Folding and intracellular transport of the yeast plasma-membrane H(+)-ATPase: effects of mutations in KAR2 and SEC65.
We have developed two independent assays to study the integration, folding, and intracellular transport of the polytopic plasma membrane H(+)-ATPase in yeast. To follow folding, controlled trypsinolysis was used to distinguish between the E1 conformation of the ATPase (favored in the presence of ADP) and the E2 conformation (favored in the presence of vanadate). By this criterion, wild-type ATPase appears to recognize its ligands and assume distinct conformations within a short time after its biosynthesis. To follow intracellular transport, we have exploited the fact that export of newly synthesized ATPase from the endoplasmic reticulum is accompanied by kinase-mediated phosphorylation, leading to a shift in electrophoretic mobility. Because proper folding is required for transport from the endoplasmic reticulum, the mobility shift also serves as a convenient bioassay for correct folding. As a first step toward identifying cell components important in folding of the nascent ATPase, we have used the dual assays to examine the role of KAR2, encoding the yeast homolog of immunoglobulin heavy chain binding protein/78-kDa glucose-regulated protein, and SEC65, encoding a subunit of the yeast signal recognition particle. Although mutation of KAR2 caused defective translocation of several secretory precursors into the endoplasmic reticulum lumen, ATPase folding and intracellular transport were unperturbed. By contrast, in a sec65 mutant, the folding and intracellular transport of newly synthesized ATPase were delayed. Our data suggest that conformational maturation of the ATPase is a rapid process in wild-type cells and that membrane integration mediated by signal recognition peptide is important for the proper folding of this polytopic protein.
Chang
A
A
Department of Genetics, Yale University School of Medicine, New Haven, CT 06510.
Rose
M D
MD
Slayman
C W
CW
D002462:Cell Membrane / Q000201:enzymology; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D018271:Signal Recognition Particle; D014357:Trypsin
D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C060153:KAR2 protein, yeast; C489551:SEC65 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D018271:Signal Recognition Particle; D014357:Trypsin; D006180:Proton-Translocating ATPases