Most lipids are formed by membrane bound enzyme systems. The membrane association with these proteins and their hydrophobic substrates present unique problems with respect to the regulation of their activity and the control of their gene expression. This proposal is to determine the mechanisms of regulation of the delta-9 fatty acid desaturase, an intrinsic membrane enzyme that catalyzes the conversion of saturated fatty acids to unsaturated species on the ER surface. In animals this enzyme is highly expressed in liver and in adipose cells it plays a role in fat deposition and adipocyte differentiation. This laboratory has shown that the S. cerevisiae OLE1 gene that encodes this enzyme is regulated at the levels of transcription and mRNA stability in response to a diverse array of nutrient, physiological and environmental stimuli. Our previous studies identified promoter elements that control transcription in response to nutrient saturated and unsaturated fatty acids and a separate regulation system that controls mRNA stability in response to unsaturated fatty acids. Both systems control OLE1 expression over a wide range and each responds to different regulatory cues. This proposal will focus on the mechanisms of function of these two systems. In the transcription regulation system we will identify critical promoter elements responsible for unsaturated fatty acid mediated transcription repression. We will use those elements to identify trans acting components of the signal transduction system by biochemical and genetic methods. In the mRNA stability regulation system we will focus on role of the membrane on its functions. This system acts during translational insertion of Ole1p at the ER surface and is most probably associated with regulating the fatty acyl composition and physical state of internal cell membranes. Our previous studies identified elements in the 5' untranslated region of the transcript that are essential for fatty acid regulated mRNA turnover. New results indicate that the intrinsic stability of the mRNA depends on elements of the translated protein that affect its structure and topology with respect to the ER surface. We will determine mechanisms responsible for controlling intrinsic and fatty acid mediated turnover by 1) identifying elements in the transcript required for its default and fatty acid regulated half-lives, 2) identifying elements in the nascent polypeptide that affect translation dependent mRNA stability, 3) determining the effects of membrane association, dimerization and protein folding of Ole1p on the default stability of the mRNA, and 4) identifying trans-acting molecules that control OLE1 mRNA stability under default and regulated conditions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM045768-07
Application #
6179346
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Chin, Jean
Project Start
1992-09-30
Project End
2003-06-30
Budget Start
2000-07-01
Budget End
2001-06-30
Support Year
7
Fiscal Year
2000
Total Cost
$285,529
Indirect Cost
Name
Rutgers University
Department
Other Basic Sciences
Type
Schools of Arts and Sciences
DUNS #
038633251
City
New Brunswick
State
NJ
Country
United States
Zip Code
08901
Martin, Charles E; Oh, Chan-Seok; Jiang, Yide (2007) Regulation of long chain unsaturated fatty acid synthesis in yeast. Biochim Biophys Acta 1771:271-85
Oh, Chan-Seok; Martin, Charles E (2006) Candida albicans Spt23p controls the expression of the Ole1p Delta9 fatty acid desaturase and regulates unsaturated fatty acid biosynthesis. J Biol Chem 281:7030-9
Kandasamy, Pitchaimani; Vemula, Muralikrishna; Oh, Chan-Seok et al. (2004) Regulation of unsaturated fatty acid biosynthesis in Saccharomyces: the endoplasmic reticulum membrane protein, Mga2p, a transcription activator of the OLE1 gene, regulates the stability of the OLE1 mRNA through exosome-mediated mechanisms. J Biol Chem 279:36586-92
Vemula, Muralikrishna; Kandasamy, Pitchaimani; Oh, Chan-Seok et al. (2003) Maintenance and regulation of mRNA stability of the Saccharomyces cerevisiae OLE1 gene requires multiple elements within the transcript that act through translation-independent mechanisms. J Biol Chem 278:45269-79
Jiang, Yide; Vasconcelles, Michael J; Wretzel, Sharon et al. (2002) Mga2p processing by hypoxia and unsaturated fatty acids in Saccharomyces cerevisiae: impact on LORE-dependent gene expression. Eukaryot Cell 1:481-90
Martin, C E; Oh, C-S; Kandasamy, P et al. (2002) Yeast desaturases. Biochem Soc Trans 30:1080-2
Jiang, Y; Vasconcelles, M J; Wretzel, S et al. (2001) MGA2 is involved in the low-oxygen response element-dependent hypoxic induction of genes in Saccharomyces cerevisiae. Mol Cell Biol 21:6161-9
Kohlwein, S D; Eder, S; Oh, C S et al. (2001) Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae. Mol Cell Biol 21:109-25
Vasconcelles, M J; Jiang, Y; McDaid, K et al. (2001) Identification and characterization of a low oxygen response element involved in the hypoxic induction of a family of Saccharomyces cerevisiae genes. Implications for the conservation of oxygen sensing in eukaryotes. J Biol Chem 276:14374-84
Chellappa, R; Kandasamy, P; Oh, C S et al. (2001) The membrane proteins, Spt23p and Mga2p, play distinct roles in the activation of Saccharomyces cerevisiae OLE1 gene expression. Fatty acid-mediated regulation of Mga2p activity is independent of its proteolytic processing into a soluble transcription act J Biol Chem 276:43548-56

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