THE MEMBRANE-PROTEIN ALKALINE-PHOSPHATASE IS DELIVERED TO THE VACUOLEBY A ROUTE THAT IS DISTINCT FROM THE VPS-DEPENDENT PATHWAY

Membrane trafficking intermediates involved in the transport of protei ns between the TGN and the lysosome-like vacuole in the yeast Saccharo myces cerevisiae can be accumulated in various vps mutants, Loss of fu nction of Vps45p, an Sec1p-like protein required for the fusion of Gol gi-derived transport vesicles with the prevacuolar/endosomal compartme nt (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the P VC since this gene is required for traffic out of this compartment, Th e vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-d erived transport vesicles, as defined by mutations in VPS45, and throu gh the PVC, as defined by mutations in VPS27, In this study we demonst rate that, whereas VPS45 and VPS27 are required for the vacuolar deliv ery of several membrane proteins, the vacuolar membrane protein alkali ne phosphatase (ALP) reaches its final destination without the functio n of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative path way to the vacuole is contained within its cytosolic tail, in the 13 r esidues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathw ay to the vacuole, Using a combination of immunofluorescence localizat ion and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS 45/27-independent pathway to the vacuole, In addition, the function of Vam3p is required for membrane traffic along the VPS-independent path way.