We study aspects of the two fundamental cell biological problems of cytosolic redoxcontrol and protein trafficking in the yeast S. cerevisiae, employing combined genetic, biochemical and cell biological methods.

Redox-sensing and signaling

During anaerobiosis, glycerol production in S. cerevisiae serves as a sink for reducing equivalents. The excess NADH generated in anabolism is thereby reoxidised to NAD⁺ through reduction of dihydroxyacetone phosphate to glycerol 3-phosphate (G3P) via the G3P dehydrogenase encoded by the two isogenes GPD1 and GPD2. A gpd1D gpd2D double mutant has a blocked glycerol production and does not grow under anaerobic conditions, unless supplied with an artificial acceptor of reducing equivalents. Only one of the GPD genes, GPD2, is induced following a shift to anaerobic conditions. This induction appears to be controlled by a novel, oxygen independent signaling pathway. To learn more about how cells sense, signal and regulate the redox-state of the cytosol, we are using both classical and transposon mediated mutagenesis to screen for mutants having defective GPD2 expression. Currently we are characterizing a subset of these mutants. We are also examining the role of a redox-controlled gene encoding a protein that interacts with enzyme(s) of the glycerol metabolism.

A yeast tumor suppressor homologue involved in protein secretion

By complementation of a salt sensitive yeast mutant, we isolated the SOP1(SRO7) gene. The gene product shows homology to the lgl - hugl family of tumor suppressors, whose inactivation produces tissue specific cancers in Drosophila (lgl) and seems associated with a rare form of brain tumor in man (hugl). Loss of SOP1 in yeast leads to a severe and specific sensitivity to Na⁺ that can be partly reversed by expression of the authentic tumor suppressor gene in yeast, pointing to a functional conservation of the yeast protein and the tumor suppressor. We and others have observed that Sop1p is associated with the plasma membrane and that SOP1 genetically interacts with components of the exocyst. The exocyst complex marks the site of docking between post-Golgi cargo vesicles and the plasma membrane, suggesting that Sop1p has a role in polarized secretion by acting in the late stages of the secretory pathway. In animal cells, some of the most crucial functions of the secretory pathway involve regulated delivery of specific proteins to the cell surface. Recent demonstration that (lgl) inactivation leads to mis-localization of particular membrane proteins in Drosophila epithelium has lead to the proposal that the defective protein-targeting is the primary reason for loss of cell polarity and subsequent tumor development. We are studying the analogous secretory process in yeast having observed that the salt sensitivity of sop1 mutants is primarily due to mis-localization of the ENA1 encoded sodium transporter. To develop an understanding of the precise role of Sop1p in these processes we are taking a multifaceted approach involving search for multicopy suppressors of sop mutants and GFP-tagging of Sop1/2p, Ena1p and other membrane associated proteins to directly visualize their fate in wild type and mutant cells. We are also using affinity purification to isolate tagged Sop1p protein complexes for mass spectrometric identification of constituent proteins. The results of these studies are expected to throw light on Sop function and suggest what elicits tumor formation in cells that lack these genes.