Basic Helix-Loop-Helix Transcription Factor Heterocomplex of Yas1p and Yas2p Regulates Cytochrome P450 Expression in Response to Alkanes in the Yeast Yarrowia lipolytica

The expression of the ALK1 gene, which encodes cytochrome P450, catalyzing the first step of alkane oxidation in the alkane-assimilating yeast Yarrowia lipolytica, is highly regulated and can be induced by alkanes. Previously, we identified a cis-acting element (alkane-responsive element 1 [ARE1]) in the ALK1 promoter. We showed that a basic helix-loop-helix (bHLH) protein, Yas1p, binds to ARE1 in vivo and mediates alkane-dependent transcription induction. Yas1p, however, does not bind to ARE1 by itself in vitro, suggesting that Yas1p requires another bHLH protein partner for its DNA binding, as many bHLH transcription factors function by forming heterodimers. To identify such a binding partner of Yas1p, here we screened open reading frames encoding proteins with the bHLH motif from the Y. lipolytica genome database and identified the YAS2 gene. The deletion of the YAS2 gene abolished the alkane-responsive induction of ALK1 transcription and the growth of the yeast on alkanes. We revealed that Yas2p has transactivation activity. Furthermore, Yas1p and Yas2p formed a protein complex that was required for the binding of these proteins to ARE1. These findings allow us to postulate a model in which bHLH transcription factors Yas1p and Yas2p form a heterocomplex and mediate the transcription induction in response to alkanes

Suppression of respiratory growth defect of mitochondrial phosphatidylserine decarboxylase deficient mutant by overproduction of Sfh1, a Sec14 homolog, in yeast.

Interorganelle phospholipid transfer is critical for eukaryotic membrane biogenesis. In the yeast Saccharomyces cerevisiae, phosphatidylserine (PS) synthesized by PS synthase, Pss1, in the endoplasmic reticulum (ER) is decarboxylated to phosphatidylethanolamine (PE) by PS decarboxylase, Psd1, in the ER and mitochondria or by Psd2 in the endosome, Golgi, and/or vacuole, but the mechanism of interorganelle PS transport remains to be elucidated. Here we report that Sfh1, a member of Sec14 family proteins of S. cerevisiae, possesses the ability to enhance PE production by Psd2. Overexpression of SFH1 in the strain defective in Psd1 restored its growth on non-fermentable carbon sources and increased the intracellular and mitochondrial PE levels. Sfh1 was found to bind various phospholipids, including PS, in vivo. Bacterially expressed and purified Sfh1 was suggested to have the ability to transport fluorescently labeled PS between liposomes by fluorescence dequenching assay in vitro. Biochemical subcellular fractionation suggested that a fraction of Sfh1 localizes to the endosome, Golgi, and/or vacuole. We propose a model that Sfh1 promotes PE production by Psd2 by transferring phospholipids between the ER and endosome

Evaluation of sterol transport from the endoplasmic reticulum to mitochondria using mitochondrially targeted bacterial sterol acyltransferase in <i>Saccharomyces cerevisiae</i>

<div><p>To elucidate the mechanism of interorganelle sterol transport, a system to evaluate sterol transport from the endoplasmic reticulum (ER) to the mitochondria was constructed. A bacterial glycerophospholipid: cholesterol acyltransferase fused with a mitochondria-targeting sequence and a membrane-spanning domain of the mitochondrial inner membrane protein Pet100 and enhanced green fluorescent protein was expressed in a <i>Saccharomyces cerevisiae</i> mutant deleted for <i>ARE1</i> and <i>ARE2</i> encoding acyl-CoA:sterol acyltransferases. Microscopic observation and subcellular fractionation suggested that this fusion protein, which was named mito-SatA-EGFP, was localized in the mitochondria. Steryl esters were synthesized in the mutant expressing mito-SatA-EGFP. This system will be applicable for evaluations of sterol transport from the ER to the mitochondria in yeast by examining sterol esterification in the mitochondria.</p></div

Crystallization and preliminary X-ray analysis of CTP:phosphoethanolamine cytidylyltransferase (ECT) from Saccharomyces cerevisiae

CTP:phosphoethanolamine cytidylyltransferase from S. cerevisiae has been expressed, purified and crystallized

Suppression of respiratory growth defect of mutant deficient in mitochondrial phospholipase A₁ by overexpression of genes involved in coenzyme Q synthesis in <i>Saccharomyces cerevisiae</i>

<p><i>DDL1</i> encodes a mitochondrial phospholipase A₁ involved in acyl chain remodeling of mitochondrial phospholipids and degradation of cardiolipin in <i>Saccharomyces cerevisiae</i>. The deletion of <i>DDL1</i> leads to respiratory growth defects. To elucidate the physiological role of <i>DDL1</i>, we screened for genes that, when overexpressed, suppress the respiratory growth defect of the <i>DDL1</i> deletion mutant. Introduction of <i>COQ8, COQ9</i>, or <i>COQ5</i>, which are involved in coenzyme Q (CoQ) synthesis, using a multicopy vector suppressed the respiratory growth defect of the <i>DDL1</i> deletion mutant. In contrast, introduction of <i>COQ8</i> using a multicopy vector did not accelerate the growth of the deletion mutants of <i>TAZ1</i> or <i>CLD1</i>, which encode an acyltransferase or phospholipase A₂, respectively, involved in the remodeling of cardiolipin. These results suggest genetic interactions between the mitochondrial phospholipase A₁ gene and the genes involved in CoQ synthesis.</p> <p><b>Abbreviations</b>: CoQ: coenzyme Q; CL: cardiolipin; ORF: open reading frame; PA: phosphatidic acid; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PG: phosphatidylglycerol; PLA1: phospholipase A1; iPLA1: intracellular PLA1; PLA2: phospholipase A2</p> <p>Genetic interaction between <i>DDL1</i> encoding a mitochondrial phospholipase A₁ and genes involved in coenzyme Q synthesis.</p