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Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229
Membrane trafficking intermediates involved in the transport of proteins between the TGN
and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment
(PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results
in an accumulation of the PVC since this gene is required for traffic out of this compartment.
The vacuolar ATPase subunit Vph1p transits to the
vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as
defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required
for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase
(ALP) reaches its final destination without the function
of these two genes. Using a series of ALP derivatives,
we find that the information to specify the entry of
ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this
sorting determinant results in a protein that follows the
VPS-dependent pathway to the vacuole.
Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis,
we demonstrate that, in addition to ALP, the vacuolar
syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function
of Vam3p is required for membrane traffic along the
VPS-independent pathway.
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