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Organelle Assembly in Yeast: Characterization of Yeast Mutants Defective in Vacuolar Biogenesis and Protein Sorting

Banta, Lois M. and Robinson, Jane S. and Klionsky, Daniel J. and Emr, Scott D. (1988) Organelle Assembly in Yeast: Characterization of Yeast Mutants Defective in Vacuolar Biogenesis and Protein Sorting. Journal of Cell Biology, 107 (4). pp. 1369-1383. ISSN 0021-9525. http://resolver.caltech.edu/CaltechAUTHORS:20120531-141222457

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Abstract

Yeast vacuole protein targeting (vpt) mutants exhibit defects in the sorting and processing of multiple vacuolar hydrolases. To evaluate the impact these vpt mutations have on the biogenesis and functioning of the lysosome-like vacuole, we have used light and electron microscopic techniques to analyze the vacuolar morphology in the mutants. These observations have permitted us to assign the vpt mutants to three distinct classes. The class A vpt mutants (26 complementation groups) contain 1-3 large vacuoles that are morphologically indistinguishable from those in the parental strain, suggesting that only a subset of the proteins destined for delivery to this compartment is mislocalized. One class A mutant (vpt13) is very sensitive to low pH and exhibits a defect in vacuole acidification. Consistent with a potential role for vacuolar pH in protein sorting, we found that bafilomycin A1, a specific inhibitor of the vacuolar ATPase, as well as the weak base ammonium acetate and the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, collapse the pH gradient across the vacuolar membrane and cause the missorting and secretion of two vacuolar hydrolases in wild-type cells. Mutants in the three class B vpt complementation groups exhibit a fragmented vacuole morphology. In these mutants, no large normal vacuoles are observed. Instead, many (20-40) smaller vacuole-like organelles accumulate. The class C vpt mutants, which constitute four complementation groups, exhibit extreme defects in vacuole biogenesis. The mutants lack any organelle resembling a normal vacuole but accumulate other organelles including vesicles, multilamellar membrane structures, and Golgi-related structures. Heterozygous class C zygotes reassemble normal vacuoles rapidly, indicating that some of the accumulated aberrant structures may be intermediates in vacuole formation. These class C mutants also exhibit sensitivity to osmotic stress, suggesting an osmoregulatory role for the vacuole. The vpt mutants should provide insights into the normal physiological role of the vacuole, as well as allowing identification of components required for vacuole protein sorting and/or vacuole assembly.


Item Type:Article
Related URLs:
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http://dx.doi.org/10.1083/jcb.107.4.1369DOIUNSPECIFIED
http://jcb.rupress.org/content/107/4/1369PublisherUNSPECIFIED
Additional Information:© 1988 Rockefeller University Press. Received for publication 26 May 1988, and in revised form 24 July 1988. We would like to thank Jean Edens for invaluable assistance with the electron microscopy, Michael W. Clark for the membrane-enhancement technique protocol and advice on the fluorescence microscopy, Paul Herman for helpful discussions and assistance with the zygote experiments, and John De Modena for technical assistance. We also thank K. Altendorf for the gift of bafilomycin A1; Cathy Elkins for typing the manuscript; and Joel Rothman, Tom Stevens, Barry Bowman, and Howard Riezrnan for communicating their results before publication. This study was supported by Public Health Service grant GM-32703 from the National Institutes of Health to S. D. Emr. L. M. Banta was supported by graduate fellowships from the National Science Foundation (NSF) and General Electric. J. S. Robinson was supported by graduate fellowships from the Evelyn Sharp Foundation, the Lucy Mason Clark fund, and the Markey Charitable Trust Fund. D. J. Klionsky was supported by a research fellowship from the Helen Hay Whitney Foundation. S. D. Emr is an NSF Presidential Young Investigator supported by NSF grant DCB-8451633.
Funders:
Funding AgencyGrant Number
NIHGM-32703
NSFUNSPECIFIED
General ElectricUNSPECIFIED
Evelyn Sharp Graduate FellowshipUNSPECIFIED
Lucy Mason Clark fundUNSPECIFIED
Markey Charitable Trust FundUNSPECIFIED
Helen Hay Whitney FoundationUNSPECIFIED
NSF Presidential Young Investigator AwardDCB-8451633
Record Number:CaltechAUTHORS:20120531-141222457
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20120531-141222457
Official Citation:Organelle assembly in yeast: characterization of yeast mutants defective in vacuolar biogenesis and protein sorting. L M Banta, J S Robinson, D J Klionsky, and S D Emr 107:1369-1383. doi:10.1083/jcb.107.4.1369
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:31754
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:31 May 2012 22:15
Last Modified:26 Dec 2012 15:17

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