Acidic di-leucine motif essential for AP-3-dependent sorting and restriction of the functional specificity of the Vam3p vacuolar t-SNARE.
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 pep12Delta 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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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, USA. semr@ucsd.edu
Burd
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D033942:Adaptor Proteins, Vesicular Transport; D000469:Alkaline Phosphatase / Q000502:physiology; D001692:Biological Transport / Q000502:physiology; D002268:Carboxypeptidases / Q000502:physiology; D043402:Cathepsin A; D011992:Endosomes / Q000502:physiology; D005656:Fungal Proteins / Q000378:metabolism; D007930:Leucine / Q000737:chemistry; D008565:Membrane Proteins / Q000235:genetics* / Q000502:physiology*; D034041:Monomeric Clathrin Assembly Proteins; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry*; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D051040:Vesicle-Associated Membrane Protein 3; D033921:Vesicular Transport Proteins
D033942:Adaptor Proteins, Vesicular Transport; D005656:Fungal Proteins; D008565:Membrane Proteins; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; C081224:PEP12 protein, S cerevisiae; D010750:Phosphoproteins; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D029701:Saccharomyces cerevisiae Proteins; D051040:Vesicle-Associated Membrane Protein 3; D033921:Vesicular Transport Proteins; C439023:clathrin assembly protein AP180; D000469:Alkaline Phosphatase; D002268:Carboxypeptidases; D043402:Cathepsin A; D007930:Leucine