TY - JOUR
T1 - Substitutions of aspartate 378 in the phosphorylation domain of the yeast PMA1 H+-ATPase disrupt protein folding and biogenesis
AU - Nakamoto, Robert K.
AU - Verjovski-Almeida, Sergio
AU - Allen, Kenneth E.
AU - Ambesi, Anthony
AU - Rao, Rajini
AU - Slayman, Carolyn W.
PY - 1998/3/27
Y1 - 1998/3/27
N2 - There is strong evidence that Asp-378 of the yeast PMA1 ATPase plays an essential role in ATP hydrolysis by forming a covalent β-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.
AB - There is strong evidence that Asp-378 of the yeast PMA1 ATPase plays an essential role in ATP hydrolysis by forming a covalent β-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.
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U2 - 10.1074/jbc.273.13.7338
DO - 10.1074/jbc.273.13.7338
M3 - Article
C2 - 9516429
AN - SCOPUS:0032571325
SN - 0021-9258
VL - 273
SP - 7338
EP - 7344
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 13
ER -