Substitutions of Aspartate 378 in the Phosphorylation Domain of the Yeast PMA1 H+-ATPase Disrupt Protein Folding and Biogenesis

Abstract

There is strong evidence that Asp-378 of the yeast PMA1 ATPase plays an essential role in ATP hydrolysis by forming a covalent ␤-aspartyl phosphate reaction intermediate. In this study, Asp-378 was replaced by Asn, Ser, and Glu, and the mutant ATPases were expressed in a temperature-sensitive secretion-deficient strain (sec6 -4) that allowed their properties to be examined. Although all three mutant proteins were produced at nearly normal levels and remained stable for at least 2 h at 37°C, they failed to travel to the vesicles that serve as immediate precursors of the plasma membrane; instead, they became arrested at an earlier step of the secretory pathway. A closer look at the mutant proteins revealed that they were firmly inserted into the bilayer and were not released by washing with high salt, urea, or sodium carbonate (pH 11), treatments commonly used to strip nonintegral proteins from membranes. However, all three mutant ATPases were extremely sensitive to digestion by trypsin, pointing to a marked abnormality in protein folding. Furthermore, in contrast to the wildtype enzyme, the mutant ATPases could not be protected against trypsinolysis by ligands such as MgATP, MgADP, or inorganic orthovanadate. Thus, Asp-378 functions in an unexpectedly complex way during the acquisition of a mature structure by the yeast PMA1 ATPase.

Structure-Function Relationships in Membrane Segment 5 of the Yeast Pma1 H+-ATPase

“…By contrast, on galactose-containing medium, where the mutant gene was also expressed, no growth was observed for cells transformed with H701A, R695A, or L706A. This dominant lethal phenotype indicates that the abnormal proteins interfere with the processing of coexpressed wild-type ATPase, as has previously been shown for H701D, H701Q, H701R, and a number of other PMA1 mutations (26,29,(31)(32)(33).…”

“…Metabolic Labeling and Immunoprecipitation-To measure the synthesis of mutant ATPases that were unable to reach the secretory vesicles, SY4 cells were shifted from galactose medium at 23°C to glucose medium at 39°C as described above and then metabolically labeled with [ 35 S]methionine (26). Total membranes were isolated and immunoprecipitated with anti-Pma1 antibody (26), and after SDS-polyacrylamide gel electrophoresis, the gels were fixed, incubated in 1 M sodium salicylate (30 min at 23°C), dried, and exposed to Hyperfilm-MP (Amersham Pharmacia Biotech).…”

“…Total membranes were isolated and immunoprecipitated with anti-Pma1 antibody (26), and after SDS-polyacrylamide gel electrophoresis, the gels were fixed, incubated in 1 M sodium salicylate (30 min at 23°C), dried, and exposed to Hyperfilm-MP (Amersham Pharmacia Biotech).…”

Epitope‐Tagged Constructs of the Yeast Plasma‐Membrane H+‐ATPase

“…ATP-dependent proton transport was determined by measuring the initial rate of acridine orange uorescence quenching (19). Protein concentrations were determined by the method of Lowry et al (20) as modi ed by Ambesi et al (15), with bovine serum albumin as a standard.…”

“…Secretory vesicles were isolated and assayed for PMA1 expression, ATP hydrolysis, and ATP-dependent H + pumping as previously described (10,15). For determination of K m values, the concentration of Na 2 ATP was varied between 0.15 and 7.5 mM, with MgCl 2 always in excess of ATP by 5 mM.…”