Gene. 88 (1990) 247-252 Elsevier

247

GENE 03479

Structure and transcriptional control of the Saccharomyces cer~isiae POXI gene encoding acylcoenzyme A oxidase (Recombinant DNA; dideoxy sequencing; homology; gene disruption; phenotype; transcriptional control; yeast)

A. Dmochowska', D. Dignard6, R. Maleszka" and D.Y, Thomas" o DepartmentofGenetics, UniversityofWarsaw, Al. Ujazdowskie4,~O-478 Warsaw(Poland)Tel. 294-011 and b Genetics Section, Bimechnolo£y Research Institute. National Research Council of Canada. Momrdal, Quebec H4P 2R2 (Canada) Received by J. Marmur: 13 July 1989 Revised: 18 October 1989 Accepted: 20 October 1989

SUMMARY

We have cloned the Saccharomyces cerevisiae gene coding for the peroxisomal enzyme: fatty acyl-CoA oxidase (POX). The gene (named POXI ) is unique in S. cerevisiaeand has been identified through homology with the POX4 and POX5 genes of Candida tropicalis. The POXI gene encodes a 84-kDa POX protein composed of 748 amino acids. The identity between the S. cerevisiae and C. tropicalis enzymes is about 40~0, and there is a greater degree of similarity between the ?4 termini than the C termini. A disruption of the POXI coding sequence diminishes the ability of yeast eells to grow on oleic acid as a sole carbon source. The expression of the POXI gene is regulated at the level of transcription, and is induced more than 25-fold by the addition of oleic acid to the medium,

INTRODUCTION

Pemxisomes are inducible orgunelles surrounded by a single membrane that forms a cellular compartment for various metabolic pathways (for a review see Lazarow and Fujiki, 1985). In the yeast Candida tropicalis, peroxisomes are the sole location for the set of enzymes involved in ~-oxidation of fatty acids (Kawamoto et el., 1978). Fatty acids and n-elkanes, which serve as agood source of carbon and energy, stimulate the proliferation of these ornanelles. Correspondence to: Dr. D.Y. Thomas, Genetics Section, BiotechnologY Research Institute, National Research Council of Canada, 6100 Royalmonnt Avenue, Montr6al, Quebec H4P 2R2 (Canada) Tel. (514)496-6t$6; Fax (514)496-6232. Abbreviations: an, amino acid(s); bp, base pair(s); C., Candida; CoA, ¢oenzyme A; kb, kilobase(s) or 1000 bp; ORF, open reading frame; POX, fatty acyl-CoA oxidase; $., Saccharomyces; tsp, transcn'ption start point(s); wt, wild type; YEP, I% yeast extraet/2~ bacto peptone. 0378-1119/90/$03.50 @ 1990Elsevier Science Publishers B.V. (BiomedicalDivision)

The most abundant induced peroxisomal protein was identified as the first enzyme of the p.oxidation pathway, fatty acyI-CoA oxidase (POX) (Kamiryo and Okazaki, 1984). The structural genes which code for the C. Iropicalis enzyme were isolated in several laboratories (Kamiryo and Okazaki, 1984; l~.achubinski et el., 1985; Okazaki et el., 1986, 1987; Murray and Rachubinski, 1987). The POXencoding gene from Candida mahosa (Hill et al., 1988) and the eDNA for the rat enzyme (Miyazawa et al., 1987) have also been cloned. In Saccharomyces cerevisiae, which is unable to utilise fatty acids and n-alkanes efficientlyas a carbon source, the induction of observable peroxisomal structures and of enzymes of the p-oxidation pathway was achieved by addition of fatty acids to the growth medium (Veenhuis et el., 1987; Skoneczny et al., 1988). Thus in S. cerevisiaea set of conditions is now identified for the induction of peroxisomes that is comparable with the induction ofperoxisomes in C. troptcalis.

248 There were no signals characteristic ofS. cerevisiae introns (Langford and Galiwitz, 1983) within the complete sequence. Comparison of the protein encoded by the cloned gene with the C. tropicalis POX enzymes PXP4, PXP5 and PXP2 (Okazaki etal., 1986; 1987; Murray and Rachubinski, 1987), the C. maltosa enzyme (Hill et al., 1988) and also with the rat acyl-CoA oxidase type 1 (Miyazawa etal., 198"/) showed areas of identity throughout the protein (Fig. 3). We thus concluded that we had cloned a copy of the S. cere~iae gene for this enzyme. Since three related genes, which code for POX have been detected in C. tropicalis, one differentially spliced gene in the rat (Osumi et ai., 1987) and a single gene in C. maltosa, we were interested in determining if the cloned gene was the only gene coding for POX in S. cerevisiae. We hybridized a DNA probe consisting of a 2.4-kb BgllI fragment (see Fig. 1) to a Southern blot orS. cerevisiae DNA at normal and reduced stringency. A single restriction fragment of the size predicted from the known physical map of this region

The aim of the present study was the isolation and characterization of the $. cerevisiae gene coding for POX. The isolation of such a gene should provide a basis for the thrther investigations of fatty acid-dependent induction of peroxisomal proteins and targeting to peroxisomes in this organism.

EXPERIMENTAL AND DISCUSSION

(a) Cloning and characterization of the POXI gene During the sequencing of a 10-kb $. cerevisiae DNA fragment in plasmid pADI7 that contained the £ E X I gene (Dmochowska et al., 1987) we detected another extended ORF (Fig. 1). Subsequent comparison of this sequence with databases revealed some identity with the POX4 and POX5 genes ofC. tropicalis(Okazaki et al., 1986). The completed sequence yielded an ORF of 2247 nt coding for a protein composed of 748 aa (approx. 84 kDa) (Fig. 2). Bg

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Fig. I. The 5. cerevLriaechromosome Vii DNA fragment carrying the POX! 8ene: restriction map ofclone pAD17. CHCI, clathrin heavy chain Sane (Lemmon and Jones, 1987). (A) A more detailed restriction map of the POX; gene. (B) The disrupted POX! gone in strain A232-4A-23. This strain was constructed by one-step gene disruption (Rothstoin, 1983) oftbe POXI locus (black boxes) by the following procedure. The Bg/lI fragment (hatched box) containing the LEU2 gene (the open bar) from plasmid pYFgl (Storms et al., 1979) was introduced into the BamHl site present in the POX! gen¢. The parentheses indicate restriction sites that were destroyed after ligntion of these two fragmants. The disrupted POXI Sane was excised and transformed into the strain A232-4A (Mate ura? trpl leu2) to evict and replace the wt copy of the POX! gene (Ire et al., 1983). B, BamHl; Bg, BS/II; E, EcoRI; H, HIndllI; K, Kpnl; P, Pstl; Pv, Pvull; S, Sail; Sp, SpAl; Xb, Xbal; Xh, XhoI. Fig. 2. The nt sequence oftbe POXI gene and deduced aa sequence. Appropriate DNA fragmants were subcloned in MI3 vectors (Yanisch-Perron et al., 1985). Sequencing was by the dideoxynucleotide chain termination method (Sanger et al., 1977). The nt sequence is numbered from the ATG start codon ofthe POXI ORF (nt + I). Some restriction sites used for this work are shown above the nt sequence. The candidates for TATA boxes are at positions -86, -49 and -22. The mapping or t~p was made by reverse transcriptase using the primar 5'-CAGAACCACCGAATCGGGA'ITA.Y and poly(A)+mRNA from oleic acid- and from acetate-grown cells (see EXPERIMENTAL AND DISCUSSION, section ¢). The tap common for acetate and oleate grown ceils are underlined with I and 2 lines for minor and major tap respectively; the tap specific for oleate are indicated with black circles. The postulated signal for transcription termination in yeast (Zaret and Sherman, 1982) is shown with a wavy line (nt 2344-2389). The GenBank accession number of this nt sequence is M27515.

249

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ATA GTG AAT rAT CCT ~TA G~t GAC CAA GTT AAC ACA TTT T~A GAG TCA TCC CCG GAG AGG AGA ACT GTG ACG CAC GCC TT& ATA GAC ~ VOl Aep G i n V i i k i n Thr ~ho Zau G l a S e t S o t Pro G l u Ar G Ar~ Thr Leu Th= tLt8 AI~, ~ t a Z l e A~p G i n Z l e Vol ASh Amp ~ro 110

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~ ATG ¢P,A GAG ~,A G~A ATT ACT GCC AN~ JUrA AT& GCT AGG CTT GCT AGT TAT ATG TTG ~ KCT CAT ACG GAC TAT TAC GAT GCT Leu Lys Thr Asp Thr A~p Ty= Ty= &sp A1O LyS Lye He~ 91~ G2u Arg G l u Z l e Tier Alo Lys L y : ~10 A18 J~G Leu /kla SOt Tim Na~;

271

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~t~L'~d~lr GAG CAC GAT &TC ~ ACA GTG CGC JUrA CAC TTT CGC GAC ACT GI~C CTG ATG AAA GAG TTG CAA GCA /tAT GAT CCA GAC NUt GCT TCG CCT 1,eu Net. Lyo GIU IAU Gln A.l.a ASh Asp L~ro JUp 14to k.l.o s e t pro Phe ArQ Asp Thr AJp GIU Ht8 AOp I"10 Ly8 Thr VO1 AZ~g l~y8 H18

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CGC GTG GGT GTA CAC TT& ACA AAC AN~ GAC CTT TTT &TA TTC GAT A~G AGA TTG TCA C1T GTA Gr.A AAT &TT GAT CC? C&A TTG GGT ~ Leu Thr Ash Lya Jusp Lea Plte Z l e FAG Asp L¥ ,t A=~ Laa 9or Lea Vol A l a Asn Z l e A l p Pro G i n Leu G l y Th;: A=g v s l Gly Vol It18

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CAG GAG AGA GGT GCC ACT TTG ATG AJ~A TTG GGG CTA TTT GGT AAT TGT ATC AAG GGC AAT GGT ACT GAT GAG CJ~ ATC CGG TAT TGG ~ Lea G l y Lea Phe G l y /urn C~,t Z l e Lys Gly Ash Gly Th= Asp Glu Gln I I 0 ArQ TJ~ Trp Lea 0 I n Gla A=q Gly A1o Thr IAtl I~r. /.y~;

541 181

GGT AT& TAT GGC TGT TTT GCA &TG ACT G~G TTA GGA CAT GGT TCC AAT GTT GCC CAG CTG CAG ACT AGG GCT GTG TAC rAY N~G r.AA A~T G l y Z l e Ty~r G l y Cya Phe A I s Ne~ T h : GlU Leu G l y tilm G l y S e : AOA V a l A l a G i n Lea G i n Ti~ A=g A l a V~I T~: Asp L¥8 Gin A~m

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GTT GGT CGT CAG C~A TTC GCA CCT AGA /tAG GGA TTG TCT GN~ &CA CA~ TTA ATC GA¢ TAT CCC CTT CP~C CA~ TAT CGT GTT TTA CCA CA~ Z l e Asp TF~ Pro 1,eu Itla G l n Ty~ Arg Val Le~ Pro Gln VaX Gl~y Azg Oln GXn Phe A1o Pro M g Lyg G l y Loa 8 o r G l a Thr G i n ~

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1711 571

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Structure and transcriptional control of the Saccharomyces cerevisiae POX1 gene encoding acyl-coenzyme A oxidase.

We have cloned the Saccharomyces cerevisiae gene coding for the peroxisomal enzyme: fatty acyl-CoA oxidase (POX). The gene (named POX1) is unique in S...
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