The transport of newly synthesized proteins through the vacuolar protein sorting pathway in the budding yeast Saccharomyces cerevisiae requires two distinct target SNAP receptor (t-SNARE) proteins, Pep12p and Vam3p. Pep12p is localized to the pre-vacuolar endosome and its activity is required for transport of proteins from the Golgi to the vacuole through a well defined route, the carboxypeptidase Y (CPY) pathway. Vam3p is localized to the vacuole where it mediates delivery of cargoes from both the CPY and the recently described alkaline phosphatase (ALP) pathways. Surprisingly, despite their organelle-specific functions in sorting of vacuolar proteins, overexpression of VAM3 can suppress the protein sorting defects of pep12Δ cells. Based on this observation, we developed a genetic screen to identify domains in Vam3p (e.g., localization and/or specific protein–protein interaction domains) that allow it to efficiently substitute for Pep12p. Using this screen, we identified mutations in a 7–amino acid sequence in Vam3p that lead to missorting of Vam3p from the ALP pathway into the CPY pathway where it can substitute for Pep12p at the pre-vacuolar endosome. This region contains an acidic di-leucine sequence that is closely related to sorting signals required for AP-3 adaptor–dependent transport in both yeast and mammalian systems. Furthermore, disruption of AP-3 function also results in the ability of wild-type Vam3p to compensate for pep12 mutants, suggesting that AP-3 mediates the sorting of Vam3p via the di-leucine signal. Together, these data provide the first identification of an adaptor protein–specific sorting signal in a t-SNARE protein, and suggest that AP-3–dependent sorting of Vam3p acts to restrict its interaction with compartment-specific accessory proteins, thereby regulating its function. Regulated transport of cargoes such as Vam3p through the AP-3–dependent pathway may play an important role in maintaining the unique composition, function, and morphology of the vacuole.
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24 August 1998
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August 24 1998
Acidic Di-leucine Motif Essential for AP-3–dependent Sorting and Restriction of the Functional Specificity of the Vam3p Vacuolar t-SNARE
Tamara Darsow,
Tamara Darsow
Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668
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Christopher G. Burd,
Christopher G. Burd
Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668
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Scott D. Emr
Scott D. Emr
Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668
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Tamara Darsow
Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668
Christopher G. Burd
Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668
Scott D. Emr
Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668
Address all correspondence to Scott D. Emr, Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093-0668. Tel.: (619) 534-6462. Fax: (619) 534-6414. E-mail: [email protected]
Received:
May 20 1998
Revision Received:
July 15 1998
Online ISSN: 1540-8140
Print ISSN: 0021-9525
1998
J Cell Biol (1998) 142 (4): 913–922.
Article history
Received:
May 20 1998
Revision Received:
July 15 1998
Citation
Tamara Darsow, Christopher G. Burd, Scott D. Emr; Acidic Di-leucine Motif Essential for AP-3–dependent Sorting and Restriction of the Functional Specificity of the Vam3p Vacuolar t-SNARE . J Cell Biol 24 August 1998; 142 (4): 913–922. doi: https://doi.org/10.1083/jcb.142.4.913
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