Purification, Identification, and Properties of a Saccharomyces cerevisiae Oleate-activated Upstream Activating Sequence-binding Protein That Is Involved in the Activation of POX1

Abstract

Peroxisomes have a central function in lipid metabolism, and it is well established that these organelles are inducible by many compounds including fatty acids. Peroxisomes are the sole site for the ␤-oxidation of fatty acids in yeast. The first and rate-limiting enzyme of this cycle is fatty acyl-CoA oxidase. The gene encoding this enzyme in Saccharomyces cerevisiae (POX1) undergoes a complex regulation that is dependent on the growth environment. When this yeast is grown in medium containing oleic acid as the main carbon source, peroxisomes are induced and POX1 expression is activated. When cells are grown in the presence of glucose, the expression of POX1 mRNA is repressed, whereas growth on a carbon source such as glycerol or raffinose causes derepression. This rigorous regulation is brought about by the complex interactions between trans-acting factors and cis-elements in the POX1 promoter. Previously, we characterized regulatory elements in the promoter region of POX1 that are involved in the repression and activation of this gene (Wang, T., Luo, Y., and Small, G. M. (1994) J. Biol. Chem. 269, 24480 -24485). In this study we have purified and identified an oleate-activated transcription factor (Oaf1p) that binds to the activating sequence (UAS1) in the POX1 gene. The protein has a predicted molecular mass of approximately 118 kDa.Peroxisomal ␤-oxidation is an important pathway in mammalian metabolism for catabolizing long and very long chain fatty acids. In many organisms, including yeasts, peroxisomal ␤-oxidation is the sole mechanism for the breakdown of fatty acids (1). The enzymes involved in this pathway are regulated according to the growth environment. Expression of genes encoding peroxisomal proteins in the yeast Saccharomyces cerevisiae is repressed when the yeast cells are grown in the presence of glucose, derepressed during growth on a nonfermentable carbon source, and activated when a fatty acid such as oleate is supplied for growth (2). This control is achieved through stringent transcriptional regulation of the genes encoding these proteins (3-8).Over the past several years we have focused our attentions toward understanding the mechanisms that regulate genes encoding peroxisomal ␤-oxidation enzymes in S. cerevisiae. In order to address this question, we have concentrated on the regulation of POX1, the gene encoding acyl-CoA oxidase, the rate-limiting enzyme of this cycle. Previously we characterized two upstream repression sequences (URS1 and URS2) 1 and one upstream activating sequence (UAS1) in the promoter region of POX1 (7, 9). We demonstrated that a protein or protein complex binds to UAS1 in an oleate-dependent fashion, and this brings about the activation of POX1. A similar UAS sequence (termed oleate response element) was identified in the upstream regions of genes encoding some of the other peroxisomal proteins (3, 4, 10).Several factors have been shown to be involved in the glucose repression of thiolase, the last enzyme in the peroxisomal ␤-oxidation cycle, which in S. cerevisiae ...

Regulation of the yeast SPS19 gene encoding peroxisomal 2,4‐dienoyl‐CoA reductase by the transcription factors Pip2p and Oaf1p: β‐oxidation is dispensable for Saccharomyces cerevisiae sporulation in acetate medium

“…Oaf1p mediates oleate induction control in a similar way to Pip2p (Luo et al, 1996;Karpichev et al, 1997;Rottensteiner et al, 1997), and we used a ⌬oaf1 deletant in order to determine whether Oaf1p plays any role in inducing SPS19. Expression of the full-length promoter SPS19-lacZ reporter construct in this mutant was compared with that in both the wild-type (in which the reporter is normally highly expressed) and the ⌬pip2 strain.…”

“…OREs are known to mediate the induction of a number of S. cerevisiae genes encoding peroxisomal proteins, including catalase A (CTA1; Filipits et al, 1993), acyl-CoA oxidase (POX1 ; Wang et al, 1994) and thiolase (FOX3 ; Einerhand et al, 1993), and are the binding target for Pip2p and Oaf1p (Luo et al, 1996;Rottensteiner et al, 1996;Karpichev et al, 1997). Peroxisomes and their biogenesis are the subject of intense research, not least because these organelles and their proliferation have been linked to several human genetic disorders, including Zellweger syndrome (Kaiser and Kramar, 1988;Masters and Crane, 1995).…”

Dynamics of Gene Expression Revealed by Comparison of Serial Analysis of Gene Expression Transcript Profiles from Yeast Grown on Two Different Carbon Sources

“…In the process we developed a statistical method for comparison of SAGE results and the planning of future experiments. We also constructed a SAGE library of mutant yeast cells in which the oleate-induced enlargement of the peroxisomal compartment was abrogated by deletion of the genes encoding transcription factors Pip2p and Oaf1p (Luo et al, 1996;Rottensteiner et al, 1996). This analysis revealed genes that are under the control of Pip2p and Oaf1p but in addition showed genes whose activity increased to survive the genetic impairment imposed to the pip2/oaf1 mutant cells.…”

The Saccharomyces cerevisiae Peroxisomal 2,4-Dienoyl-CoA Reductase Is Encoded by the Oleate-inducible GeneSPS19

“…The induction of a number of other S. cerevisiae genes encoding peroxisomal proteins such as catalase A (CTA1) and thiolase (FOX3) is mediated via a positive gene control sequence called the oleate response element (ORE 1 ; 20, 21). OREs have recently been shown to act as the binding target for the products of PIP2 (20) and OAF1 (21), and strains deleted at either locus were unable to grow on oleate (cis-C 18:1 (9) ) as the sole carbon source.…”