Abstract
There is strong evidence that Asp-378 of the yeast PMA1 ATPase plays an essential role in ATP hydrolysis by forming a covalent beta-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 degrees 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 wild-type 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.
Highlights
§ Departmento de Bioquimica, Instituto de Quimica, Universidade de Sao Paulo, Avenida Prof
The ␥-phosphoryl group of ATP is transferred to an aspartyl residue embedded in a strongly conserved sequence motif (CSDKTGTLT) near the beginning of the central hydrophilic loop
The membranes could be labeled with antibody against an epitope-tagged D378N construct, consistent with the idea that newly synthesized mutant ATPase became arrested at this early step in the secretory pathway
Summary
§ Departmento de Bioquimica, Instituto de Quimica, Universidade de Sao Paulo, Avenida Prof. In the PMA1 Hϩ-ATPase of the fission yeast Schizosaccharomyces pombe, a phospho-intermediate with the same properties has been detected and carefully documented [12,13]. It has been studied in the closely related Hϩ-ATPase of Neurospora crassa (14 –15) and in the Hϩ-ATPase of Saccharomyces cerevisiae by (16 –17). In galactose medium at 23 °C, the wild-type gene is expressed, and the normal form of the ATPase is delivered to the plasma membrane where it can support growth. In glucose medium at 37 °C, on the other hand, expression shifts to the mutant gene, and newly synthesized mutant ATPase becomes trapped in secretory vesicles. The vesicles are abundant, isolated [24], and lend themselves well to assays of ATP hydrolysis and ATP-driven Hϩ translocation [22]
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