomes have re- ceived much less attention, and only recently have the cellular events involved in peroxisome biogenesis started to be eluci- dated.

Peroxisomes appear to arise by growth and fission of preex- isting peroxisomes (reviewed in Lazarow and Fujiki (1985) and Borst (1986,1989)). Peroxisomes do not contain DNA(Kamiry0 et al., 1982) and do not possess any independent protein syn- thetic machinery as do mitochondria and chloroplasts. Peroxi- somal proteins are encoded by nuclear genes, synthesized on free polysomes, and imported posttranslationally into the per- oxisome in an energy-dependent manner. Two sequence motifs have been identified which can act to target proteins to the peroxisome matrix. A carboxyl-terminal peroxisomal targeting signal (PTS),l having the generalized structure Ser/Ala/Cys- LydArgMis-Leu/Metle/Phe (F’TS-11, has been shown to be sufficient to target proteins to peroxisomes (for reviews see Subramani, 1992; Aitchison et al., 1992a; de Hoop and Ab, 1992). F’TS-1 sequences have also been found in proteins tar- geted to glyoxysomes and glycosomes, which along with peroxi- somes form the microbody family of organelles, and in diver- gent organisms (mammals, plants, yeasts, trypanosomes, and insects; Gould et al., 1990a; Keller et al., 1991; Aitchison et al., 1991; Blattner et al., 1992). A second peroxisomal targeting signal (PTS-2) has also been identified in rat liver 3-ketoacyl- CoA thiolase, which is present as an amino-terminal extension on the newly synthesized protein, and cleaved upon import into the peroxisome (Swinkels et al., 1991).

Recently the power of yeast genetics has been applied to the study of peroxisome biogenesis. Peroxisome assembly mutants have been isolated in Saccharomyces cerevisiae (Erdmann et al., 1989; van der Leij et al., 19921, Hansenula polyrnorpha (Cregg et al., 1990), and Pichia pastoris (Liu et al., 1992; Gould et al., 1992). These mutants are characterized by a lack of morphologically identifiable peroxisomes and by a mislocaliza- tion of peroxisomal matrix proteins to the cytosol. Both of these features have been described for cells of individuals afflicted with the human autosomal recessive peroxisomal disorder, Zellweger syndrome (Schutgens et al., 1986; van den Bosch et al., 1992). Therefore, yeast peroxisome assembly mutants rep- resent a n excellent model system to study the genetic defects involved in this disease.

We have recently isolated peroxisomal assembly (pay) mu- tants in the yeast Yarrowia lipolytica (Nuttley et al., 19931, which is ideally suited for studies of peroxisomal assembly. First, it is amenable to both classical and molecular genetic

2OkgP, 20,000 x g pellet (primarily peroxisomes and mitochondria); The abbreviations used are: PTS, peroxisomal targeting signal;

2OkgS, 20,000 x g supernatant (primarily cytosol); bp, base pair; kbp, kilobase pair; PAGE, polyacrylamide gel electrophoresis.

556

Characterization of PAY4 gene of E: lipolytica 557

TABLE I E: lipolytica strains

Strains"

E122' 22301-3l pay2' PBY4'

~4-223

PAY43 P4-K03

Genotype

MATA, ura3-302,leu2-270, l y s S l l MATB, ura3-302,leu2-270, his1 U T A , pay2, ura3-302,leu2-270, ly&-11 MATA, pay4, ura3302,1eu2-270,1ys8-11 MATA, pay4, ura3-302,leu2-27O,lys8-11 pOl(LEU2) MATA, leu2-270, ura3-302,lysSll, pay4::LEUa MATA/MATB ura3-302/ura3-302,leu2-270Aeu2-270,

lys%ll/+, hid+, pay4:LEU2/+

a Source: C. Gaillardin, Thiverval-Grignon; Nuttley et al. (1993); This study.

techniques, thereby permitting the dissection of the mecha- nism of peroxisome biogenesis by a genetic approach. Second, Y Eipolytica grows well on oleic acid relative to a yeast like S. cerevisiae, with a concomitant strong induction of peroxisomes and peroxisomal proteins. This feature facilitates experimental procedures such as the identification of peroxisomal assembly mutants and the isolation of affected genes by functional complementation that involve the observation of growth on oleic acid, as well as the analysis of proteins required for per- oxisomal assembly.

Herein we report the detailed morphological and biochemical characterization of one pay mutant, pay4, and the cloning and analysis of the functionally complementing PAY4 gene. The mutant pay4 strain fails to assemble normal peroxisomes and mislocalizes peroxisomal matrix enzymes to the cytosol. The PAY4 gene encodes a polypeptide, Pay4p, of -112 kDa. Pay4p is a novel member of a superfamily of putative ATPases and shows similarity to proteins involved in processes as diverse as vesicle-mediated transport, control of cell cycle, and gene ex- pression in human immunodeficiency virus. Pay4p also shows similarity to Paslp and Pas5p, putative ATPases shown to be essential for peroxisome assembly in S. cerevisiae (Erdmann et al., 1991) and I! pastoris,2 respectively. Together these proteins form a family of putative ATPases involved in peroxisome bio- genesis in yeast.

MATERIALS AND METHODS Strains and Media-Yeast strains used in this study are given in

Table I. Yeast were grown in complete (YEPD) or minimal (YNO, YND, and induction) media, as required. YEPD medium contained 1% yeast extract, 2% peptone, 2% glucose; YNO medium contained 0.67% yeast nitrogen base without amino acids, 0.05% (w/v) Tween 40, 0.1% (w/v) oleic acid; YND medium contained 0.67% yeast nitrogen base without amino acids, 0.1% yeast extract, 2% glucose; induction medium con- tained 0.67% yeast nitrogen base without amino acids, 0.5% yeast ex- tract, 0.5% peptone, 0.1% glucose, 0.1% (wh) oleic acid, 0.5% (w/v) Tween 40 (Erdmann et al., 1989). Media were supplemented with uracil, leucine, lysine, and histidine each at 50 pgm-', as required. Growth was at 30 "C unless specified differently.

Isolation of Pay4 Mutants-Pay4 mutants were isolated after muta- genesis of E122 cells with 1-methyl-3-nitro-1-nitrosoguanidine (Gleeson and Sudbery, 1988). The screening protocol included selection for an inability to utilize oleic acid as a carbon source, fractionation into 2OkgP (primarily peroxisomes and mitochondria) and 2OkgS (primarily cyto- sol) fractions of yeast cells (Aitchison et al., 1991), and immunofluores- cence microscopy (Aitchison et al., 1992b) with anti-SKL antibodies. Fixation and preparation for electron microscopy was performed as described previously (Waterham et al., 1992) Mutants were character- ized by standard genetic techniques for E: lipolytica (Gaillardin et al., 1973).

Marker Enzyme Analyses-The following marker enzyme activities were measured: peroxisomes, catalase (Baudhuin et al., 1964) and p-hy- droxyacyl-CoA dehydrogenase (Osumi and Hashimoto, 1979); mitochon- dria, cytochrome c oxidase (Cooperstein and Lazarow, 1951).

Cloning and Characterization of the PAY4 Gene-To isolate the PAY4 gene, the strain pay4 was transformed by electroporation with a ge-

' S. Subramani, personal communication.

nomic DNA library of E: lipolytica contained in the Escherichia coli shuttle vector pINA445 (Nuttley et al., 1993). Leu' transformants were screened on YNO-agar plates for their ability to utilize oleic acid as a sole carbon source. Complementing plasmids were recovered by trans- formation of E. coli.

Standard recombinant DNA methodology including enzymatic modi- fication of DNA, DNA fragment purification, and plasmid isolation was performed essentially as described in Ausubel et al. (1989).

DNA Sequencing-Various restriction endonuclease fragments of the PAY4 gene were inserted into the vector pGEM"IZfl+) (Promega, Madi- son, WI) for dideoxynucleotide sequencing of both strands from double- stranded templates (Sanger et al., 1977; Zhang et al., 1988). The de- duced Pay4p sequence was used to search the SWISS-PROT Protein Sequence Data Bank release of August 23, 1992, for similarities with other known protein sequences using the FSTPSCAN program of PC/ GENE (IntelliGenetics, Mountain View, CA).

Integrative Disruption of the PAY4 Gene-The LEU2 gene of E: lipo- Zytica was isolated as an Eco47IIYEglII fragment from pINA445 and inserted into the PAY4 gene digested with StuI and BglII. This con- struction replaced 809 base pairs (bp) of the PAY4 gene open reading frame with an approximately 2.1-kbp fragment containing the entire LEU2 gene. This construct was digested with SphI and ScaI to liberate the LEU2 gene flanked by 720 and 280 bp of the PAY4 gene at its 5' and 3' ends, respectively. This linear molecule was used to transform E: lipolytica E122 to leucine prototrophy. Leu' transformants were screened for the ole- phenotype and mated to 22301-3. Diploids were sporulated, and random spore analysis performed. The segregation of the Leu+ and ole- phenotypes was analyzed by replica plating. pay4::LEUa segregants were mated to pay4 and the resultant diploids checked for complementation.

Antisera-For the production of antibodies to Pay4p, a 1302-bp frag- ment of the PAY4 gene open reading frame encoding amino acids 24- 458 of Paylp was excised with Ecl 13611 and EgZII and inserted into the pMALc2 vector (New England Biolabs) in-frame and downstream of the open reading frame encoding the maltose binding protein. Expres- sion of the maltose binding protein gene is under the control of the "tac" promoter, which is induced in the presence of isopropyl-P-D-thiogalac- topyranoside. A lysate of E. coli synthesizing the maltose binding pro- tein-Pay4p fusion was prepared essentially as described by Ausubel et al. (1989), and the fusion protein was purified further by SDS-PAGE (Laemmli, 1970; Fujiki et al., 1984) on a 10% preparative gel. The fusion protein was electroeluted into dialysis tubing, dialyzed against 50 rn ammonium bicarbonate, lyophilized, and dissolved in a minimal amount of distilled, deionized water. Antibodies were raised by primary injections of 100 pg of fusion protein into New Zealand White rabbits at multiple subcutaneous sites. m e r 5 weeks, booster injections of 50 pg were administered subcutaneously at multiple sites. Six weeks after the primary injections, the rabbits were sacrificed, their blood collected, and sera prepared. The specificities of antisera were determined by western blotting (Burnette, 1981) of yeast cell lysates (Needleman and Tzagoloff, 1975; Nuttley et al., 1990). Antigen-antibody complexes were detected by enhanced chemiluminescence (Amersham Corp.) and quantitated with a GS-300 scanning densitometer and the GS-350 data analysis system (Hoefer Scientific Instruments, San Francisco, CA).

Anti-SKL serum was a gift of Dr. S. Subramani, University of Cali- fornia, San Diego. Antiserum to S. cerevisiae peroxisomal thiolase was a gift of Dr. W.-H. Kunau, Ruhr-University.

RESULTS

Characterization of the Pay4 Mutant-The Y lipolytica mu- tant strain pay4 has been shown to be incapable of growth on oleic acid (Fig. 1, pay4; Nuttley et al., 1993), Pay4 has been shown to be a mutant in peroxisome assembly by two criteria: the lack of characteristic punctate peroxisomal staining with anti-SKL antibodies in double-labeling immunofluorescence microscopy and the mislocalization of peroxisomal marker en- zymes t o the cytosol (Nuttley et al., 1993). Analysis of the pa- rental and mutant strains by electron microscopy supports the classification of the pay4 mutant as peroxisome-deficient. Pa- rental cells grown in the presence of oleic acid, show many peroxisomes (microbodies) scattered through out the cell (Fig. 2, B and C; Mb). In contrast, no peroxisomes could be detected in a survey of mutant pay4 cells grown under the same condi- tions (Fig. 2, A and D ) . These results extend our results from immunofluorescence analysis with anti-SKL antibodies (Nut-

558 Characterization of PAY4 gene of X lipolytica

FIG. 1. Growth of various yeast strains on oleic acid medium. Appear- ance of the complemented strain PAY4 in comparison to the parental strain E122, the mutant strain pay4, the gene disrup- tion strain P4-K0, the diploid D4-22 (P4-KO x 22301-3), and a second parental strain 22301-3 (not supplemented with the correct auxotrophic requirements). Growth was for 5 days on YNO-agar.

E122

PAY4

P4”KO

22301-3

D4--22

tley et al., 1993; see also Fig. 4, below) which revealed a lack of peroxisomal staining in the mutant cells. Peroxisomal ghosts, aberrant membrane structures containing peroxisomal mem- brane proteins, have been described in a number of peroxisome- deficient mutants from various sources (Santos et al., 1988; Zoeller et al., 1989; Gould et al., 1992). Analysis of micrographs of oleic acid-induced pay4 cells has revealed no evidence of such structures in this strain.

A PTS-2-containing Protein Is Mislocalized to the Cytoplasm in the pay4 Mutant-We have previously demonstrated that peroxisomal proteins containing PTS-1 motifs are mislocalized to the cytoplasm in pay mutants (Nuttley et al., 1993). We investigated whether a peroxisomal protein containing a PTS-2 motif, i.e. thiolase, is also mislocalized to the cytoplasm in the mutant pay4. Antibodies to peroxisomal thiolase of S. cerevisiae recognize thiolase from E: lipolytica. Western blot analysis of 2OkgP (primarily peroxisomes and mitochondria) and 2OkgS (primarily cytosol; Aitchison et al., 1991) fractions of the paren- tal E122 strain showed a single polypeptide band of molecular weight -43,000 exclusively in the 2OkgP fraction (Fig. 3, lane P ) , reflecting the peroxisomal localization of thiolase. Analysis of the pay4 mutant showed that thiolase is not localized to peroxisomes (2OkgP, lane P ) in this mutant but mislocalized to the cytoplasm (20kgS, lane S). Moreover, the thiolase in the 2OkgS (lane S ) of the pay4 mutant showed reduced electropho- retic mobility relative to that in the 2OkgP fraction of the pa- rental E122 strain. Combining the E122 20kgP and pay4 2OkgS fractions showed that this difference in molecular weight was not an artifact of electrophoresis and is most likely evidence of proteolytic cleavage of an amino-terminal PTS-2 sequence upon import of thiolase into the peroxisomal matrix. Cleavage of an amino-terminal PTS-2 has been shown for rat liver peroxisomal thiolase (Swinkels et al., 1991), and our results represent the first demonstration, to our knowledge, that such a process may also occur in yeast peroxisomes.

Isolation of the PM4 Gene-Transformation of the pay4 mu- tant strain with a library of E: lipolytica genomic DNA yielded 42 transformants capable of restored growth on oleic acid. Three independent recombinant plasmids, designated pol , p02, and p03, were rescued into E. coli. A combination of

restriction mapping and subcloning analysis showed that the complementing activity of the inserts was localized to a com- mon 4.5-kbp segment (data not shown). Retransformation of pay4 with any one of pol , p02, or p03 confirmed that the recombinant plasmids were responsible for the complementing activity. A transformant, hereafter called PAY4 (Fig. 11, harbor- ing the plasmid p o l was used for further study.

The putative PAY4 gene was used for a gene disruption ex- periment with the E: lipolytica LEU2 gene, as described under “Materials and Methods.” A leu+/ole- transformant, designated P4-K0, was isolated, and integration of the LEU2 gene into the PAY4 locus was confirmed by Southern blot analysis (data not shown). The recessive nature of the ole- phenotype was dem- onstrated by the ability of the P4-KO x 22301-3 diploid to grow on oleic acid (Fig. 1,04-22). Sporulation of the diploid D4-22 showed cosegregation of the ole- and leu+ phenotypes. When an ole-, matB isolate from sporulation of D4-22 was back-crossed to the original pay4 mutant, the resulting diploids were inca- pable of growth on oleic acid, thereby confirming that the au- thentic PAY4 gene had been cloned.

Antibodies raised to the carboxyl-terminal PTS-1 sequence Ser-Lys-Leu (SKL) were used to investigate the targeting of F’TS-1 containing proteins by double-labeling immunofluores- cence microscopy. These antibodies reveal a punctate pattern characteristic of peroxisomes in parental E122 cells (Aitchison et al., 1992b; Fig. 4, top panel ). This pattern was absent in the pay4 mutant strain (middle panel) but was restored in the pay4 strain transformed with p o l to give the PAY4 strain (bottom panel). The presence of peroxisomes was also demon- strated by examination of the PAY4 transformant by electron microscopy. As shown in Fig. 5, Panels A and B, transformed cells display a morphology indistinguishable from that of the parental strain, including the presence of numerous peroxi- somes (Mb). Examination of the P4-KO strain (Fig. 5, Panel C ) , in which the PAY4 gene has been disrupted by insertion of the LEU2 gene, revealed that this strain lacked any morphologi- cally detectable peroxisomes.

Transformation of pay4 with p o l to yield PAY4 also restored the correct localization of peroxisomal marker enzyme activi- ties. In the parental strain E122, approximately 30% of the

Characterizatioh of PAY4 gene of X lipolytica 559

. _ , .

r

FIG. 2. Ultrastructure of the parental E122 (Paneb B and C ) and pay4 mutant (PanelsA andD) atrains. Cells were pwn to saturation in YEPD medium, diluted 1:4 into YNO medium, and grown for a further 24 h. The cells were then fixed with KMnO, and processed for electron micr08~0py. M b , microbody (peroxisome); N, nucleus; V, vacuole. Bur = 1 p m .

catalase activity and 60% of the p-hydroxybutyryl-CoA dehy- drogenase activity were found in the 2OkgP fraction upon sub- cellular fractionation (Fig. 61, reflecting the peroxisomal loca- tion of these enzymes. The activities of these enzymes recovered in the 2OkgS fraction were due, at least partially, to leakage from peroxisomes broken during the fractionation pro- cedure (Aitchison et al., 1991). The relatively larger amounts of catalase in the 2OkgS may be due to preferential leakage of catalase from the peroxisome (Alexson et al., 1985). There may also be cytosolic and peroxisomal isoforms of catalase in I: lipolytica, as there are in S. cereuisiue (Hartig and Ruis, 1986; Cohen et al., 1988); however, this has not been investigated further. In the pay4 mutant strain (as well as in the disrupted strain P4-K0), less than 3% of catalase activity and approxi- mately 10% of dehydrogenase activity were recovered in the 2OkgP. Transformation of pay4 with p o l corrected this defect, resulting in the recovery of catalase and dehydrogenase activi- ties in the 2OkgP fraction at levels comparable to those found in the parental strain E122. The localization of the mitochondrial marker enzyme cytochrome c oxidase to the 2OkgP fraction was

not affected by the pay4 mutation, as comparable levels of cytochrome c oxidase activity were found in the 20kgP fractions of E122, pay4, PAY4, and P4-KO.

Nucleotide Sequence of the PW4 Gene and the Deduced Amino Acid Sequence of Pay4pSequencing of the 4567-bp BamHI fragment common to the three complementing plas- mids pol , p02, and p03 revealed an open reading frame en- coding a protein of 1025 amino acids and having a predicted molecular weight of 112,258 (Fig. 7). This predicted molecular weight is in agreement with the relative molecular weight of Pay4p determined by SDS-PAGE and western blot analysis (see Figs. 11 and 12). The nucleotides surrounding the proposed initiating codon conform to the consensus sequence for trans- lation initiation in yeast, with a conserved A at position -3 and a conserved C a t position +5 relative to the A of the initiation codon (Cigan and Donahue, 1987).

A putative TATA element (TATATA’ITA) in the 5’-untrans- lated region of the PAY4 gene is found between positions -461 and -453 relative to the A of the proposed initiation codon. This relatively large distance between the putative TATA element

560 Characterization of PAY4 gene of E: lipolytica

€122 pay4

S" P pay4S S + " "

P

106-

00-

49.5 .c

32.5-

27.5 +

h o . 3. Localization of thiol- in the pusntal E122 and mu- tant pay4 strains grown on induction medium. The equivalent cellular fractions of t h r nupernabant t S ) and prllrt CP) fractions from a 20.000 x R crntrifugntion of postnuclear suprrnatanta were srparated hy SIX-PAGE, transfrrrrd to nitmcellulone. and probed with antiserum to S. rrwuisine thiolasr. The numbers a t left indicate the mivations of molecular mans standards (in k h l .

and the init iator codon makes it unlikely that this element plays a role in the transcriptional regulation of the PAY4 gene (Xuan et al., 1990; Heslot and Gaillardin, 1992). It is possible tha t t he expression of the PAY4 gene is controlled by a "TATA- less" promoter. Such a scenario probably exists for the Y fipo- iytica LYS5 gene, which does not display an obvious TATA box. Transcription of this gene was shown to initiate at a 13-bp CA-rich region (position -50 relative to the A of the initiator codon) immediately downstream of a n 11-bp CT-rich region. This CT-rich region is preceded by a second 11-bp CT-rich re- gion located approximately 70 bp upstream of the first CT-rich region (Xuan et al.. 1990). A strikingly similar situation exists in the region upstream of the initiator codon of the PAY4 gene. A 14-bp CA-rich region between -20 and -7 is immediately preceded by a 12-bp CT-rich region between -32 and -21. A second CT-rich region of 11 bp occurs between -77 and -67.

In Y lipolytica, the TAG . . . TGAT . . . TTT transcription ter- mination motif (Zaret and Sherman, 1982; Sutton and Broach, 1985) is found at the 3' ends of four genes whose sequences have been published (Davidow et al . , 1985, 1987; Xuan et al . , 1990; Kattig et al., 1991). This motif is also found in the PAY4 gene starting at position +3152. 73 nucleotides downstream of the stop codon. A " T A T A transcription termination motif (Henikoff and Cohen. 1984) is also found in the PAY4 gene starting a t nucleotide +3264.

Analysis of the Pay4p Sequence-A number of ATP-binding proteins have been shown by comparative studies using pri- mary sequence and crystallographic data to share certain con- sewed motifs. The sequence motif (A or G)-X-X-X-X-G-KG or T). known as the "P-loop" or "A-motif," represents the consen- sus sequence for the most conserved of these motifs and is believed to form a flexible phosphate binding loop between a p-sheet and an a-helix (Walker et al., 1982; Saraste et al., 1990). This motif was found twice in Pay4p at amino acid po- sitions 477-484 and 760-767. This arrangement of duplicated

ATP-binding domains separated by 200-300 amino acids is characteristic of a recently described family of presumptivr MgY+-dependent Amases that include the peroxisomal assrm- bly proteins Paslp of S. cerwisine f Erdmann et ai . . 1991 and Pas5p of I? pastoris.' Other members of this family includr S . cerevisiae SeclAp and i h mammalian counterpart NSF, which have been shown to be involved in vesicular fusion along the secretory pathway fEakle et al . , 1988; Wilson ~t af., 19WN; S . cerevisiae Cdc48p (Frnhlich et ai. . 1991 I, involved in cell cyclt- regulation; and two vertebrate homologues of Cdc4Rp. VCP (porcine vasolin-containing protein; Koller and Rrownstrin, 1987) and P97 from Xenopus inet-is f Prters et a / . , 19901. In contrast, a single.4TP binding motif is found in t h r TAT-hinding protein (TRP-1) of human immunodeficiency virus that is in- volved in viral gene expression (Nrlhock et ai . , 19901.

Characterization of PAY4 gene of Y: lipolytica 561

FIG. 5. ultrastructure of the PAY4 transformant (Pane& A and B ) and P4KO mutant (Panel C ) strains. Cells were induced in YNO medium, fixed with KMn04, and processed for electron microscopy. Mb, microbody (peroxisome); N, nucleus. Bar = 1 pm.

The ATP-binding domains of all the above proteins show a high degree of sequence conservation over a region of approxi- mately 175 amino acids; however, in direct comparisons with Pay4p, the second domain of Pay4p shows the highest degree of sequence conservation with the ATP-binding domains of the different proteins. As seen in Fig. 8, the second ATP-binding domain of Pay4p is strongly conserved in Paslp (Panel A 1, NSF (Panel B) , and Secl8p (Panel C). Both of the ATP-binding do- mains of VCP (Panel D) and Cdc48p (Panel E ) show a high degree of conservation with the second domain of Pay4p. The single ATP-binding domain of TBP-1 shows higher identity with the second domain of Pay4p (Panel F ) . Comparison of the Pay4p and Pas5p sequences also revealed a high degree of sequence conservation over the ATP-binding domains. These two sequences also display a significant sequence conservation along the entire length of the proteins (Fig. 9). The nine most highly conserved domains are aligned in Fig. 10. The high degree of sequence conservation among the domains is evi- denced by the fact that 16% of the amino acid residues are

identical in all nine sequences, and an additional 29% of the amino acids are similar.

The similarities in Pay4p, Paslp, and Padp, together with the fact that these proteins were identified through their role in peroxisome biogenesis, might suggest that these proteins are functional homologues in the yeasts k: lipolytica, S. cerevisiae, and Rpastoris, respectively. However, comparison of the Pay4p and Paslp reveals that the similarity between the two se- quences is limited primarily to the second ATP-binding do- mains of the two proteins (49.4% identity/l6.7% similarity), with overall sequence identity of 21.5% and sequence similarity of 14.9%. The primary sequence of Pas5p required for peroxi- some assembly in the yeast R pastoris2 has been found to exhibit a high degree of conservation with Pay4p, with 59.0% identity and 15.3% similarity between their carboxyl-terminal ATP-binding domains and 46.0% identity and 15.3% similarity over the entire length of the polypeptides. Pas5p shows a lesser degree of conservation with Paslp, with 24.1% identity and 15.1% similarity, with the alignment being concentrated in the

562 Characterization of PAY4 gene of Y lipolytica catalase dehydrogenase @Tj?Jj cyt. c oxidase

80 ~~ ." - ." ~ ~~ ~~ ~~

70 1

E l 22 pay4 PAY4 PAY4-KO

lipolytica strain^ grown in induction medium. The supernatant Fro. 6. Fractionation charactariatics of marker enzymes in E

(2OkgS) and pellet (2OkgP) fractions from a 20,000 x g centrifugation of postnuclear supernatants were assayed for catalase, p-hydroxybutyryl- CoA dehydrogenase, and cytochrome c oxidase activities. The percent- ages of total activities recovered in the pellet fractions are shown.

ATP-binding domain. Therefore, while Pay4p and Pas5p are closely related at the primary sequence level and may be func- tional homologues, Paslp appears to be a related, but non- homologous, member of a superfamily of putative ATPases in- volved in peroxisome biogenesis.

Immunological Detection and Characterization of Pay4p "Rabbit polyclonal antibodies raised against a maltose bind- ing protein-Pay4p fusion were used to identify the product of the PAY4 gene. The antibodies detected a polypeptide of -112 kDa in Y lipolytica grown in oleic acid that was not recognized by the preimmune serum (data not shown). The anti-Pay4p antibodies were used in a Western blot analysis to probe cell lysates of oleic acid-grown parental strain E122, the pay4 mu- tant strain, the PAY4 gene disruption strain P4-KO, and a second peroxisome assembly mutant strain pay2 (Fig. 11). The immunoreactive 112-kDa polypeptide was detected in cell ly- sates of the E122 parental strain and the pay2 mutant strain but was absent in cell lysates of the pay4 mutant strain and the knockout strain P4-KO. These data suggest that the mutation in the pay4 strain does not result in the production of a non- functional form of Pay4p but is most probably a nonsense mu- tation resulting in premature termination of translation.

Immunological detection of Pay4p was used to investigate the regulation of the PAY4 gene. Parental E122 cells were transferred from glucose medium to medium containing oleic acid as the sole carbon source. The presence of oleic acid as the sole carbon source allows for the proliferation of peroxisomes and the induction of peroxisomal enzymes in various yeast species. Cell lysates were prepared at various time points after transfer of E122 cells to oleic acid medium. The lysates were analyzed by western blotting with anti-Pay4p and anti-SKL antibodies (Fig. 12). In E122 cells grown in glucose (T = 01, a small amount of Pay4p could be detected in the cell lysate, but no anti-SKI, immunoreactive polypeptides were seen. Pay4p was detectably induced (5-fold) after 2 h in oleic acid medium and was induced a maximum 20-fold after 3.5 h in oleic acid medium. This induction of Pay4p was mirrored by the induc- tion of the major anti-SKI, immunoreactive polypeptides after the shift of E122 cells into oleic acid medium.

DISCUSSION

A genetic approach using yeast has led to the identification of a number of genes called sec genes that encode proteins in- volved in the secretory process (for a review, see Pryer et al.

of the PAY4 gene. A presumptive consensus TATA sequence is shad- FIG. 7. Nucleotide sequence and deduced amino acid sequence

owed. The putative TATA-less promoter sequences are underlined. The putative transcription termination signals in the 3'-untranslated se- quence are double underlined.

(1992)). A similar approach has now been applied to the iden- tification of genes encoding proteins required for the assembly of peroxisomes in yeast. The Y: lipolytica pay4 mutant displays a phenotype indicative of a disruption in peroxisome assembly.

Characterization of P’4 gene of Y lipolytica 563

488 I I - lI1I,

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I

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\ \

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I I I I 1 D

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I I1 ”

\

I

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Analysis Program (version 7.0, Beckman Instruments). The parameters used for the analysis were set as follows: minimum match 6, minimum FIG. 8. Matrix comparison. Matrix comparison of sequences was performed using the Matrix Comparison program of the MicroGenie Sequence

percentage 60, allowable loop 7. The positions of consensus sequences for ATP binding are marked by the black boxes and are designated I and I1 according to their positions from the amino terminus of the protein. The abscissa in each case is Pay4p. The ordinate corresponds to A, Paslp; B, NSF; C, Secl8p; D, VCP E , Cdc48p; F, TBP-1.

Characterization of PAY4 gene of k: lipolytica 564

ZBB

490

beB

me

1888

I I I I I

\ . \

\

\

P \

I I1 \ "

M, E122 pay4 P4-KO pay2

116 +

80 +

FIG. 11. Immunological detection of Pay4p. Cells of the parental strain E122, the mutant strain pay4, the gene disruption strain P4-K0, and a second independent peroxisome assembly mutant strain pay2 were precultured in YEPD medium and then transferred to induction medium for 5 h. Cell lysates were prepared, separated by SDS-PAGE, and transferred to nitrocellulose. The nitrocellulose was probed with anti-Pay4p antibodies by western blotting. The numbers at left indicate the migrations of molecular mass standards (in m a ) . * = Pay4p.

Antl-PAY4p

TIME(h) 0 2 3.5 4 4.5 5 5.5 6 6.5

116+

80+

ZBB 490 688 me 1-

FIG. 9. Matrix comparison of the Pay4p and Pas5p sequences. The abscissa corresponds to Pay4p. The ordinate corresponds to Pas5p. Analysis was performed as described in Fig. 8.

PaY4P Pas5p Paalp

SeclSp VCP(1) VCP(I1) Cdc4Sp(I) CdcQBp(I1) TBP-1

NSF

x 2 3 NSF Paalp

SeclSp VCP(1) VCP(I1) Cdc4Sp(I) Cdc4Sp(II) TBP-1

PaY4P Passp Paelp NSF SeclSp VCP(1) VCP(I1) Cdc4Sp(I) Cdc4Sp(II) TBP-1

P ~ Y Q P Pae5p Paslp

sec1sp VCP(1) VCP(I1) CdcQSp(1) Cdc4Sp(II)

NSF

TBP-1

I---MWPIP A--I I "_" bXLFYGPWTGKrrLAKRIATNF~NFFSV-KGPELLN~I~ESE~ 902 GILLYGYPGCCXTLLASAVAWCGLNFXSV-KGPEILNKPIWEQNIRE 781

FsRWYS!8- 803

~ILLYUPPGCGKTLLARQIGKMLNAREPKVVNCPEILNKWGBSERNIRK 304 G L L t P G PPOTGRTLIARXIGTMLN~EPKIVNGPEILSKW 325 GILLYGPPGTGKTLIAETGAFFFLX-NGPE1HSKUGESESNLP.X 288 GVLFYGPWCGKTLLAXAIANECQANFISI-KGPELLTMWFGESEANVRE 561 GVLWGPPGTGKTLHARAVANETGAFFFLr-NGPEVMSKMAGSSESNLRK 297 G V L P Y O P P O T C K T L L A K A V A T E V S ~ F I S V - K G P E L L S E 570 GVLKYGPPGTGKTLLARACAAQTKATFL~-AGPCtLVQ~rGDGAKL~ 235

LPADAEEEQRR~OANSGLHTTIFDEIDAICKQRGSX-AGSTGVHDT~Q 353

~FDKARQAAPC--------VLFFDELDSIAKARGGNIGDGC~W.DRVINQ so3

.~FDKARAAAPT--------WFLDELDSIAKARGGSLCDAGGASDRWNQ 612

LFKDREAEY8RKGEESSLHIIIFDELDSVFKQRGSR-GDGT~GD~Q 374 AFEEAEKNAPA--------IIFIDELDAIAPKREKTHGE---VEP.RIVSQ 327

APEEAEKNAPA--------1fFIDEIDSfAPKRDKTNGE---VERRWSQ 336

AFALAKEKAPS--------IXFIDELDAIGTKRFDSEKAGDP.EVQRTKLE 277

~ s T A ~ E G ~ ~ ~ ~ LLAEL~GHSGODGGDGVFWOATKRPDLLDEACLRWRFDKH 993

*TsEFT 893

LLTQKDC--AE-GLDGVYILAAAATGRPDLIDSALLRPGRLDKSVICNIPT~ 867 LLSKIDGVEQ---LNNILVIGMTNRPDLIDEiULRPGRLEVKMEIGLPDE 400 LLAKMD-VDQ---LNNILVIGMTNRKDLIDSALLRPGRFEVQVEIHLPDE 420 LLTLKDGLKQRA---HVIWAATNRPNSIDPAL3RFGRFDREVDIGIPDA 374 ILTEKDGHSTK---KNVFIIGATNRPDIIDPAILRFGRLDQLIYYPLPDE 650 LLTLMDGHKARS---NWVIATNRPNSIOPAZ;RRFGRPDREVDIGIPDA 353 LLTEKDGHNAK---KNVFVIGATNRPDQIDPAILRPCRLDQLIYVPLPDE 659 LLNQLDGFQPNT---QVKVIAATNRVDILDPRLLRSGRLDP.KXEFPKPNE 324

KG~-ILHIHTAR"----RGHQLLSADVDI 426 KGRLQ-XFDIQTKKn-----RENNMKSDDVNL 446 TGRLE-ILQIHT-------"KNHKWLDDVDL 396 KSRY-AILW\NL---------RKSPVAKDVDL 672

KNKKLADDVDL 405 NARL-SILNAQL------"-RKTPLEPGLEL 681 EAR-AP.IKQIHS--------- RKMNVSPDVNY 346

TGW-VLRIHT--"""-

FIG. 10. Amino acid alignment of the conserved ATP-binding regions of Pay4p, PasSp, Paslp, NSF, SeclSp, VCP, Cdc48p, and TBP-1. Motifs A and B of the consensus sequence for ATP-binding are indicated. Six or more identical or similar amino acid residues are highlighted. Similarity rules: G = A = S ; V = I = L = M = F = Y = W , K = R = H ; D = E = Q = N ; S = T = Q = N .

Morphologically identifiable peroxisomes are absent in pay4 cells, and both PTS-1 and PTS-2 containing proteins are mis- localized to the cytoplasm. In these respects, pay4 cells appear

Antl-SKL nME(h) 0 2 3.5 4 4.5 5 5.5 6 6.5

c

49+

33+

FIG. 12. Induction of Pay4p by growth of cells in oleic acid. E122 cells were precultured in YEPD medium and then transferred to induction medium. Cell lysates were prepared at the times indicated

time point were separated by SDS-PAGE and transferred to nitrocellu- after transfer to induction medium. Equal amounts of protein from each

lose. The nitrocellulose was probed with either anti-Pay4p antibodies (top panel) or anti-SKL antibodies (bottom panel) by Western blotting. The numbers a t the left of each panel indicate the migrations of mo- lecular mass standards (in kDa).

to share many characteristics ascribed to cells of patients with inborn errors affecting peroxisome assembly, like Zellweger syndrome, and therefore represent an excellent model system to investigate the genetic basis of such disorders.

Three genes encoding proteins required for peroxisome as- sembly in s. cerevisiae have been isolated and characterized (Erdmann et al., 1991; Hohfeld et al., 1991; Wiebel and Kunau, 1992). In this paper we report the cloning and characterization of the PAY4 gene, the first description of a gene required for peroxisome assembly in I: Zipolytica. Functional complementa- tion of the pay4 mutant strain with the PAY4 gene reestab- lished peroxisome assembly in the mutant as evidenced by morphological (Figs. 4 and 5) and biochemical (Fig. 6) criteria. Genetic proof for the authenticity of the cloned gene was ob- tained from gene disruption and genetic analysis of the result- ing strain. Gene disruption experiments also demonstrated that the PAY4 gene was not required for cell viability and that

Characterization of PAY4 gene of I? lipolytica 565

peroxisomes are therefore not required for cell viability. Nucleotide sequence analysis of the PAY4 gene identified a

large open reading frame that could encode a polypeptide of 112,258 Da (Fig. 7). There is substantial evidence indicating that the product of the PAY4 gene is indeed a polypeptide of -112 kDa. Firstly, antibodies raised against a fusion between maltose binding protein and the putative PAY4 gene product recognize a protein with an M, of 112,000 in Western blots of cell lysates of the parental strain E122 and of a second pay mutant strain pay2, which is in a different complementation group from that of pay4 (Fig. 11). This protein of -112 kDa was absent in the pay4 mutant strain and the gene disruption strain P4-KO. Secondly, the synthesis of the -112-kDa poly- peptide was induced by oleic acid and repressed by glucose in a manner similar to that of peroxisomal proteins recognized by anti-SKL antibodies (Fig. 12). Many yeast peroxisomal proteins (Kamiryo and Okazaki, 1984; Fujiki et al., 1986; Nuttley et al., 19901, and also proteins required for peroxisome assembly in S. cerevisiae (Erdmann et al., 1991; Hohfeld et al., 1991), have been shown to be induced by growth on oleic acid.

The hydropathy profile of Pay4p did not provide strong evi- dence for any membrane-spanning region and indicated rather a hydrophilic polypeptide (Kyte and Doolittle, 1982). Western blot analysis of subcellular fractions showed that Pay4p was not localized to the fraction enriched for peroxisomes (data not shown). This would suggest a cytoplasmic localization for Paylp, although at this time we cannot exclude the possibility of a peripheral association of this polypeptide with the peroxi- somal membrane.

Pay4p shows remarkable partial sequence similarity with a number of proteins involved in important and diverse biological functions such as vesicle-mediated protein transport (NSF and SeclSp), control of cell cycle (Cdc48p, VCP, and P97), modula- tion of gene expression (TBP-11, and peroxisome biogenesis (Paslp and Pas5p) (Figs. 8 and 10). All these proteins have in common a stretch of approximately 175 amino acids that con- tains a consensus sequence for ATP binding. Therefore, it would appear that the PAY4 gene represents a member of a multigene family that has evolved to encode a large number and variety of proteins that control a diversified set of biological functions. The only common function that can be attributed to all these proteins appears to be the hydrolysis of ATP. ATPase activity has been demonstrated for the VCP-like protein of Xenopus oocytes (Peters et al., 1990) and has been suggested to be important for the functioning of NSF (Clary et al., 1990). Paslp has also been suggested to be an ATPase based on the conservation of its ATP binding sites (Erdmann et al., 1991). We therefore propose that Pay4p is also a putative ATPase re- quired for peroxisome assembly and that its tentative localiza- tion to the cytosol suggests that it acts at an early step in peroxisome assembly.

A comparison of sequence conservation would suggest that Pay4p of k: lipolytica, Paslp of S. cerevisiue, and Pas5p of I! pastoris represent members of a multigene family encoding putative ATPases required for peroxisome assembly. Whether or not these proteins are true functional homologues can best be addressed by heterologous complementation experiments. It will be interesting to determine whether other such ATPases are found in these yeasts and whether such ATPases are in- volved in peroxisome assembly in other yeast species and in mammals. An approach based on the polymerase chain reaction might be used to address this question most easily, with prim- ers derived from regions conserved in the genes encoding these three proteins. An alternative approach is to attempt to comple- ment the yeast lesions using mammalian cDNA libraries ex- pressed in the mutants. Such an analysis opens up the possi- bility of rapidly identifying ATPases required for peroxisome

assembly in humans and potential mutations of these proteins that could lead to genetic diseases of peroxisome assembly such as Zellweger syndrome.

Acknowledgments-We thank Gillian Regoeczi for excellent technical assistance and Al Spong and Suresh Subramani for sharing data on Pas5p prior to publication.

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