Sequence of the PAS8 gene, the product of which is essential for biogenesis of peroxisomes in Saccharomyces cerevisiae

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Biochimica et Biophysica Acta, 1216 (1993) 325-328 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00

BBAEXP 90587

Short Sequence-Paper

Sequence of the PAS8 gene, the product of which is essential for biogenesis of peroxisomes in Saccharomyces cerevisiae Tineke Voorn-Brouwer, Inge van der Leij, Wieger Hemrika, Ben Distel and Henk F. Tabak * Department of Biochemistry, E.C. Slater Institute, University of Amsterdam, Academic Medical Centre, Meibergdreef 15, 1105 AZ Amsterdam (The Netherlands) (Received 15 July 1993)

Key words: Peroxisome, pas8 gene; AAA-protein family; Organelle assembly; Gene sequence; (Yeast)

In a genetic screen for mutants disturbed in peroxisomal functions we found that the laboratory 'wild type' strain YP102 behaved like a typical peroxisome assembly mutant. Here, we report the sequence of the complementing gene (PAS8), coding for a protein of 1030 amino acids that appears to be a novel member of the AAA-protein family which also includes NSFp and PASlp.

Most eukaryotic cells contain small spherical organelles bounded by a single membrane called peroxisomes which contain enzymes for the /3 oxidation of (very-long-chain) fatty acids, HzO2-producing oxidases and catalase to decompose H 2 0 2. The presence of additional enzymes is often dependent on species, cell type, or differentiation state. Their importance is underlined by the occurrence of serious human diseases, such as the cerebro-hepato-renal or Zellweger syndrome, due to the abnormal function a n d / o r biogenesis of peroxisomes (reviewed in Refs.l-3). We and others have started a genetic analysis in Saccharomyces cerevisiae as a model eukaryote in an effort to understand the function, maintenance and proliferation of these organelles [4,5]. During this work we encountered a laboratory strain that surprisingly was not able to grow on oleate as sole carbon source. After fractionation of spheroplast homogenates, peroxisomal matrix enzymes remained in the supernatant fraction, while in control cells these enzymes were present in an organellar pellet. Electron-microscopical observation of cells grown for a short time on oleatecontaining medium to induce peroxisomes did not reveal peroxisomes or structures that could be labeled by immuno-gold cytochemistry using an antibody against thiolase [4]. In all respects this 'wild type' strain be-

* Corresponding author. Fax: + 31 20 6915519. The sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number L20789.

haved like a typical peroxisome assembly (or pas) mutant (it is designated pas8 in our mutant collection). Crossing pas8 with an oleate-proficient strain followed by sporulation of the diploids resulted in 2:2 segregation of the oleate-minus phenotype, indicating a single gene deficiency in pas8 [4]. We therefore used the pas8 strain to clone the corresponding wild type gene by functional complementation. Here, we report the D N A sequence of the PAS8 gene and discuss the properties of the encoded protein. The smallest complementing clone of 4.2 kb was sequenced and found to contain an open reading frame of 3.1 kb encoding a protein of 1030 amino acids with a calculated molecular mass of 116 kDa (Fig. 1). The upstream region - 1 4 7 to - 1 3 6 resembles the consensus O R E (oleate response element), an UAS found in the promotor of a number of genes encoding peroxisomal proteins [6], its functional significance in regulation of transcription of the PAS8 gene remains to be established however. Disruption of the found O R F in vivo, using a LEU2 disruption construct, resulted in the same loss of functions as observed in the pas8 mutant. Diploids of a cross between the pas8 mutant and the disruption strain were not able to grow on oleate. This excludes the possibility that a suppressor of the pas8 mutation was cloned (experiment not shown). The protein encoded by the pas8 gene contains a number of interesting features: (i) the carboxy-terminal part of PAS8p contains two sequence motifs characteristic for ATP-binding or hydrolysing proteins: the Walker motifs A and B [7];

326 1 61 121 181 241 301 361 421

AAG ACC TCA AGA AAG CAC CAT TAT

CTC GCG CTT CAG AAG TIE CGG GTG

ACT AAT GGA AGG TGT CTT AGA TTT

GAT GAC TGA AGG TTT ACA CTT GCA

AGA AGA AAA GAG ACT CCA TCA TAC

CGA GCC ATT TCC AAG CAT AAC CCT

AGA CGA ACT CTA AAA AGT AAG CCA

ACT AGC GAA CAT GAG ATA TCA AAA

GAA CTT AAA GGC ATA CGG TTA GAA

AAA GCT AAA GGC AAA CGA GAA AGC

TAG CAT GCA CAA GAA TAA TTA GAT

ATT CTT CAG GAT CTT TCG GAA TAT

AAA GGA GAA AAA TAT TCA CAA AGT

CCA TAT GGC TGA ATA ACA AGA AAC

GTA CTC TAT GGC TCA CTT ACA ATT

CCA CGC GGT ~ TGT CCG GTA AAT

481

CTT L GAA E CCA P AGC S TGC C ATT I TTT F TTA L TTT F TAT Y ATT I ATC I GTC V TTT F ATA I GAT D AAA

ACG T TAC Y GGT G AT? I TAT Y AAT N TTG L ACA T ACA T GCT A GAT D AAT N ACA T GTT V GCA A ATT I TGC

TT'f A G T F S GGC GAT G D TGC ACG C T GTT GTA V V TTT GAA F E G A A CAA E Q C A T TAC H Y TCT GGT S G GAA ACC E T AAC GAA N E CTT GAA L E GAT GAT D D AGT GGA S G TTA CTA L L AGC TTT S F CCC ATT P I T T T CAA

CTC L AAA K CCT P CCT P CCC P TTG L AAA K CTT L CAA Q GAT D TCT S AAT N TCG S CTC L CCA P GCT A AAT N AAT N AAT N TGG W ATT I TTG L CAT H CAA Q AAG K TGC C A~'f I CAT H CAA Q GGA G CAT H CAG Q TAC Y

TCC S AAA K GGT G TCC S ATA I TAC Y TAT Y TGC C TTG L GAA E CTC L TCA S TIT F CCC P GAC D AAT N ATT I GAT D GAT D TTC F GAT D AGT S ATC I AGG R GCT A CTr L AGG R TTG L AAA K ACT T ATG M TGG W ATG M

GGA G GCT A AAA K AAA K TTA L TCA S AAC N CAA Q ATA I GAA E CCC P ATA I ATA I AAC N TGC C CAA Q ATT I TTA L GAG E TTT F CCC P AGA R TTC F GGA G ACA T TCG S GCT A GAT D TCT S ACA T AGA R TAC Y GAT D

ATA I GAA E ATT I CTT L GAC D AAA K ATC I ATT I TTA L TAT Y TGC C TAT Y ACA T GGC G AGC S GTT V CTA L ATC I TCT S GTC V AAT N TCC S CCC P ATC I ATG M CTC L AAA K TCA S ATA I TTT F TTT F TTA L AAT N

TAC Y TGT C GTA V TIT F AAT N CTA L AAC N TrA L GTA V GCA A ACA T GCC A GTG V TrT F GTC V TTC F ACA T CCA P GAT D ACA T CGG R AAT N TAT Y ACC T GTG V ACT T TGT C ATT I AAC N GTT V GAG E TCT S ATT I

GCC A TTA L CAT H GGG G GTA V ATG M TCC S AAC N AAT N TTA L ATT I TTT F TCA S AAA K GTG V ATC I ACC T ATC I GAC D AGC S ACG T CTT L GTA V CTA L AGA R TCA S GAA E TTG L TTC F GGC G ATT I TCA S TCA S

CCC P TAT Y TGT C TTT F GTA V GAC D ATG M TGC C GAT D CCT P TCA S ACA T AAT N AAA K ACA T TCA S TriG L GCT A GAA E GCA A AAG K CAG Q AGA R AAT N TTT F AAT N AAT N CTT L GAA E TCC S CTC L CAT H TTT F

TGT C GGG G GTC V ATG M CCT P CTA L GAA E TCA S ACT T AAA K AGA R GAC D ATG M AGA R ATA I AGA R AAA K TTT F GAT D GAG E CTA L AGA R CAA Q GCC A GCT A TCA S GTC V GAC D ATG M GTA V GTA V GAA E TCA S

AGC A T T S I ACA ATT T I CTG G A T L D CCG A C A P T G T A CTG V L CCG C A A P Q ACC G T G T V CCT T T C P F GAG C A G E Q A T A GGG I G GAT C T T D L GCT G A A A E GGT TGC G C ACA ATA T I TCA A A G S K G~T G G A V G AAG T T T K F GAT T C A D S GAG C T A E L CTC G A T L D ATA ACA I T TAT T A C Y Y TTG G T T L V TCT G T G S V TCA A A A S K AGA CAA R Q C T A CCG L P GTA A A C V N TCT AAA S K AAT AAC N N CCC G T T P V CTA AAC L N TC_A CTC S L

TCT S AGA R GAT D CAG Q GAT D GAG E GTT V CCG P AAA K ACA T TTG L ACT T GTC V TAC Y TCC S GGC G TTT F AGT S GGG G TGC C ACT T GGT G AAC N TTA L TAT Y CTG L TAC Y GCT A CTT L ATA I CCG P AGA R TCT S

AGG R T~fG L TCA S CCC P AGC S ATG M CAT H CAG Q CTA L AAT N CGA R C~fA L AGG R GCA A AAT N TGG W AGC S ATG M CAG Q 7TC F AAT N T~fT F ATA I CTT L CTT L GAT D GCA A AAT N CTG L GAT D TCT S GAT D TCA S

541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401

K

C

F

Q

ATT I ATA I GAT D TCT S AGA R TAC Y Tq~C F AAC N CTG L ATT I ATT I GCA A TTT F AGC S CTG L GTT V

CTT L GCA A TCC S CAT H CCA P TAT Y AAT N AAT N GAG E ATT I TIT F ATC I AAA K TIT F CGT R CCA P

TGT C GAG E CTT L TTT F TTA L GAT D TGT C GTG V ATA I GGT G TTG L AAA K TTC F AGA R ATC I GTA V

CAA Q GAA E GTA V ATT I CCC P TTG L TCT S GGC G GAT D TAC Y GCT A TTA L CCA P TCA S TTT F TCT S

GGG A G G CAG A G A TCG T A A AGA AGT AGC C A A GGA AAG GTG G A G ATG A A G M K GAT ATA D I CCT CAG P Q CTG C C C L P ACT ATG T M GTA A C G V T CAA C A A Q Q TCT CGA S R GGC C T T G L AGT GCT S A TCT GCT S A CCT GCA P A CTG C G C L R CTA GTT L V CCT CCC P P ATA GGC I G CTT CAA L Q GAG A G C E S GCT GAT A D TAC T A C Y Y TCT AAA S K ATC A C T I T GCT G A A A E TTA GAA L E CAT TCC H S GGT A T A G I TCA A C A S T TCA CCT S P CAA G A T Q D GAC G A T D D AAC GTG N V GAA GCA E A Gq'F C A A V Q TAT TCT Y S

CTT GAT CCA TCT TTG TGC AGC GCA A TAT Y TAC Y TTC F GAT D TTT F ATA I GAT D GTT V CTA L TTA L CCT P CTG L AAG K AAA K CAT H TCT S AAA K CTC L AAG K GAT D AAC N ACT T ACT T ACT T CAT H TCT S GCT A CCA P TIT F CCT P CAG Q CAA Q GCT A

TGC TAA CCC CGA CTA GGC ATA TCG S crc L GGT G TGT C TIT F CTG L CAA Q ATT I GAT D AAA K TCA S CAC H GAT D CTG L ATA I ACA T CAG Q AGG R AAT N AAC N AAT N AGA R TTr F TCC S ACA T CTA L AAG K GTG V GAG E ACG T TCC S AGA R AAG K GGT G

327

(ii) two short hydrophobic regions (between amino acids 245-269 and 540-555) could represent membrane-spanning elements, but whether or not the protein is indeed associated with membranes remains to be demonstrated. The remainder of the protein has a hydrophilic character. A search in protein data banks revealed that the Walker motifs A and B are part of a conserved domain of at least 185 amino acids which is present once or twice in a set of proteins collectively called the AAA family (ATPases associated with diverse cellular activities) by Kunau et al. [8]. This protein family includes, amongst others, proteins involved in the secretory pathway (NSFp [9] and SEC18p [10]), proteins contributing to cell-cycle progression (CDC48p [11]), a protein involved in the formation of the bc 1 complex of the respiratory chain (BCSlp [12]), and proteins involved in gene expression (SUGlp [13]). PASlp is another protein of this triple A family [14] which, like PAS8p, is required for the biogenesis of peroxisomes in S. ceredsiae. The participation of both

2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541 3601 3661 3721 3781

ETA L TIT F GAT D CAA Q TTG L AGA R ATT I TAC Y CCT P GAT D AGT S GAA E GAT D GTA V GCT A AAA K AAG K CAA Q GCT A TAT GTA TTG GCT

ACT T TAC Y TTA L ATC I GAC D AGC S GCA A AT? I TGT C TCG S ACC T GCA A ACC T AAG K CTC L GT? V AT? I GAG E AAT N GTC TCT AAG ACA

CCT P CAA Q TCA S CCT P ACA T GGT G ACA T GGT G GTC V GGT G GAC D CTA L AAA K CTT L TGC C TCT S GCG A CAG Q ~ F CCA ATC TAA ACT

TTA L GAA E AAA K AAC N ATA I ATT I AAT N GAG E ATA I GGT G GCT A CTA L CAA Q ATT I TCA S CAG Q ACA T CTC L GAA E TAT GAA TAA CGA

GAC D TCA S GCT A GTA V GAC D CTT L TTT F AGT S TIT F GTT V GAC D AGA R T?G L GAG E GAT D CAT H AAA K ACC T GGT G TIT AGG CCA TAT

ATC I AAG K ACT T ACT T ATG M TTT F TCT S GAA E ~ F ATG M GGT G CCA P AAT N TTG L GCA A AAC N GAG E CCA P GCT A GCC GAA TAA CCG

AAA K AAG K TCG S TGG W CCG P TAT Y TTA L GCT A GAT D GAT D GTA V GGA G ATT I GCA A ATG M GAA E GAT D AGT S TAA * ~ AAA AGG TGC

PASlp and PAS8p in this process is not reflected in the similarity of their amino acid sequences since outside the conserved 185 amino acid domains no significant sequence similarity is observed. Furthermore, PASlp has two copies of the conserved domain, while no membrane-spanning elements are predicted. Its location in the cell has not been determined due to its low abundance. Loss of PASlp function results in a phenotype thus far indistinguishable from that of the p a s s mutant and a number of other pas mutants. Although binding of a nucleotide or its subsequent hydrolysis has only in some cases been demonstrated for the proteins of the triple A family, a possible function of this module could be to ensure the processivity or the vectorial nature of the mediated process by coupling it to energy consumption. Unfortunately, the similarity between the various proteins is limited to this accessory function and it drops rapidly outside the region of the conserved domain. This severely hampers speculation on the possible function of PAS8p by comparing it with its family members. It may explain why

TCA S TGT C AAA K GAT D CTA L GGT G AAT N AAT N GAA E CGT R TTT F CGA R TTA L AAG K CTT L TTG L ACT T GTG V GTA

ATT I GGA G GCT A GAT D AAA K CCA P TI~ F GTG V ATC I ATC I GTT V

AAA CAG TTC AGA

7TT A T C T T T T T A T I T A C T TAC C C T CCC C G G C T C A T A A A A A G A TAC TAG TAG T T G A T T A T A C A T A A T A A A A A T A T A A A A C A A 7TG A C T GCC A T A C ~ A A A A C C A T T C GTG T

F GAG E CTA L AAC N ACG T AAG K TCG S ATA

GTG V TGG W AGG R ATA I CAT H CCG P TTT F CGC R GAT D GTT V ATC I GAT D GCT A TGT C GCC A GGA G GTT V CGG R TCA

GAA E CTT L AAC N GGT G CCT P GGT G AGT S AGA R TCA S TCA S GGA G AAA K CTG L CCA P ATG M GAG E GTC V GCT A ACT

ACG T CCG P GAA E GGT G GAA E ACA T GTT V GTG V GTA V CAG Q GCA A TTG L ACT T TIT F TCA S AAT N GTA V GAA E CAG

GCA A CAA Q TTC F ATT I TTA L GGT G AAA K TIT F GCA A TTA L ACA T TTA L CGC R AAT N AGA R ATT I AAA K CTG L AGC

CGA R TCA S TCG S GAT D TTT F AAA K GGC G CAA Q CCC P CTA L AAC N TAT Y AAA K TAC Y ATT I TCT S ATG M AAT N GTA

ATG M ATT I GTT V TTT F ACC T ACT T CCT P AAG K AAA K GCA A AGA R TTA L TTC F ACC T GCA A ACA T GAA E CAT H TAG

ACG T ~ L TCG S GTT V TCA S CTA L GAA E GCG A CGT R GAG E CCA P GGC G GTG V GGG G CGC R CGT R GAC D TAT Y GTA

GCT A ATC I ATT I AAA K GGT G ATG M CTG L AGA R GGT G TTA L GAC D ATT I CTC L GCA A ATG M CGC R TTC F GAA E AAT

ACT 'T ACT T GGT G GGT G ATG M GCT A TTG L GAG E AAT N GAT D TTA L CCG P GAC D GAT D GTA V TGG W TTG L GCG A TTG

GCG A CAG Q GCC A GAA E AAA K AAG K AAT N GCT A CAA Q GGT G TTG L GAT D AAC N TTT F GAA E TTT F AAA K GTG V TAA

CGT R GAG E CCA P ATA i AAG K GCC A ATG M AAA K GGT G ATG M GAC D ACG T GAC D TAT Y AAA K GAT D GCA A AGA R ATA

Fig. 1. Nucleotide sequence of the PASS gene and the predicted amino acid sequence of the encoded protein.

328 t h e s e p r o t e i n s c o n t r i b u t e to such diverse p r o c e s s e s as cell-cycle c o n t r o l a n d p r o t e i n - c o m p l e x f o r m a t i o n , because (except for the c o n s e r v e d d o m a i n ) the p r o t e i n s show h a r d l y any r e s e m b l a n c e in the substantial r e m a i n ing p a r t s of their a m i n o acid chains. A l t h o u g h the biological role of the PAS1 a n d PAS8 g e n e p r o d u c t s is still u n k n o w n , t h e i r p h e n o t y p e s u p o n loss o f function ( a b s e n c e of m o r p h o l o g i c a l l y disting u i s h a b l e p e r o x i s o m e s ) suggest that t h e s e roles are essential to the b i o g e n e s i s o r m a i n t e n a n c e of t h e s e organelles. It is surprising, t h e r e f o r e , that loss of functional p e r o x i s o m e s in pas8 d o e s not c o m p r o m i s e its life in the l a b o r a t o r y since it is in use as a c o m m o n wild type strain (YP102). In m a n a similar p h e n o t y p e , observed in fibroblasts from Z e I l w e g e r p a t i e n t s , is only c o m p a t i b l e with a very short life span. References 1 Borst, P. (1989) Biochim. Biophys. Acta 1008, 1-13. 2 Lazarow. P.B. (1993) Trends Cell Biol. 3, 89-93.

3 Subramani, S. (1993) J. Membr. Biol. 125, 99-106. 4 Van der Leij, I., Van den Berg, M., Boot, R., Franse, M. M., Distel, B. and Tabak, H.F. (1992) J. Cell Biol. 119, 153-162. 5 Erdmann, R., Veenhuis, M., Mertens, D. and Kunau, W.H. (1989) Proc. Natl. Acad. Sci. USA 86, 5419-5423. 6 Einerhand, A.W.C., Kos, W.T., Distel, B. and Tabak, H.F. (1993) Eur. J. Biochem. 214, 323-331. 7 Walker, J.E., Saraste, M., Runswick, M.J. and Gay, N.J. (1982) EMBO J. 1, 945-951. 8 Kunau, W.H., Beyer, A., Franken, T., Gotte, K,, Marzioch, M., Saidowsky, J., Skaletz-Rorowski, A. and Wiebel, F.F. (1993) Biochimie 75, 209-224. 9 Wilson, D.W., Wilcox, C.A., Flynn, G.C., Chen, E., Kuang, W.J., Henzel, W.J., UIIrich, A. and Rothman, J. (1989) Nature 339, 355-359. 10 Eakle, K.A., Bernstein, M. and Emr, S.D. (1988) Mol. Cell. Biol. 8, 4089-4109. 11 Froehlich, K.U., Fries, H.W., Erdmann, R., Bothstein, D. and Mecke, D. (1991) J. Cell Biol. 114, 443-453. 12 Nobrege, F.G., Nobrega, M.P. and Tzagoloff, A. (1992) EMBO J. 11, 3821-3829. 13 Swaffield, J.C., Bromberg, J.F. and Johnston, S.A. (1992) Nature 357, 698-700. 14 Erdmann, R., Wiebel, F.F., Flessau, A., Rytka, J., Beyer, A., Froehlich, K.U. and Kunau, W.H. (1991) Cell 64, 499-510.