Quality control in the yeast secretory pathway: a misfolded PMA1 H+-ATPase reveals two checkpoints.
The yeast plasma-membrane H(+)-ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway and has recently emerged as an excellent system for identifying quality control mechanisms along the pathway. In the present study, we have tracked the biogenesis of Pma1-G381A, a misfolded mutant form of the H(+)-ATPase. Although this mutant ATPase is arrested transiently in the peripheral endoplasmic reticulum, it does not become a substrate for endoplasmic reticulum-associated degradation nor does it appear to stimulate an unfolded protein response. Instead, Pma1-G381A accumulates in Kar2p-containing vesicular-tubular clusters that resemble those previously described in mammalian cells. Like their mammalian counterparts, the yeast vesicular-tubular clusters may correspond to specific exit ports from the endoplasmic reticulum, since Pma1-G381A eventually escapes from them (still in a misfolded, trypsin-sensitive form) to reach the plasma membrane. By comparison with wild-type ATPase, Pma1-G381A spends a short half-life at the plasma membrane before being removed and sent to the vacuole for degradation in a process that requires both End4p and Pep4p. Finally, in a separate set of experiments, Pma1-G381A was found to impose its phenotype on co-expressed wild-type ATPase, transiently retarding the wild-type protein in the ER and later stimulating its degradation in the vacuole. Both effects serve to lower the steady-state amount of wild-type ATPase in the plasma membrane and, thus, can explain the co-dominant genetic behavior of the G381A mutation. Taken together, the results of this study establish Pma1-G381A as a useful new probe for the yeast secretory system.
Ferreira
Thierry
T
Department of Genetics and the Center for Cell and Molecular Imaging, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Mason
A Brett
AB
Pypaert
Marc
M
Allen
Kenneth E
KE
Slayman
Carolyn W
CW
D002462:Cell Membrane / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D016253:Microscopy, Immunoelectron; D011233:Precipitin Tests; D017510:Protein Folding; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D011786:Quality Control; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology
D029701:Saccharomyces cerevisiae Proteins; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases