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Isolation of yeast mutants defective in protein targeting to the vacuole

Bankaitis, Vytas A. and Johnson, Lianna M. and Emr, Scott D. (1986) Isolation of yeast mutants defective in protein targeting to the vacuole. Proceedings of the National Academy of Sciences of the United States of America, 83 (23). pp. 9075-9079. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:20150127-091612638

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Abstract

We have constructed a PRC1-SUC2 gene fusion that directs the synthesis in Saccharomyces cerevisiae of a hybrid polypeptide consisting of a 433-residue amino-terminal domain derived from the yeast vacuolar protease carboxypeptidase Y (CPY; EC 3.4.16.1) and a 511-residue carboxyl-terminal domain derived from the secreted yeast enzyme invertase (EC 3.2.1.26). Fractionation data indicated that this amount of CPY primary sequence is sufficient to quantitatively divert invertase to the yeast vacuole. The phenotypic consequence of localizing active invertase to the vacuole has enabled us to select for mutants that "mislocalize" the hybrid protein to the cell surface. The corresponding mutations that lead to this effect are all trans-acting and recessive, and they define at least eight complementation groups. These vacuolar protein targeting (vpt) mutants also exhibit hybrid protein independent defects in wild-type CPY delivery to the yeast vacuole. Precursor forms of CPY accumulate in the mutants and are secreted into the yeast periplasm and extracellular medium. The vpt mutants should provide useful information pertaining to the mechanisms by which yeast cells regulate vacuolar protein traffic.


Item Type:Article
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http://www.pnas.org/content/83/23/9075.shortPublisherArticle
Additional Information:© 1986 National Academy of Sciences. Communicated by Giuseppe Attardi, July 16, 1986. We are grateful to Jane Goldsborough and Miles Chang for expert technical assistance and to Cathy Elkins for typing the manuscript. We also thank Joel Rothman and Tom Stevens for many helpful discussions and the communication of their results prior to publication. The advice of Greg Payne and Randy Schekman on the use of bovine serum albumin to stabilize mislocalized p2 CPY is similarly appreciated. The work was supported by the National Institutes of Health (GM32703), by the Searle Scholars Program, and by a Presidential Young Investigator Award from the National Science Foundation (to S.D.E.). V.A.B. and L.M.J. were supported by Research Fellowships from the Helen Hay Whitney Foundation and the National Institutes of Health (GM09646), respectively.
Funders:
Funding AgencyGrant Number
NIHGM32703
Searle Scholars ProgramUNSPECIFIED
NSFUNSPECIFIED
Helen Hay Whitney FoundationUNSPECIFIED
NIHGM09646
Record Number:CaltechAUTHORS:20150127-091612638
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20150127-091612638
Official Citation:Bankaitis, V. A., Johnson, L. M., & Emr, S. D. (1986). Isolation of yeast mutants defective in protein targeting to the vacuole. Proceedings of the National Academy of Sciences, 83(23), 9075-9079.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:54126
Collection:CaltechAUTHORS
Deposited By: Jason Perez
Deposited On:27 Jan 2015 21:19
Last Modified:27 Jan 2015 21:19

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