In vitro reconstitution of cytoplasm to vacuole protein targeting in yeast.
Although the majority of known vacuolar proteins transit through the secretory pathway, two vacuole-resident proteins have been identified that reach this organelle by an alternate pathway. These polypeptides are targeted to the vacuole directly from the cytoplasm by a novel import mechanism. The best characterized protein that uses this pathway is aminopeptidase I (API). API is synthesized as a cytoplasmic precursor containing an amino-terminal propeptide that is cleaved off when the protein reaches the vacuole. To dissect the biochemistry of this pathway, we have reconstituted the targeting of API in vitro in a permeabilized cell system. Based on several criteria, the in vitro import assay faithfully reconstitutes the in vivo reaction. After incubation under import conditions, API is processed by a vacuolar-resident protease, copurifies with a vacuole-enriched fraction, and becomes inaccessible to the cytoplasm. These observations demonstrate that API has passed from the cytoplasm to the vacuole. The reconstituted import process is dependent on time, temperature, and energy. ATP gamma S inhibits this reaction, indicating that API transport is ATP driven. API import is also inhibited by GTP gamma S, suggesting that this process may be mediated by a GTP-binding protein. In addition, in vitro import requires a functional vacuolar ATPase; import is inhibited both in the presence of the specific V-ATPase inhibitor bafilomycin A1, and in a yeast strain in which one of the genes encoding a V-ATPase subunit has been disrupted.
Scott
S V
SV
Section of Microbiology, University of California, Davis 95616, USA.
Klionsky
D J
DJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000502:physiology; D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D002451:Cell Compartmentation / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000502:physiology; D006868:Hydrolysis; D009154:Mutation / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D013997:Time Factors; D014617:Vacuoles / Q000378:metabolism*
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D000626:Aminopeptidases; C019689:APE1 protein, S cerevisiae; D000251:Adenosine Triphosphatases; D019204:GTP-Binding Proteins