Mutations at the phosphorylation site (Asp-378) of the yeast plasma-membrane H ؉ -ATPase have been shown previously to cause misfolding of the ATPase, preventing normal movement along the secretory pathway; Asp-378 mutations also block the biogenesis of co-expressed wild-type ATPase and lead to a dominant lethal phenotype. To ask whether these defects are specific for Asp-378 or whether the phosphorylation region as a whole is involved, alanine-scanning mutagenesis has been carried out to examine the role of 11 conserved residues flanking Asp-378. In the sec6 -4 expression system (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940 -7949), the mutant ATPases displayed varying abilities to reach the secretory vesicles that deliver plasma-membrane proteins to the cell surface. Indirect immunofluorescence of intact 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). Although the extent of ER retention varied among the mutants, the endoplasmic reticulum appeared to be the only secretory compartment in which the mutant ATPases accumulated. All of the mutant proteins that localized either partially or fully to the ER were also malfolded based on their abnormal sensitivity to trypsin. Among them, the severely affected mutants had a dominant lethal phenotype, and even the intermediate mutants 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 by double label immunofluorescence. Taken together, the results indicate that the residues surrounding Asp-378 are critically important for ATPase maturation and transport to the cell surface.

Soluble calmodulin‐binding proteins from Saccharomyces carlsbergensis were analyzed in cells grown on glucose, maltose and galactose as carbon source. A large number of polypeptide chains showed affinity for calmodulin by affinity chromatography and overlay techniques. Amongst these, polypeptides of 115, 67 and 45 kDa were only detected during the second exponential phase of growth on glucose or non‐fermentative carbon sources, suggesting that they might be subjected to catabolite repression. Polypeptides of 195 and 22 kDa were only observed in cells grown on maltose, whereas 88 kDa polypeptide was only observed in galactose‐grown cells. Among the calmodulin ‐binding polypeptides, eight were phosphorylated in a Ca2+ /calmodulin ‐dependent manner (220, 200, 175, 100, 62, 55, 31 and 16 kDa). Ca2+/calmodulin dependent [γ‐32P] incorporation was dramatically decreased in yeast cells submitted to a heat treatment.

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