Global regulatory functions of Oaf1p and Pip2p (Oaf2p), transcription factors that regulate genes encoding peroxisomal proteins in Saccharomyces cerevisiae

Abstract

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.

FIG. 1

Expression of genes encoding peroxisomal proteins in wild-type (w.t.), oaf1Δ (ΔO1), pip2Δ (ΔP2), and oaf1Δ pip2Δ (ΔO1P2) yeast strains. Poly(A)⁺ RNA fractions from cells grown in 50-ml cultures were resolved in a 1% formaldehyde agarose gel (see Materials and Methods). Cells were grown either in glucose (YPD), glycerol (YPG), or glycerol-oleate (YPGO) medium. Levels of mRNA for POX1 (YGL20Sw) (a), FOX2 (YKR009c) (b), FOX3 (YIL160c) (c), TES1 (YJR019c) (d), SPS19 (YNL202w) (e), and PEX11 (YOL147w) (f) were quantitated from a Northern blot, and the values were normalized with actin levels as an internal control for loading. Expression in our wild-type strain grown in the presence of oleate was taken to be 100%.

FIG. 2

Expression of the six genes, MDH3 (YDL078c) (a), PEX5 (YDR244w) (b), FAA2 (YER015w), (c), PXA2 (YKL188c) (d), YCAT (YML042w) (e), and IDP3 (YNL009w) (f), that encode peroxisomal membrane or matrix proteins is induced two- to threefold in glycerol- and oleate (Gly/Ole)-grown cells. The induction is completely abolished when the OAF1 and PIP2 genes are both disrupted, but there is partial induction in cells lacking only one of these genes. w.t., wild type. Levels of expression were quantitated and normalized as described in the legend to Fig. 1.

FIG. 3

Oleate-mediated induction of peroxisomal catalase requires Oaf1p but not Pip2p. w.t., wild type; Gly/Ole, glycerol and oleate. CTA1 mRNA expression was measured and quantitated as described in the legend to Fig. 1.

FIG. 4

Northern analysis of PIP2 expression in a wild-type strain (w.t.) and strains in which OAF1 or PIP2 have been disrupted. Sixty micrograms of total RNA isolated from cells grown in YPD (glu), YPG (gly), or YPGO (g/o) medium was resolved in a 1% formaldehyde agarose gel, transferred to a nylon membrane, and hybridized as described in Materials and Methods.

FIG. 5

Oleate-dependent induction of genes encoding two different isoforms of citrate synthase. (a) CIT1 (YNR001c), encoding mitochondrial citrate synthase, is induced by oleate and is regulated by Oaf1p and Pip2p. (b) The oleate-dependent induction of CIT2 (YCR005c), encoding a peroxisomal isoform of this enzyme, is not mediated by these transcription factors. Growth media and method of quantitation are described in the legend to Fig. 1, except that cells were also grown in raffinose (YPR) or raffinose-oleate (YPRO [Raff/Ole]) medium for the analysis of CIT2 expression.

FIG. 6

mRNA expression of the ORFs that are regulated by Oaf1p and/or the Oaf1p Pip2p heterodimer. YJL218w (a) and YPL095c (b) are induced by oleate and regulated by Oaf1p and Pip2p in a similar fashion to genes encoding peroxisomal β-oxidation enzymes (Fig. 1). YBR159w (c) is induced by oleate and regulated by Oaf1p and Pip2p, whereas partial induction of YIL120w (d) is mediated by Oaf1p alone. High expression of YOL002c (e) in the presence of glucose is abolished in oaf1Δ and pip2Δ strains. The oleate-dependent induction of YOR100c (f) requires both Oaf1p and Pip2p, whereas glucose repression of this gene is abolished in an oaf1Δ pip2Δ strain. w.t., wild type; Gly/Ole, glycerol and oleate. Growth media and method of quantitation are described in the legend to Fig. 1.

FIG. 7

PXA1, encoding a peroxisomal membrane protein, lacks a consensus ORE in its promoter region, but is induced by oleate in an Oaf1p- and Pip2p-dependent fashion. w.t., wild type; Gly/Ole, glycerol and oleate. Expression was quantitated and normalized as described in the legend to Fig. 1.