The membrane protein alkaline phosphatase is delivered to the vacuole by a route that is distinct from the VPS-dependent pathway.
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.
Piper
R C
RC
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229, USA.
Bryant
N J
NJ
Stevens
T H
TH
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D019084:Fluorescent Antibody Technique, Indirect; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D050765:Qa-SNARE Proteins; D011956:Receptors, Cell Surface / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014617:Vacuoles / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins; C087335:PEP1 protein, S cerevisiae; D050765:Qa-SNARE Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C092934:VPS45 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D000469:Alkaline Phosphatase; C022325:aspartic proteinase A; D016282:Aspartic Acid Endopeptidases