PHOSPHORYLATION REGION OF THE YEAST PLASMA-MEMBRANE H-ATPASE - ROLE IN PROTEIN-FOLDING AND BIOGENESIS()

Mutations at the phosphorylation site (Asp-378) of the yeast plasma-me mbrane H+-ATPase have been shown previously to cause misfolding of the ATPase, preventing normal movement along the secretory pathway; Asp-3 78 mutations also block the biogenesis of co-expressed wild-type ATPas e and lead to a dominant lethal phenotype. To ask whether these defect s are specific for Asp-378 or whether the phosphorylation region as a whole is involved, alanine-scanning mutagenesis has been carried out t o examine the role of 11 conserved residues flanking Asp-378, In the s ec6-4 expression system (Nakamoto, R.K., Rao, R., and Slayman, C. W. ( 1991) J. Biol. Chem. 266, 7940-7949), the mutant ATPases displayed var ying abilities to reach the secretory vesicles that deliver plasma-mem brane proteins to the cell surface. Indirect immunofluorescence of int act cells also gave evidence for a spectrum of behavior, ranging from mutant ATPases completely arrested (D378A, K379A, T380A, and T384A) or partially arrested in the endoplasmic reticulum to those that reached the plasma membrane in normal amounts (C376A, S377A, and G381A). Alth ough the extent of ER retention varied among the mutants, the endoplas mic reticulum appeared to be the only secretory compartment in which t he mutant ATPases accumulated. All of the mutant proteins that localiz ed either partially or fully to the ER were also malfolded based ore t heir abnormal sensitivity to trypsin. Among them, the severely affecte d mutants had a dominant lethal phenotype, and even the intermediate m utants caused a visible slowing of growth when co-expressed with wild- type ATPase. The effects on growth could be traced to the trapping of the wild-type enzyme with the mutant enzyme in the ER, as visualized b y double label immunofluorescence, Taken together, the results indicat e that the residues surrounding Asp-378 are critically important for A TPase maturation and transport to the cell surface.