title: Phosphatidic acid and phosphoinositides facilitate liposome association of Yas3p and potentiate derepression of ARE1 (alkane-responsive element one)-mediated transcription control.
abstract: In the n-alkane assimilating yeast Yarrowia lipolytica, the expression of ALK1, encoding a cytochrome P450 that catalyzes terminal mono-oxygenation of n-alkanes, is induced by n-alkanes. The transcription of ALK1 is regulated by a heterocomplex that comprises the basic helix-loop-helix transcription activators, Yas1p and Yas2p, and binds to alkane-responsive element 1 (ARE1) in the ALK1 promoter. An Opi1 family transcription repressor, Yas3p, represses transcription by binding to Yas2p. Yas3p localizes in the nucleus when Y. lipolytica is grown on glucose but localizes to the endoplasmic reticulum (ER) upon the addition of n-alkanes. In this study, we showed that recombinant Yas3p binds to the acidic phospholipids, phosphatidic acid (PA) and phosphoinositides (PIPs), in vitro. The ARE1-mediated transcription was enhanced in vivo in mutants defective in an ortholog of the Saccharomyces cerevisiae gene PAH1, encoding PA phosphatase, and in an ortholog of SAC1, encoding PIP phosphatase in the ER. Truncation mutation analyses for Yas3p revealed two regions that bound to PA and PIPs. These results suggest that the interaction with acidic phospholipids is important for the n-alkane-induced association of Yas3p with the ER membrane.
authors.affiliation: Department of Biotechnology, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Hirakawa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Horiuchi
authors.forename: Ryouichi
authors.initials: R
authors.lastname: Fukuda
authors.forename: Akinori
authors.initials: A
authors.lastname: Ohta
mesh_terms: D000473:Alkanes / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D010712:Phosphatidic Acids / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D014158:Transcription, Genetic; D025062:Yarrowia / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000473:Alkanes; D004271:DNA, Fungal; D010712:Phosphatidic Acids; D010716:Phosphatidylinositols; D012097:Repressor Proteins
keywords: 1-naphthaleneacetic acid; 2D-TLC; AID; ARE1; DAG; DAGPP; DTT; EGFP; ER; FFAT; GST; IPTG; NAA; NLS; ORF; P450; PA; PC; PCR; PE; PI; PI(3,4,5)P(3); PI(4)P; PI(4,5)P(2); PIP; PLO assay; PS; Phospholipid; RLM-RACE; RNA ligase-mediated rapid amplification of cDNA ends; SDS-PAGE; Transcriptional repressor; Yarrowia lipolytica; alkane-responsive element 1; auxin-inducible degron; bHLH; basic helix-loop-helix; cytochrome P450; diacylglycerol; diacylglycerol pyrophosphate; dithiothreitol; endoplasmic reticulum; enhanced green fluorescent protein; glutathione S-transferase; isopropyl β-D-1-thiogalactopyranoside; n-Alkane; nuclear localization signal; open reading frame; phosphatidic acid; phosphatidylcholine; phosphatidylethanolamine; phosphatidylinositol; phosphatidylinositol 3,4,5-trisphosphate; phosphatidylinositol 4,5-bisphosphate; phosphatidylinositol 4-phosphate; phosphatidylserine; phosphoinositide; polymerase chain reaction; protein lipid overlay assay; sodium dodecyl sulfate-poly acrylamide gel electrophoresis; two phenylalanines in an acidic tract; two-dimensional thin layer chromatography