Herman, Paul K. and Emr, Scott D. (1990) Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. Molecular and Cellular Biology, 10 (12). pp. 6742-6754. ISSN 0270-7306 http://resolver.caltech.edu/CaltechAUTHORS:HERmcb90
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VPS34 gene function is required for the efficient localization of a variety of vacuolar proteins. We have cloned and sequenced the wild-type VPS34 gene in order to gain a better understanding of the role of its protein product in this intracellular sorting pathway. Interestingly, disruption of the VPS34 locus resulted in a temperature-sensitive growth defect, indicating that the VPS34 gene is essential for vegetative growth only at elevated growth temperatures. As with the original vps34 alleles, vps34 null mutants exhibited severe vacuolar protein sorting defects and possessed a morphologically normal vacuolar structure. The VPS34 gene DNA sequence identifies an open reading frame that could encode a hydrophilic protein of 875 amino acids. The predicted protein sequence lacks any apparent signal sequence or membrane-spanning domains, suggesting that Vps34p does not enter the secretory pathway. Results from immunoprecipitation experiments with antiserum prepared against a TrpE-Vps34 fusion protein were consistent with this prediction: a rare, unglycosylated protein of approximately 95,000 Da was detected in extracts of wild-type Saccharomyces cerevisiae cells. Cell fractionation studies indicated that a significant portion of the Vps34p is found associated with a particulate fraction of yeast cells. This particulate Vps34p was readily solubilized by treatment with 2 M urea but not with Triton X-100, suggesting that the presence of Vps34p in this pelletable structure is mediated by protein-protein interactions. vp34 mutant cells also exhibited a defect in the normal partitioning of the vacuolar compartment between mother and daughter cells during cell division. In more than 80% of the delta vps34 dividing cells examined, no vacuolar structures were observed in the newly emerging bud, whereas in wild-type dividing cells, more than 95% of the buds had a detectable vacuolar compartment. Our results suggest that the Vps34p may act as a component of a relatively large intracellular structure that functions to facilitate specific steps of the vacuolar protein delivery and inheritance pathways.
|Additional Information:||Copyright © 1990 by the American Society for Microbiology. Received 17 July 1990/Accepted 17 September 1990 We thank Elliot Altman, Todd Graham, and Jeff Stack for many helpful discussions and critical reading of the manuscript. John De Modena for technical assistance, Kurt Eakle for use of his sequence analysis software, Janet Shaw and Bill Wickner for communicating results prior to publication, and Cathy Blagg for expert assistance with preparation of the manuscript. This work was supported by a Presidential Young Investigator Award from the National Science Foundation (to S.D.E.), Public Health Service grant GM-32703 from the National Institutes of Health (to S.D.E.), and a Natural Sciences and Engineering Research Council of Canada postgraduate scholarship (to P.K.H.).|
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