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.
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