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Published September 1, 1990 | public
Journal Article Open

Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis


vps33 mutants missort and secrete multiple vacuolar hydrolases and exhibit extreme defects in vacuolar morphology. Toward a molecular understanding of the role of the VPS33 gene in vacuole biogenesis, we have cloned this gene from a yeast genomic library by complementation of a temperature-sensitive vps33 mutation. Gene disruption demonstrated that VPS33 was not essential but was required for growth at high temperatures. At the permissive temperature, vps33 null mutants exhibited defects in vacuolar protein localization and vacuole morphology similar to those seen in most of the original mutant alleles. Sequence analysis revealed a putative open reading frame sufficient to encode a protein of 691 amino acids. Hydropathy analysis indicated that the deduced product of the VPS33 gene is generally hydrophilic, contains no obvious signal sequence or transmembrane domains, and is therefore unlikely to enter the secretory pathway. Polyclonal antisera raised against TrpE-Vps33 fusion proteins recognized a protein in yeast cells of the expected molecular weight, approximately 75,000. In cell fractionation studies, Vps33p behaved as a cytosolic protein. The predicted VPS33 gene product possessed sequence similarity with a number of ATPases and ATP-binding proteins specifically in their ATP-binding domains. One vps33 temperature-sensitive mutant contained a missense mutation near this region of sequence similarity; the mutation resulted in a Leu-646----Pro substitution in Vps33p. This temperature-sensitive mutant strain contained normal vacuoles at the permissive temperature but lacked vacuoles specifically in the bud at the nonpermissive temperature. Our data suggest that Vps33p acts in the cytoplasm to facilitate Golgi-to-vacuole protein delivery. We propose that as a consequence of the vps33 protein-sorting defects, abnormalities in vacuolar morphology and vacuole assembly result.

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Copyright © 1990 by the American Society for Microbiology. Received 27 March 1990/Accepted 15 June 1990 We thank Raffi Aroian for the YCp5O library DNA, Dan Klionsky for many helpful discussions, and Elliot Altman, Editte Gharakhanian, and Bruce Horazdovsky for critically reading the manuscript. We also thank Yoh Wada and Yasuhiro Anraku for communicating results prior to publication. This work was supported by Public Health Service grant GM-32703 from the National Institutes of Health (to S.D.E.). Partial support was also provided by General Electric Co. (to L.M.B.) and the Natural Sciences and Engineering Research Council of Canada (to P.K.H.) T.A.V. is supported by a postdoctural fellowship from the American Cancer Society.


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