Cellular & Molecular Physiology: Membrane Protein Sorting and Trafficking | Membrane Proteins - Pumps and Transporters
Research in Carolyn Slayman’s laboratory uses the plasma-membrane H+-ATPase of yeast (Saccharomyces cerevisiae) as a simple model for studies on P-type cation pumps, a physiologically important family that includes the Na+,K+-, H+,K+-, and Ca2+-ATPases of animal cells. These pumps control the ionic composition of cells; many of them also serve as important drug targets (e.g., for cardiac glycosides in the case of Na+,K+-ATPase and anti-ulcer drugs in the case of gastric H+,K+-ATPase).
Extensive Research Description
Over the past several years, the Slayman laboratory has constructed a large collection of site-directed mutants and used them to explore structure-function relationships in key regions of the 100 kDa H+-ATPase. Results of particular significance include: (1) discovery of a 13-amino acid stretch in stalk segment 4 within which mutations disrupt the interaction between the catalytic domain of the ATPase (located in the cytoplasm) and the proton channel (embedded in the membrane); and (2) clear implication of one face of stalk segment 5 in the metabolic activation of the ATPase by glucose. Cysteine residues have now been introduced into both regions, and methods are being developed to track conformational changes by means of fluorescent sulfhydryl reagents. With a recently published high resolution structure of sarcoplasmic reticulum Ca2+-ATPase as a template, biophysical data from the H+-ATPase mutants should yield helpful insights into the molecular mechanism by which ATP hydrolysis is coupled to cation transport.
In parallel, the H+-ATPase is being used as a tool to probe the way in which newly synthesized plasma membrane proteins are delivered from the endoplasmic reticulum to the cell surface, as well as the quality control mechanisms that remove poorly folded or otherwise defective proteins. During the past year, a nested series of truncations has been constructed and has provided intriguing evidence for an oligomerization step at the C-terminus of the ATPase; oligomerization appears to occur early in the secretory pathway and to be essential for normal biogenesis.
- Lecchi, S., Nelson, C.J., Allen, K.E., Swaney, D.L., Thompson, K.L., Coon, J.J., Sussman, M.R., and Slayman, C.W. (2007) Tandem phosphorylation of Ser-911 and Thr-912 at the C-terminus of yeast plasma membrane H+-ATPase leads to glucose-dependent activati
- Guerra, G., Petrov, V.V., Allen, K.E., Miranda, M., Pardo, J.P., and Slayman, C.W. (2007) Role of transmembrane segment M8 in the biogenesis and function of yeast plasma-membrane H+-ATPase. Biochim.Biophys.Acta 1768:2383-2392. (PMID: 17573037)
- Mason, A. B., Allen, K. E., and Slayman, C. W. (2006) Effects of C-terminal truncations on trafficking of the yeast plasma membrane H+-ATPase. Biological Chemistry 281:23887-23898
- Lecchi, S., Allen, K. E., Pardo, J. P., Mason, A. B., and Slayman, C. W. (2005) Conformational changes of yeast plasma membrane H+-ATPase during activation by glucose: role of Thr912 in the carboxy-terminal tail. Biochemistry 44:16624-16632