Endosomal transport function in yeast requires a novel AAA-type ATPase, Vps4p.
In a late-Golgi compartment of the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y (CPY) are actively sorted away from the secretory pathway and transported to the vacuole via a pre-vacuolar, endosome-like intermediate. The vacuolar protein sorting (vps) mutant vps4 accumulates vacuolar, endocytic and late-Golgi markers in an aberrant multilamellar pre-vacuolar compartment. The VPS4 gene has been cloned and found to encode a 48 kDa protein which belongs to the protein family of AAA-type ATPases. The Vps4 protein was purified and shown to exhibit an N-ethylmaleimide-sensitive ATPase activity. A single amino acid change within the AAA motif of Vps4p yielded a protein that lacked ATPase activity and did not complement the protein sorting or morphological defects of the vps4 delta1 mutant. Indeed, when expressed at normal levels in wild-type cells, the mutant vps4 gene acted as a dominant-negative allele. The phenotypic characterization of a temperature-sensitive vps4 allele showed that the immediate consequence of loss of Vps4p function is a defect in vacuolar protein delivery. In this mutant, precursor CPY was not secreted but instead accumulated in an intracellular compartment, presumably the pre-vacuolar endosome. Electron microscopy revealed that upon temperature shift, exaggerated stacks of curved cisternal membranes (aberrant endosome) also accumulated in the vps4ts mutant. Based on these and other observations, we propose that Vps4p function is required for efficient transport out of the pre-vacuolar endosome.
Babst
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Division of Cellular and Molecular Medicine and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla 92093-0668, USA [comment]
Sato
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TK
Banta
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LM
Emr
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SD
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001692:Biological Transport; D002268:Carboxypeptidases / Q000378:metabolism; D043402:Cathepsin A; D003001:Cloning, Molecular; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D005800:Genes, Fungal / Q000235:genetics*; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014617:Vacuoles / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D002268:Carboxypeptidases; D043402:Cathepsin A; D000251:Adenosine Triphosphatases