J Biol Chem, Vol. 274, Issue 35, 24514-24521, August 27, 1999

Alternatives to the Isomerase-dependent Pathway for the -Oxidation of Oleic Acid Are Dispensable in Saccharomyces cerevisiae
IDENTIFICATION OF YOR180c/DCI1 ENCODING PEROXISOMAL ^3,5-^2,4-DIENOYL-CoA ISOMERASE^*

Aner Gurvitz^§, Anu M. Mursula^§, Ahmed I. Yagi^§, Andreas Hartig, Helmut Ruis, Hanspeter Rottensteiner^¶, and J. Kalervo Hiltunen^§

From the  Institut für Biochemie und Molekulare Zellbiologie der Universität Wien and Ludwig Boltzmann-Forschungsstelle für Biochemie, Vienna Biocenter, Dr Bohrgasse 9, A-1030 Wien, Austria and the ^§ Biocenter Oulu, Department of Biochemistry, University of Oulu, FIN-90570 Oulu, Finland

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Fatty acids with double bonds at odd-numbered positions such as oleic acid can enter -oxidation via a pathway relying solely on the auxiliary enzyme ³-²-enoyl-CoA isomerase, termed the isomerase-dependent pathway. Two novel alternative pathways have recently been postulated to exist in mammals, and these additionally depend on ^3,5-^2,4-dienoyl-CoA isomerase (di-isomerase-dependent) or on ^3,5-^2,4-dienoyl-CoA isomerase and 2,4-dienoyl-CoA reductase (reductase-dependent). We report the identification of the Saccharomyces cerevisiae oleic acid-inducible DCI1 (YOR180c) gene encoding peroxisomal di-isomerase. Enzyme assays conducted on soluble extracts derived from yeast cells overproducing Dci1p using 3,5,8,11,14-eicosapentenoyl-CoA as substrate demonstrated a specific di-isomerase activity of 6 nmol × min¹ per mg of protein. Similarly enriched extracts from eci1 cells lacking peroxisomal 3,2-isomerase additionally contained an intrinsic 3,2-isomerase activity that could generate 3,5,8,11,14-eicosapentenoyl-CoA from 2,5,8,11,14-eicosapentenoyl-CoA but not metabolize trans-3-hexenoyl-CoA. Amplification of this intrinsic activity replaced Eci1p since it restored growth of the eci1 strain on petroselinic acid for which di-isomerase is not required whereas Eci1p is. Heterologous expression in yeast of rat di-isomerase resulted in a peroxisomal protein that was enzymatically active but did not re-establish growth of the eci1 mutant on oleic acid. A strain devoid of Dci1p grew on oleic acid to wild-type levels, whereas one lacking both Eci1p and Dci1p grew as poorly as the eci1 mutant. Hence, we reasoned that yeast di-isomerase does not additionally represent a physiological 3,2-isomerase and that Dci1p and the postulated alternative pathways in which it is entrained are dispensable for degrading oleic acid.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the yeast Saccharomyces cerevisiae, saturated fatty acids are degraded through a peroxisomal -oxidation process (1) catalyzed by the enzymes acyl-CoA oxidase (Pox1p), 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA dehydrogenase (Fox2p; peroxisomal multifunctional enzyme type 2; peroxisomal MFE¹ type II), and 3-ketoacyl-CoA thiolase (Pot1p/Fox3p) as shown in Fig. 1A (2-5). To metabolize even-numbered cis-double bonds in unsaturated fatty acids such as petroselinic acid (cis-C_18:1(6)), yeast rely on peroxisomal Sps19p (6) which corresponds to the auxiliary enzyme 2,4-dienoyl-CoA reductase (2,4-reductase) and peroxisomal Eci1p (7, 8) representing the auxiliary enzyme ³-²-enoyl-CoA isomerase (3,2-isomerase; Fig. 1B). Eci1p is also crucial for yeast to degrade odd-numbered cis-double bonds in fatty acids such as oleic acid (cis-C_18:1(9)) via the isomerase-dependent pathway (dashed arrows; Fig. 1C).

In mammals, odd-numbered cis-double bonds are thought to be degraded in both peroxisomes and mitochondria by an additional route (solid arrows; Fig. 1C) that is mediated sequentially by 3,2-isomerase, ^3,5-^2,4-dienoyl-CoA isomerase (di-isomerase), 2,4-reductase, and again 3,2-isomerase, termed the reductase-dependent pathway (9-12). A recently proposed second alternative di-isomerase-dependent pathway (13) operates in the absence of 2,4-reductase (bent arrow; Fig. 1C). Di-isomerase activity that transfers double bonds from the ³-⁵ to the ² -⁴ positions has hitherto not been identified in yeast, and it was not known whether fungi possess di-isomerase- or reductase-dependent pathways. Although the cDNA of the rat ECH1 gene encoding both peroxisomal and mitochondrial di-isomerase has previously been cloned and characterized (14), a knockout mouse model devoid of this enzyme activity is not yet available. Therefore, the in vivo requirement in eukaryotes for di-isomerase during the breakdown of fatty acids with double bonds at odd-numbered positions remains unknown.

This study is concerned with whether yeast cells possess all of the enzymes entrained in the alternative pathways for degrading oleic acid postulated to exist in mammals and whether these alternatives are physiologically significant. We report on the oleic acid-inducible DCI1 gene encoding yeast peroxisomal di-isomerase. Identification of DCI1 had meant that S. cerevisiae could serve as a model system for studying the requirement of alternative routes for growth on fatty acids with double bonds at odd-numbered positions. We provide growth data on mutants devoid of the three auxiliary enzymes Dci1p, Sps19p, and Eci1p, and we discuss the in vivo significance of the alternative pathways.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Strains and Gene Disruptions-- Escherichia coli strain DH10B was used for all plasmid amplifications and isolations. The S. cerevisiae strains constructed here were derived from BJ1991 (15) and are listed in Table I. Construction of strains BJ1991pex6 and BJ1991pox1 has been described elsewhere (16). Construction of DCI1-disrupted strains was performed according to published methods (17). The amplification product (dci1::kanMX4) generated by a polymerase chain reaction (PCR) performed on pFA6a-kanMX4 template DNA (17) with the 58-mer and 59-mer oligonucleotides YOR180C-S1 and YOR180C-S2 was used to transform strain BJ1991. The deletion of DCI1 in geneticin-resistant transformants was verified by PCR using the oligonucleotide pairs YOR180C-A1/K2 and K3/YOR180C-A4 (17). Additional PCR verification of the DCI1 deletion was performed using the oligonucleotide pairs YOR180C-A1/A2 or -A3/A4 which yielded the expected product with wild-type genomic DNA as template but not with that of the mutant.

The BJ1991dci1eci1 strain was generated by transforming the BJ1991dci1 deletant with an SpeI-SphI fragment from pAG916 containing an eci1::kanMX4::URA3 deletion. The eci1 deletion was verified in URA3 transformants that failed to grow on oleic acid by PCR using the YLR284C-A1/A4 oligonucleotides (7) which gave rise to a single band representing the deleted gene. The BJ1991eci1sps19 double disruptant was generated by transforming a BJ1991eci1 (7) strain with an ScaI-StuI fragment from pAG129 (6) containing the sps19::LEU2 deletion. Alteration of SPS19 as a result of incorporating the sps19::LEU2 disruption fragment was verified by Southern analysis of HindIII-digested genomic DNA using a 1.4-kb SphI-XbaI SPS18/19 fragment from pAG454 (6).

Media and Growth Conditions-- RNA was isolated from logarithmic cultures grown for 16 h in medium containing 1% (w/v) yeast extract, 2% (w/v) bactopeptone, and either 2% (w/v) D-glucose, 2% (v/v) ethanol, or 0.2% (w/v) oleic acid and 0.02% (w/v) Tween 80 (adjusted to pH 7.0 with NaOH) to A_600 nm = 1.0. Plates or liquid media used to assess utilization of fatty acids were prepared as described (6, 7) and contained as the sole carbon source 0.125% (w/v) of either palmitic acid, arachidonic acid (cis-C_{20:4(5,8,11,14)}), or oleic acid and 0.5% (w/v) Tween 80. Propagation of cells for growth assays on solid or in liquid media or for enzyme assays was performed as described previously (6, 7). YPD-G418 plates used to select for yeast transformants carrying the kanMX4 deletion cassette contained 200 µg × ml¹ G418 (geneticin) as described (17).

Plasmid Constructions-- Plasmids and oligonucleotides used are listed in Table II. The plasmid pSK::YOR180c containing DCI1 was constructed by inserting the XbaI- and PstI-digested PCR product generated with oligonucleotides H0393 and H0394 using genomic yeast DNA as template into a similarly digested pBluescript®SK(+) vector (pSK; Stratagene, La Jolla, CA). The plasmid overexpressing DCI1 (pAG774) was generated by ligating the amplified product of oligonucleotides YOR180C-A1 and YOR180C-A4 to an SmaI-digested YEplac181 (18).

For subcellular localization, Dci1p was fused to the carboxyl terminus of green fluorescent protein (GFP) as described for GFP-Eci1p (7). A triple ligation was performed with the following DNA fragments: an NotI-DCI1-SalI PCR product (generated using oligonucleotides NotI-180C-F and SalI-180C-R); an EcoRI-NotI restriction digest fragment containing GFP behind the ADH2 promoter (from plasmid pADH2-GFP-ECI1; Ref. 7); and an EcoRI- and SalI-digested YIplac204 (18), resulting in pADH2-GFP-DCI1. The EcoRV-linearized plasmid was integrated in the trp1 locus of strains BJ1991 wild type and BJ1991pex6, and integrants were designated yAG933 and yAG934, respectively.

The URA3-based eci1 disruption plasmid was constructed by ligating the YLR284C-A1/A4 (7)-primed PCR product using BJ1991eci1 (7) genomic DNA as template to pGEM®-T (Promega Corp., Madison, WI), resulting in pGEM::eci1. A 1.2-kb HindIII fragment containing the URA3 gene from plasmid pJJ244 (19) was then used to disrupt the kanMX4 gene at the respective restriction site, resulting in pAG916. Construction of the plasmid expressing rat di-isomerase (ECH1) was performed as follows: oligonucleotides ECH1-F and ECH1-R were used in a PCR applied to ECH1 template cDNA to generate an XbaI- and XhoI-delimited product. The amplification product was ligated to pSK, and following XhoI- and XbaI-double digestion the released insert was purified and ligated to the promoter of the oleic acid-inducible catalase A gene (CTA1) in pYE352-CTA1 (20), yielding plasmid pKA-ECH1.

Enzyme Assays-- Assays were performed on soluble protein extracts prepared from homogenized oleic acid-induced yeast cells (6, 7). The assay for di-isomerase was performed as described (14, 21) using either 5-cis-3-hexadienoyl-CoA or 3,5,8,11,14-eicosapentenoyl-CoA as substrates. Candida sp. acyl-CoA oxidase (Sigma) was used to generate the latter substrate from arachidonoyl-CoA (14). The assay system for 3,2-isomerase activity in Dci1p was performed in the absence of exogenous acyl-CoA oxidase using excess purified recombinant rat di-isomerase and arachidonoyl-CoA as substrate. In this assay system, which monitored metabolism of 2,5,8,11,14-eicosapentenoyl-CoA via 3,5,8,11,14-eicosapentenoyl-CoA to 2,4,8,11,14-eicosapentenoyl-CoA, the rate-limiting step was the ²-³ isomerization. 2,5,8,11,14-Eicosapentenoyl-CoA was generated from arachidonoyl-CoA by endogenous acyl-CoA oxidase (Pox1p) in the lysates of yeast cells grown on oleic acid and was not rate-limiting. To 30 µM arachidonoyl-CoA was sequentially added 2 µg of purified recombinant rat di-isomerase, 10 µg of soluble protein extract from BJ1991eci1 harboring the plasmid vector, followed by 10 µg of protein from isogenic cells overexpressing Dci1p. The assay for 2-enoyl-CoA hydratase 1 was conducted according to published methods (22) using trans-2-hexenoyl-CoA as substrate. The assay for peroxisomal MFE type II (combined 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA dehydrogenase activities) was performed with trans-2-decenoyl-CoA as substrate (23).

Miscellaneous-- The following procedures were performed according to published methods: nucleic acid manipulations, formaldehyde gel electrophoresis, blotting and hybridization (24), DNA fragment isolation (25), yeast transformation (26), yeast RNA preparation for Northern analysis (27), and determination of protein concentration (28). The DNA fragments consisting of DCI1, ECI1, SPS19, POX1, or ACT1 that were used to probe the Northern blot were obtained from pSK::YOR180c, pSK::YLR284c (7), pAG113 (6), pAD17 (2), and pYA301 (29), respectively. The [-³²P]dATP-labeled probes were generated with a random primer labeling kit (Prime-a-Gene, Promega, Madison, WI) according to the manufacturer's instructions. Fluorescence of ethanol-derepressed yeast cells fixed in 3.7% (v/v) formaldehyde was performed as described (7). DNA staining was done with 4',6-diamidino-2-phenylindole (DAPI) at a final concentration of 2 µg × ml¹. Electrophoretic mobility shift assays were performed with crude extracts from cells grown on oleic acid as described (30). Excision and end labeling of the fragment containing the ECI1 ORE has previously been described (7). For competition, a 25-fold excess of double-stranded oligonucleotides (listed in Table II) comprising the oleate response elements of DCI1 (YOR180C-ORE1/2), as well as those of ECI1 (YLR284C-ORE1/2), SPS19, POX1, and CTA1/CTA1mut was added to the reaction mixture.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

YOR180c Encodes an Oleic Acid-inducible Peroxisomal Protein-- Current understanding of mammalian -oxidation postulates that oleic acid can be metabolized via alternative pathways (solid arrows; Fig. 1C). Yeast cells have been shown previously to require 3,2-isomerase (Eci1p) for breaking down oleic acid (7, 8); however, it was not known whether they also possessed a di-isomerase that could enable them to degrade this fatty acid via the reductase-dependent or the di-isomerase-dependent routes. A previous search of the S. cerevisiae data bases for open reading frames with promoters containing an oleate response element (ORE) (31, 32) that could encode peroxisomally targeted proteins entrained in -oxidation revealed two novel homologous (46% identity) genes YLR284c (ECI1) and YOR180c² (7). In line with other genes encoding -oxidation enzymes up-regulated in cells grown in oleic acid medium compared with those propagated in either glucose or ethanol media (30, 33), YOR180c, designated here as DCI1, was found to be induced (Fig. 2A).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Pathways for degrading fatty acids. A, the -oxidation spiral for breaking down saturated fatty acids. The yeast enzymes Pox1p (acyl-CoA oxidase), Fox2p (2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA dehydrogenase), and Pot1p/Fox3p (3-ketoacyl-CoA thiolase) are noted. Enzymes are indicated to the left of the dashed arrows, and metabolites are noted above them in italics. B, the position of the auxiliary enzymes Sps19p (2,4-dienoyl-CoA reductase) and Eci1p (3-2-enoyl-CoA isomerase) in the degradation pathway of unsaturated fatty acids with double bonds at even-numbered positions. Double arrowhead represents the two steps of Fox2p. C, the three possible pathways for degrading fatty acids with double bonds at odd-numbered positions. The isomerase-dependent pathway (dashed arrows) has been shown previously to be required for growth of yeast on oleic acid medium. The postulated mammalian reductase- and di-isomerase-dependent pathways are indicated by solid arrows.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Peroxisomal Dci1p is induced in oleic acid medium. A, carbon source-dependent transcriptional activation of DCI1 was determined by Northern analysis using RNA from the wild-type strain BJ1991 (WT) and a pip2oaf1 mutant grown in the indicated media (7). The filter was probed with labeled DCI1, ECI1, SPS19, POX1, and the constitutively transcribed ACT1. B, formation of a Pip2p- and Oaf1p-dependent protein-DNA complex with ECI1 ORE (7) is competed by excess DCI1 ORE (indicated by an asterisk). Labeled ECI1 ORE was incubated with crude extracts from oleic acid-induced cells, and bound DNA was resolved from free probe using a 4% (w/v) polyacrylamide gel. The retarded complex representing Pip2p-Oaf1p is indicated by an arrow. For competition, a 25-fold excess of unlabeled double-stranded oligonucleotides comprising the OREs of ECI1, SPS19, POX1, and CTA1, CTA1mut, and DCI1 was added to the reaction mixture prior to the wild-type crude extracts. C, Dci1p is located in peroxisomes. The BJ1991 wild type and the otherwise isogenic pex6 strain propagated in YP medium containing 2% (v/v) ethanol and expressing GFP-Dci1p (strains yAG933 and yAG934, respectively) were fixed in 3.7% (v/v) formaldehyde and examined for GFP fluorescence. The DNA in nuclei and mitochondria was detected by staining cells with DAPI. The structural integrity of cells was recorded as Nomarski images.

Appropriately, levels of DCI1 transcript were very low in a similarly propagated pip2oaf1 mutant (34) devoid of the DNA-binding proteins mediating the transcriptional response to oleic acid. The induction of DCI1 was consistent with the presence in its promoter of an ORE matching the consensus (CGGN₃TNA-N_3-6-TNAN₃CCG where N represents any nucleotide; see Ref. 30). Subsequent electrophoretic mobility shift assays revealed that the DCI1 ORE could compete protein interacting with the ECI1 ORE (7) that was present in a soluble protein extract derived from homogenized wild-type cells but that was absent from pip2 or oaf1 mutant extracts (Refs. 34 and 35; Fig. 2B). During the course of this work, YOR180c was reported elsewhere as an unidentified oleic acid-inducible open reading frame (36-38), serving to support our conclusion that since both transcriptional up-regulation and protein-DNA interaction depended on PIP2 and OAF1, the up-regulation of DCI1 in oleic acid medium was due to the DCI1 ORE.

Dci1p resembles peroxisomal Eci1p (as well as other peroxisomal proteins) also at the carboxyl terminus, where an HRL tripeptide is thought to represent a novel variant of peroxisomal targeting signal type I (PTS1) (39, 40). In accordance with recent findings (36-38), fluorescence microscopy demonstrated that a GFP-Dci1p fusion appeared as punctate fluorescence in wild-type cells propagated in ethanol medium; however, in peroxisome-deficient pex6 cells that grow normally under these conditions (41), this fluorescence was diffuse (Fig. 2C). Control DAPI staining for DNA demonstrated a similar mitochondrial (as well as nuclear) fluorescence pattern in both strains. This indicated that Dci1p was compartmentalized in peroxisomes. Since in addition to being oleic acid-inducible as well as peroxisomal, Dci1p is also a member of the hydratase/isomerase family of proteins (42) with the potential to utilize acyl-CoA substrates, it possessed the characteristics of yeast proteins involved in -oxidation of fatty acids.

Overexpression of Dci1p in an eci1 Strain Restores Growth on Oleic Acid-- This study is concerned with the identification of yeast di-isomerase. A search of the data bases revealed three yeast genes encoding proteins with similarity to rat di-isomerase, these being ECI1, YOR180c/DCI1, and YDR036c. Eci1p has been shown previously not to contain di-isomerase activity (7), whereas YDR036c, which does not encode a protein with an obvious PTS and did not show up in our ORE search, is probably not involved in -oxidation of fatty acids. To determine whether YOR180c/DCI1 represented the gene for di-isomerase, it was overexpressed in an eci1 strain that was impaired for growth on oleic acid. We reasoned that since the two 3,2-isomerase activities entrained in the reductase-dependent pathway (diagonal arrows; Fig. 1C) may not necessarily be encoded by ECI1, amplification of a di-isomerase could relieve the requirement for Eci1p during growth on oleic acid.

Mutants overexpressing Dci1p were compared with those enriched with Eci1p or harboring the plasmid vector for utilization of oleic acid using a zone-clearing assay (Fig. 3). In this and subsequent assays, fatty acid plates were used that additionally contained Tween 80 which acted to form an emulsion but also as a poor carbon source. Thus, cells could form small colonies on these plates but zones of clearing (i.e. opaque halos in the medium), such as those generated by the mutant expressing Dci1p or Eci1p (Fig. 3), indicated utilization of the fatty acid substrate. Hence, this demonstrated that Dci1p could have a direct role in -oxidation of fatty acids. Cells harboring the respective plasmid vector did not form a clearing zone in the medium and were, therefore, considered impaired for utilization of oleic acid. Growth of the eci1 strain on oleic acid could have been restored due to at least two possibilities as follows: (i) overexpression of Dci1p channeled the carbon flow from oleic acid via the di-isomerase and/or reductase-dependent pathways (Fig. 1C), or (ii) Dci1p represented a second 3,2-isomerase.

View larger version (85K): [in this window] [in a new window]   Fig. 3.   Dci1p restores growth of an eci1 mutant devoid of 3,2-isomerase on oleic acid medium. Formation of clearing zones by a mutant strain BJ1991eci1 overexpressing Dci1p was compared with those overexpressing Eci1p or harboring the plasmid vector. The strains used were yAG922 (eci1 [DCI1]), yAG957 (eci1 [ECI1]), and yAG958 (eci1 [Vector]).

To determine the mechanism of the relief of the requirement for Eci1p, an eci1sps19 double disruptant was generated that was additionally devoid of 2,4-reductase activity (Fig. 1C). In this mutant, the reductase-dependent pathway would be blocked. In agreement with current knowledge of the breakdown of fatty acids with double bonds at even-numbered positions which requires Sps19p (Fig. 1B), amplification of Dci1p did not re-establish growth of the mutant on petroselinic acid but did on oleic acid (performed with strains yAG952 and yAG953). Assuming that Sps19p represented the 2,4-reductase in Fig. 1C, this indicated that the reductase-dependent pathway did not represent the exclusive route by which Dci1p could have acted to restore growth of the eci1 mutant on oleic acid.

Dci1p Is Dispensable for Yeast Growth on Oleic Acid-- To elucidate the extent to which Dci1p was involved in the metabolism of fatty acids, a DCI1-deleted strain was constructed and examined for growth on fatty acid media using a zone-clearing assay. Wild-type and dci1 strains were applied as serially diluted culture spots onto plates containing either palmitic, oleic, or arachidonic acid (Fig. 4). In this zone-clearing assay, utilization of the fatty acids was represented by thin opaque rings around the colonies. These plate assays showed that the dci1 strain was capable of forming a clear zone in all solid media examined (including in cis-12-octadecenoic acid; cis-C_18:1(12), not shown). A pox1 strain lacking acyl-CoA oxidase (Fig. 1A) which is defective for -oxidation (2) was added to the plates to demonstrate lack of zone formation on any of the fatty acids tested. A similarly applied eci1 strain with impaired growth on unsaturated fatty acids (Fig. 1, B and C) but not on saturated ones (7, 8) formed wild-type clear zones only on palmitic acid. Although some clearing was also produced by the eci1 strain on oleic acid, albeit below that by the dci1 strain, it failed to utilize arachidonic acid. The sps19 disruptant (yAG141) lacking 2,4-reductase (Fig. 1B) and impaired for growth on fatty acids with double bonds at even-numbered positions was unable to utilize arachidonic acid but was otherwise unaffected on oleic acid and palmitic acid.

View larger version (79K): [in this window] [in a new window]   Fig. 4.   Growth of a DCI1-deleted strain is not affected on fatty acids. Formation of a clearing zone by the mutant strain BJ1991dci1 was examined on solid media containing oleic acid, arachidonic acid, or palmitic acid. Strains BJ1991 wild type (WT), BJ1991pox1, BJ1991sps19 (yAG141), BJ1991eci1, and BJ1991dci1 were grown in rich glucose medium to A600 nm = 1.0, and serially diluted culture aliquots of 2 µl were spotted on plates. Following incubation for 5 days, the plates were recorded photographically.

Vital counts following growth in liquid oleic acid medium were used to corroborate the observations made with the plates (Table III). In a representative experiment, at the time point (75 h) when the pox1 and eci1 strains were clearly affected since their respective number of surviving colonies had reached only 9 and 18% that of wild type, those of the dci1 and sps19 mutants were as high as 110 and 116%, respectively. This indicated that, like with the sps19 disruption, deletion at DCI1 did not affect the number of surviving cells following prolonged propagation in liquid medium containing oleic acid and supported the observations made using solid media.

Dci1p-enriched Cell Extracts Contain Di-isomerase and Isomerase Activities-- To determine the enzymatic properties of Dci1p, it was overexpressed in the corresponding dci1 strain from a multi-copy plasmid. Soluble protein extracts derived from homogenized oleic acid-induced cells were used in assays for possible hydratase/isomerase enzyme activities, including those of 2-enoyl-CoA hydratase 1 (with trans-2-hexenoyl-CoA as substrate) and di-isomerase (using 3,5-hexadienoyl-CoA or 3, 5,8,11,14-eicosapentenoyl-CoA), as well as peroxisomal MFE type II. By using 3,5,8,11,14-eicosapentenoyl-CoA as substrate, a specific di-isomerase activity was measured in the Dci1p-enriched extracts (Fig. 5, arrow 2) as an increase in A_300 nm. This activity was comparable to that contained in pure recombinant rat di-isomerase (Fig. 5, arrow 3) and was equivalent to 6 nmol × min¹ per mg of protein. Extracts from a mutant harboring the plasmid vector did not contain detectable levels of this activity (Fig. 5, arrow 1). It has been shown previously that the increase in A_300 nm under these experimental conditions is due to the transfer of ³-⁵-conjugated double bonds of dienoyl-CoAs to the ²-⁴ positions (14). Di-isomerase activity could also be measured in the Dci1p-enriched extracts using 3,5-hexadienoyl-CoA as substrate (not shown).

View larger version (8K): [in this window] [in a new window]   Fig. 5.   Dci1p is a di-isomerase. A, addition of 20 µg of protein (arrow 1) from a soluble extract derived from an oleic acid-induced BJ1991dci1 strain harboring the plasmid vector to an assay containing 3,5,8,11,14-eicosapentenoyl-CoA as substrate did not result in di-isomerase activity. B, soluble extract (20 µg of protein; arrow 2) from an oleic acid-induced BJ1991dci1 disruptant overexpressing Dci1p (yAG851) contained di-isomerase activity. The increase in A300 nm was due to accumulation of the conjugated 2,4-double bond in enoyl-CoA thioesters (14, 22). C, addition of 1 µg of purified recombinant rat di-isomerase (arrow 3) resulted in similar absorbance increase.

Assays for 2-enoyl-CoA hydratase 1 or peroxisomal MFE type II activities performed on the control and Dci1p-enriched extracts did not demonstrate any differences. The similarity between Dci1p and Eci1p also prompted the determination of whether Dci1p additionally possessed a detectable level of 3,2-isomerase activity. By using trans-3-hexenoyl-CoA as substrate, the activity in soluble protein extracts from homogenized eci1 cells overexpressing Dci1p was below the detection limit of the assay; however, these extracts could generate 3,5,8,11,14-eicosapentenoyl-CoA from 2,5,8,11,14-eicosapentenoyl-CoA and therefore contained an intrinsic ³-²-enoyl-CoA isomerase activity (performed according to the method under "Experimental Procedures" using strains yAG922 and yAG958, not shown).

Overexpressed Dci1p Functionally Replaces Eci1p Due to Amplification of a 3,2-Isomerase Activity-- Detection of the intrinsic 3,2-isomerase activity in Dci1p raised the question of whether re-establishment of the eci1 strain's growth on oleic acid by overexpressing Dci1p (Fig. 3) could have been mediated not only by engagement of the alternative routes but also by replacing the missing 3,2-isomerase activity (Fig. 1C). Therefore, the effect of overexpressing Dci1p in the eci1 strain was examined also on petroselinic acid for which the alternative pathways are not thought to be engaged, whereas Eci1p is (Fig. 1B). Since enrichment with Dci1p restored the ability of the eci1 strain to utilize petroselinic acid in a similar manner to Eci1p (Fig. 6), this underscored the enzyme assay result that Dci1p possessed some 3,2-isomerase activity. Hence, in the previous growth assay on oleic acid (Fig. 3), in addition to possibly activating the two alternative routes, overexpressed Dci1p could have also acted to restore the growth of the mutant by supplying the missing 3,2-isomerase activity.

View larger version (98K): [in this window] [in a new window]   Fig. 6.   3,2-Isomerase activity in Dci1p restores growth of the eci1 mutant on petroselinic acid. Formation of clearing zones by the mutant strain BJ1991eci1 over-expressing Dci1p was compared with those overexpressing Eci1p or harboring the plasmid vector on petroselinic acid medium. Strains used are listed in the legend to Fig. 3.

Dci1p Is Not a Redundant Eci1p Isoenzyme-- To determine whether Dci1p represented a physiological 3,2-isomerase, a BJ1991dci1eci1 double mutant was generated and compared with the BJ1991eci1 single mutant for utilization of oleic acid, on which the latter strain grows to some extent (Fig. 4). If deletion of both ECI1 and DCI1 were to block completely degradation of unsaturated fatty acids, then the double mutant would be expected to appear as a pox1 strain, where no zone was detected even at the highest density of cells spotted on solid oleic acid medium (Fig. 4). However, close scrutiny of similarly dense dci1eci1 double mutant and eci1 single mutant cells demonstrated that both utilized this fatty acid to some extent since a faint ring could be seen (Fig. 7). This minimal zone was probably due to their ability to undergo three rounds of -oxidation prior to encountering the ⁹ double bond in oleic acid. Growth of the single and double mutants was also examined in liquid oleic acid medium. Following propagation for 48 h, the number of viable colonies of the double mutant was comparable to that of the corresponding single deletant (11 and 13% of the wild type, respectively). This implied that as a single genomic copy, DCI1 probably did not act as a gene for a redundant Eci1p isoenzyme despite containing an intrinsic 3,2-isomerase activity that could replace ECI1 following amplification.

View larger version (87K): [in this window] [in a new window]   Fig. 7.   Growth of a BJ1991eci1 single mutant and a BJ1991dci1eci1 double mutant on oleic acid is comparably impaired. The formation of clearing zones on solid oleic acid medium by the BJ1991dci1eci1, BJ1991eci1 or BJ1991dci1 mutants was compared with the BJ1991 wild type.

However, the finding that Dci1p could generate 3,5,8,11,14-eicosapentenoyl-CoA from 2,5,8,11,14-eicosapentenoyl-CoA indicated that it could represent a novel isomerase necessary for generating the 3,5-dienoyl-CoA substrate required for the di-isomerase (upper diagonal arrow; Fig. 1C). In this case, overexpression of a di-isomerase without an intrinsic 3,2-isomerase activity in the eci1 mutant (in which Dci1p is intact) should re-establish growth on oleic acid due to amplification of the di-isomerase pathway. Rat di-isomerase could be used for this purpose since the purified recombinant protein does not contain 3,2-isomerase activity (14) and since heterologous expression of rat peroxisomal proteins has been shown previously to work in yeast cells, e.g. rat peroxisomal MFE type I can replace Fox2p and Eci1p (7, 20).

Rat ECH1 encoding di-isomerase (14) was expressed in the eci1 mutant. Enzyme assays performed on extracts derived from yeast cells using 3,5,8,11,14-eicosapentenoyl-CoA as substrate verified that the rat di-isomerase was active, and subsequent immunoelectron microscopy demonstrated that the rat protein entered their peroxisomes (not shown). However, in a zone-clearing assay on oleic acid or petroselinic acid media using strains yAG977 to yAG980 (Table II) expression of rat di-isomerase did not result in the restoration of the eci1 mutant's growth. Hence an amplified rat di-isomerase could not act to relieve the requirement for Eci1p probably because Dci1p did not represent the 3,2-isomerase activities acting in concert with di-isomerase in the alternative pathways (diagonal arrows; Fig. 1C). We reason that although overexpressed Dci1p could have acted to re-establish growth of the eci1 mutant on oleic acid (Fig. 3) through each of the three routes (Fig. 1C), it probably did so solely via the isomerase-dependent pathway as a result of a grossly amplified intrinsic 3,2-isomerase activity (as with petroselinic acid; Fig. 5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Here we report the identification of the oleic acid-inducible S. cerevisiae DCI1 gene encoding peroxisomal di-isomerase. Extracts derived from Dci1p-enriched yeast cells contained both di-isomerase and 3,2-isomerase activities, and although overexpression of Dci1p in the eci1 mutant acted to replace the missing 3,2-isomerase activity on petroselinic acid, we chose to designate the novel gene as a di-isomerase and not as a 3,2-isomerase for the following reasons. First, a previous cell fractionation study revealed that 3,2-enoyl-CoA isomerase activity was not detected in any of the fractions derived from an eci1 mutant, including the peak peroxisomal fractions (8). Therefore, if Dci1p represented a minor 3,2-isomerase in a partially redundant system with the major 3,2-isomerase Eci1p, then the eci1 mutant should have been able to grow at least slowly on unsaturated fatty acids. This was clearly not the case on arachidonic acid (Fig. 4). Second, in such a system the double eci1dci1 mutant would have been expected to be more impaired than the single eci1 mutant for growth on oleic acid, which it wasn't (Fig. 7).

By using enzyme assays with trans-3-hexenoyl-CoA as substrate, 3,2-isomerase activity in extracts enriched with Dci1p was below the detection limit, whereas those similarly enriched with Eci1p contained a 3,2-isomerase activity of 60 nmol × min¹ per mg of protein (7). An in vitro assay based on excess purified recombinant rat di-isomerase could demonstrate that Dci1p contained a 3,2-isomerase activity. Hence, since 3,2-isomerase activity was too low to be detected through a conventional assay, this provided a third compelling reason not to designate the product of YOR180c as an Eci1p-like 3,2-isomerase. This situation is similar to that of rat di-isomerase which also contains a low level of hydratase 1 activity (14) and is in line with the catalytic properties of several other hydratase/isomerase family members that represent a hydratase 1 and also a 3,2-isomerase, including mammalian peroxisomal MFE type I (22), -subunit of bacterial -oxidation complex (46), and mammalian mitochondrial hydratase 1, in which the latter activity is low (47).

We also report on the dispensability for growth of yeast on oleic acid of the putative mammalian alternative pathways in which di-isomerase is entrained. Deletion of DCI1 did not affect growth of the corresponding mutant on any of the fatty acids tested. Although this cannot be entirely ruled out, the possibility that a second di-isomerase might exist in S. cerevisiae is low. Eci1p, the closest homologue of Dci1p, has been purified previously to homogeneity as a recombinant protein but was found to be without di-isomerase activity (7). YDR036c representing the gene for the third and last unidentified S. cerevisiae member of the hydratase/isomerase protein family is probably also not an oleic acid-inducible, peroxisomal di-isomerase isoenzyme.

The reductase-dependent pathway in which di-isomerase is implicated is the subject of intensive research (9-13, 43, 44), and the contribution of this pathway to the degradation of fatty acids with cis-double bonds at odd-numbered positions is controversial. One study using intact liver and heart mitochondria reported that cis-4-decenoic acid (cis-C_10:1(4)) was completely metabolized by the reductase-dependent pathway (12), whereas more recent work with soluble extracts of rat mitochondria using 2,5-octadienoyl-CoA as substrate demonstrated that most (80%) of this intermediate was metabolized via the isomerase-dependent pathway (13). Since rat peroxisomes have all the enzymes required for the reductase-dependent pathway (45), it was reasonable to assume that these routes could also occur in yeast peroxisomes.

Generation of yeast cells devoid of di-isomerase activity implied that it was possible to determine the in vivo requirement of these alternative pathways for peroxisomal degradation of fatty acids. The ability of the dci1 and sps19 strains with defective reductase pathway enzyme activities to grow on unsaturated fatty acids with cis-double bonds at odd-numbered positions was examined on solid as well as in liquid oleic acid media. Under the conditions tested, both the dci1 and sps19 strains grew much faster than the eci1 and pox1 mutants. This was in agreement with data generated during the course of this work in which a yor180c disruptant was compared with a defective pex mutant for growth on oleic acid (38). Although in our BJ1991 strain background the dci1 and sps19 mutations did not impair growth on oleic acid, those generated in a BY4733 strain caused the resulting disruptants to be partially defective (38). It would have been interesting to see whether the reported deficiency could be detectable in a vital count, was appropriately absent during growth in palmitic acid control medium, or be rectified following complementation with the corresponding gene.

We also showed that although a combination of Dci1p and rat di-isomerase could have acted as a di-isomerase-dependent route for breaking down oleic acid, overexpression of the rat protein did not restore growth of the eci1 mutant. Taken together, the data presented here serve to underscore our conclusion that yeast di-isomerase is dispensable rather than redundant and that the reductase- and di-isomerase-dependent pathways are not physiologically relevant for growth of yeast on unsaturated fatty acids such as oleic acid. Although this conclusion could be cautiously extrapolated to mammalian peroxisomal -oxidation, the role of these alternative pathways in mammalian mitochondrial -oxidation could be more important.

	ACKNOWLEDGEMENTS

We thank Hannelore Wrba, Marika Kamps, and Marion Repitz for excellent technical assistance and Andreas Firzinger for conducting the data base search and for plasmid pSK::YOR180c.

	FOOTNOTES

^* This work was supported in part by Fonds zur Förderung der Wissenschaftlichen Forschung, Vienna, Austria, Grants P12061 (to B. Hamilton) and P12118 (to A. H.), by Jubiläumsfonds der Österreichischen Nationalbank, Austria, Grant 6517 (to H. R.), and by the Sigrid Juselius Foundation, Finland, and the Academy of Finland (to J. K. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Present address: Inst. für Biochemie, Limonenstrasse 7, 12203 Berlin, Germany. Tel.: 49-30-838-2937; Fax: 49-30-838-2936; E-mail: hpr@zedat.fu-berlin.de

² Reserved in the Saccharomyces Genome Data Base as an enoyl-CoA hydratase homologue; I. V. Karpichev, J. Lopez, and G. M. Small, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: MFE, multifunctional enzyme; 2,4-reductase, 2,4-dienoyl-CoA reductase; 3,2-isomerase, ³-²-enoyl-CoA isomerase; di-isomerase, ^3,5-^2,4-dienoyl-CoA isomerase; PCR, polymerase chain reaction; kb, kilobase; GFP, green fluorescent protein; DAPI, 4',6-diamidino-2-phenylindole; ORE, oleate response element; PTS, peroxisomal targeting signal.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES