THE VESICLE TRANSPORT PROTEIN VPS33P IS AN ATP-BINDING PROTEIN THAT LOCALIZES TO THE CYTOSOL IN AN ENERGY-DEPENDENT MANNER

Molecular mechanisms of vesicle transport between the prevacuolar comp artment and the vacuole in yeast or the lysosome in mammalian cells ar e poorly understood. To learn more about the specificity of this inter compartmental step, we have examined the subcellular localization of a SEC1 homologue, Vps33p, a protein implicated to function in transport between the prevacuolar compartment and the vacuole, Following short pulses, 80-90% of newly synthesized Vps33p cofractionated with a cytos olic enzyme marker after making permeabilized yeast cells, However, du ring a chase, 20-40% of Vps33p fractionated with permeabilized cell me mbranes in a time-dependent fashion with a half-time of similar to 40 min. Depletion of cellular ATP increased the association rate to a hal f-time of similar to 4 min and caused 80-90% of newly synthesized Vps3 3p to be associated with permeabilized cell membranes. The association of Vps35p with permeabilized cell membranes was reversible after rest oring cells with glucose before permeabilization. The N-ethylmaleimide -sensitive fusion protein homologue, Sec18p, a protein with known ATP binding and hydrolysis activity, displayed the same reversible energy- dependent sedimentation characteristics as Vps33p. We determined that the photosensitive analog 8-azido-[alpha-P-32]ATP, could bind directly to Vps33p with low affinity. Interestingly, excess unlabeled ATP coul d enhance photoaffinity labeling of 8-azido-[alpha-P-32]ATP to Vps33p, suggesting cooperative binding, which was not observed with excess GT P. Importantly, we did not detect significant photolabeling after dele ting amino acid regions in Vps33p that show similarity to ATP interact ion motifs, We visualized these events in living yeast cells after fus ing the jellyfish green fluorescent protein (GFP) to the C terminus of full-length Vps33p, In metabolically active cells, the fully function al Vp33p-GFP fusion protein appeared to stain throughout the cytoplasm with one or two very bright fluorescent spots near the vacuole, After depleting cellular ATP, Vps33p-GFP appeared to localize with a puncta te morphology, which was also reversible upon restoring cells with glu cose. Overall, these data support a model where Vps33p cycles between soluble and particulate forms in an ATP-dependent manner, which may fa cilitate the specificity of transport vesicle docking or targeting to the yeast lysosome/vacuole.