| In yeast as in mammals, a large number of genes involved in sterol biosynthesis and other processes are feedback regulated by sterol levels. In mammalian cells, this regulation is mediated by the Sterol Regulatory Element Binding Proteins (SREBPs). The SREBPs activate the expression of a number of genes involved in sterol uptake and sterol and fatty acid biosynthesis in response to a need for more sterols.; This dissertation describes the identification of two sterol regulatory element binding proteins, Ecm22p and Up2p, that mediated sterol-regulation in Saccharomyces cerevisiae. These proteins were necessary for the regulation of the sterol biosynthetic genes ERG2 and ERG3. The expression of ERG2 and ERG3 increased in response to reduced sterol levels, and this change was dependent on the presence of either Ecm22p or Upc2p. Ecm22p and Upc2p were homologous proteins and members of the Zn[2]-Cys[6] binuclear cluster family of fungal transcription factors. They therefore shared no homology with their mammalian counterparts, the SREBPs.; Both Ecm22p and Upc2p were transcriptional activators that bound directly to a sterol regulatory element in the promoter of their target genes. I identified all critical positions in this regulatory element for binding in vitro and regulation in vivo. This sterol regulatory element could be found in the promoter of a large number of genes that are regulated by sterol levels, many of which are involved in the biosynthesis of sterols. This suggested that numerous genes may be coordinately regulated by Ecm22p and Upc2p in response to changes in sterol levels.; Both Ecm22p and Upc2p were membrane-associated proteins, presumably associated with the nuclear envelope/endoplasmic reticulum (ER) membrane. Ecm22p was a peripheral membrane protein, suggesting that the mechanisms of sterol-regulation in yeast and mammals were different. Membrane dissociation of the transcription factor was likely the critical step in sterol regulation, but in yeast this step may not require proteolysis. Surprisingly, the membrane association of Ecm22p was not mediated by the carboxyl-terminal domain which has been suggested to have a role in regulation. Rather, my data indicated that this domain was important for activation. |
