Genetic Study of Ion Transport in Yeast

Slayman, Carolyn

Abstract

This project focuses on the plasma-membrane H+-ATPase of yeast, a P-type cation pump that serves as a useful model for the Na+,K+-, H+,K+-, and Ca2+-ATPases of animal cells. Drawing upon the powerful array of genetic and cell biological methods that are available in yeast, we will pursue four groups of experiments: (1) A novel secretory vesicle expression system has made it possible to identify a set of mutationally sensitive amino acid residues in membrane-spanning segments 4, 5, and 6, which appear to lie at the heart of the transport mechanism. This work will continue, and fluorescent SH reagents will be used in conjunction with newly engineered Cys residues to track ligand-dependent conformational changes involved in the coupling between ATP hydrolysis and proton transport. (2) Mutants have been isolated with apparent changes in H+/ATP stoichiometry; they will be studied further, and collaborations will be launched with two other laboratories to develop patch-clamp techniques that can determine the quantitative characteristics of the transport cycle. (3) Other mutants have been identified that behave in a dominant lethal fashion, acting to trap co- expressed wild-type ATPase in the endoplasmic reticulum. We have constructed two different epitope-tagged versions of the ATPase and will use them to look for (and then to study) dimers or larger oligomers that may form during biogenesis. (4) Finally, previous work has shown that multiple Ser/Thr residues become phosphorylated during biogenesis; at least one of the phosphorylation events correlates well with an up- regulation of ATPase activity in the presence of glucose. These residues will be mapped and their functional role will be explored by mutagenesis. Taken together, the results of these studies should help to shed light on the mechanism and regulation of cation transport in eukaryotic cells.