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J. Cell Biol.,
Volume 142, Number 4, August 24, 1998 913-922
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
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 pep12
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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