Two transcription factors, Oaf1p and Pip2p (Oaf2p), are key components in the pathway by which several Saccharomyces cerevisiae genes encoding peroxisomal proteins are activated in the presence of a fatty acid such as oleate. By searching the S. cerevisiae genomic database for the consensus sequence that acts as a target for these transcription factors, we identified 40 genes that contain a putative Oaf1p-Pip2p binding site in their promoter region. Quantitative Northern analysis confirmed that the expression of 22 of the genes identified is induced by oleate and that either one or both of these transcription factors are required for the activation. In addition to known peroxisomal proteins, the regulated genes encode novel peroxisomal proteins, a mitochondrial protein, and proteins of unknown location and function. We demonstrate that Oaf1p regulates certain genes in the absence of Pip2p and that both of these transcription factors play a role in maintaining the glucose-repressed state of one gene. Furthermore, we provide evidence that the defined consensus binding site is not required for the regulation of certain oleate-responsive genes.

Expression of the POX1 gene, which encodes peroxisomal acyl coenzyme A oxidase in the yeast Saccharomyces cerevisiae, is tightly regulated and can be induced by fatty acids such as oleate. Previously we have shown that this regulation is brought about by interactions between trans-acting factor(s) and an upstream activating sequence (UAS1) in the POX1 promoter. We recently identified and isolated a transcription factor, Oaf1p, that binds to the UAS1 of POX1 and mediates its induction. A screening strategy has been developed and used to identify eight S. cerevisiae mutants, from three complementation groups, that are defective in the oleate induction of POX1. Characterization of one such mutant led to the identification of Oaf2p, a protein that is 39% identical to Oaf1p. Oaf1p and Oaf2p form a protein complex that is required for the activation of POX1 and FOX3 and for proliferation of peroxisomes. We propose a model in which these two transcription factors heterodimerize and mediate this activation process. The mutants that we have isolated, and further identification of the corresponding defective genes, provide us with an opportunity to characterize the mechanisms involved in the coordinate regulation of peroxisomal ␤-oxidation enzymes.Peroxisomes are organelles that play important roles in cellular respiration and lipid metabolism. In most organisms, peroxisomes contain enzymes involved in fatty acid oxidation (␤-oxidation) and catalase which decomposes the hydrogen peroxide generated from this process (for a review, see reference 21). Peroxisomes are essential for human survival, as demonstrated by the fact that the genetic disorder Zellweger syndrome, in which there is a lack of functional peroxisomes, is lethal (15,22).The size, abundance, and enzyme content of peroxisomes vary according to the cell type, organism, and metabolic requirements. In the yeast Saccharomyces cerevisiae, levels of peroxisomal ␤-oxidation enzymes are regulated by the available carbon source. The rate-limiting enzyme in the ␤-oxidation pathway is acyl coenzyme A (acyl-CoA) oxidase, which is encoded by a single-copy gene, POX1 (9). We have previously shown that POX1 expression is induced when S. cerevisiae is grown in the presence of oleic acid and that this transcriptional regulation is brought about by a protein, or proteins, binding to a specific upstream activating sequence (UAS1) in the POX1 promoter (41, 42). UAS1-like sequences (also called oleate response elements [OREs]) (11,14) are present in genes encoding many peroxisomal proteins, including the other enzymes of the peroxisomal ␤-oxidation cycle (10).We recently purified one protein, Oaf1p, that binds to UAS1 when cells are grown in oleate medium (25). Furthermore, by cloning and subsequently disrupting the gene encoding Oaf1p, we demonstrated that this protein is required for the oleate induction of POX1. The deduced amino acid sequence of Oaf1p reveals a C 6 zinc cluster motif, placing it in the same family of transcription factors as Gal4p and Cyp1p (7,20).The genes R...

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 ...

The yeast open reading frame YOL002c encodes a putative membrane protein. This protein is evolutionarily conserved across species, including humans, although the function of each of these proteins remains unknown. YOL002c is highly expressed in yeast cells that are grown in the presence of saturated fatty acids such as myristate. Furthermore, cells in which the YOL002c gene is disrupted grow poorly on this carbon source. These mutant cells are also resistant to the polyene antibiotic, nystatin. Gene chip analysis on yol002c⌬ cells revealed that a variety of genes encoding proteins involved in fatty acid metabolism and in the phosphate signaling pathway are induced in this mutant strain. In addition, our studies demonstrated that in the disruption strain acid phosphatase activity is expressed constitutively, and the cells accumulate polyphosphate to much higher levels than wild-type cells. A homologous human protein is able to partially rescue these defects in phosphate metabolism. We propose that YOL002c encodes a Saccharomyces cerevisiae protein that plays a key role in metabolic pathways that regulate lipid and phosphate metabolism.

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