The Vesicle Transport Protein Vps33p Is an ATP-binding Protein That Localizes to the Cytosol in an Energy-dependent Manner

Abstract

Molecular mechanisms of vesicle transport between the prevacuolar compartment and the vacuole in yeast or the lysosome in mammalian cells are poorly understood. To learn more about the specificity of this intercompartmental 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 cytosolic enzyme marker after making permeabilized yeast cells. However, during a chase, 20–40% of Vps33p fractionated with permeabilized cell membranes in a time-dependent fashion with a half-time of ∼40 min. Depletion of cellular ATP increased the association rate to a half-time of ∼4 min and caused 80–90% of newly synthesized Vps33p to be associated with permeabilized cell membranes. The association of Vps33p with permeabilized cell membranes was reversible after restoring cells with glucose before permeabilization. TheN-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-[α-32P]ATP, could bind directly to Vps33p with low affinity. Interestingly, excess unlabeled ATP could enhance photoaffinity labeling of 8-azido-[α-32P]ATP to Vps33p, suggesting cooperative binding, which was not observed with excess GTP. Importantly, we did not detect significant photolabeling after deleting amino acid regions in Vps33p that show similarity to ATP interaction motifs. We visualized these events in living yeast cells after fusing the jellyfish green fluorescent protein (GFP) to the C terminus of full-length Vps33p. In metabolically active cells, the fully functional Vps33p-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 punctate morphology, which was also reversible upon restoring cells with glucose. Overall, these data support a model where Vps33p cycles between soluble and particulate forms in an ATP-dependent manner, which may facilitate the specificity of transport vesicle docking or targeting to the yeast lysosome/vacuole. Molecular mechanisms of vesicle transport between the prevacuolar compartment and the vacuole in yeast or the lysosome in mammalian cells are poorly understood. To learn more about the specificity of this intercompartmental 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 cytosolic enzyme marker after making permeabilized yeast cells. However, during a chase, 20–40% of Vps33p fractionated with permeabilized cell membranes in a time-dependent fashion with a half-time of ∼40 min. Depletion of cellular ATP increased the association rate to a half-time of ∼4 min and caused 80–90% of newly synthesized Vps33p to be associated with permeabilized cell membranes. The association of Vps33p with permeabilized cell membranes was reversible after restoring cells with glucose before permeabilization. TheN-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-[α-32P]ATP, could bind directly to Vps33p with low affinity. Interestingly, excess unlabeled ATP could enhance photoaffinity labeling of 8-azido-[α-32P]ATP to Vps33p, suggesting cooperative binding, which was not observed with excess GTP. Importantly, we did not detect significant photolabeling after deleting amino acid regions in Vps33p that show similarity to ATP interaction motifs. We visualized these events in living yeast cells after fusing the jellyfish green fluorescent protein (GFP) to the C terminus of full-length Vps33p. In metabolically active cells, the fully functional Vps33p-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 punctate morphology, which was also reversible upon restoring cells with glucose. Overall, these data support a model where Vps33p cycles between soluble and particulate forms in an ATP-dependent manner, which may facilitate the specificity of transport vesicle docking or targeting to the yeast lysosome/vacuole. The compartmentalized nature of eukaryotic cells demands high fidelity mechanisms to ensure maintenance of each organelle's unique content. Organelles of the secretory and endocytic system rely on vesicle-mediated transport pathways not only to move protein cargo throughout the cell but also to preserve their specific protein and lipid compositions. The SNARE hypothesis describes fundamental molecular mechanisms that give rise to the specificity of vesicle-mediated protein transport in eukaryotic cells (1Sollner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2627) Google Scholar). The basic tenet of this notion is that interactions between integral membrane proteins in transport vesicles (v-SNAREs) and in their cognate target organelles (t-SNAREs) direct the specificity of all intercompartmental events (2Sollner T. FEBS Lett. 1995; 369: 80-83Crossref PubMed Scopus (45) Google Scholar). Although substantial evidence exists to support the existence of many v- and t-SNARE proteins, precisely how they function in vesicle transport specificity is continually being revised as more details are uncovered for different systems (3Nichols B.J. Ungermann C. Pelham H.R. Wickner W.T. Haas A. Nature. 1997; 387: 199-202Crossref PubMed Scopus (380) Google Scholar, 4von Mollard G.F. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar). However, a fact remaining clear and central to the SNARE hypothesis is that multimeric interactions of v- and t-SNAREs with cytosolic proteins play an essential role in the precision of vesicle-mediated transport (5Sudhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1768) Google Scholar, 6Sogaard M. Tani K. Ye R.R. Geromanos S. Tempst P. Kirchhausen T. Rothman J.E. Sollner T. PubMed Scopus Google Scholar). The of the cytosolic protein are to vesicle docking one of not only was the secretory P. S. A. PubMed Scopus Google but was also the of this of proteins that are in eukaryotic J. J. PubMed Scopus (45) Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). The of proteins in yeast of K. S. PubMed Scopus Google J. Cell Google Scholar, Stevens T.H. J. Cell Biol. Google C. Biol. PubMed Scopus Google and Vps33p Biol. PubMed Scopus Google K. T. K. Biol. PubMed Scopus Google Scholar). The was in a for yeast with in protein Biol. PubMed Scopus Google Scholar). function in is of soluble and membrane proteins, and vacuole Although the vacuole is very is not as as the C J. Cell Biol. PubMed Scopus Google Scholar). The Vps33p, is amino and The of Vps33p two regions that are to the and and Biol. PubMed Scopus Google suggesting that may bind or ATP, which In this we have examined ATP binding in the function of Vps33p. data that Vps33p bind ATP and that of a that the in the of ATP binding and a Vps33p localization the to a particulate ATP was in yeast cells, which was also observed for the green fluorescent fusion homologue, on yeast and for and maintenance was J. Google with yeast The yeast in this Biol. PubMed Scopus Google a of Biol. PubMed Scopus Google and of green fluorescent protein (GFP) fusion a for a S. PubMed Scopus Google S. A. PubMed Scopus Google and of the unique of and P. PubMed Google Scholar, M. P. 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The for Vps33p, to and to or of was not and was for the proteins that as a the 8-azido-[α-32P]ATP before the of the yeast and the labeling of with 8-azido-[α-32P]ATP was with We observed photoaffinity labeling of Vps33p the 8-azido-[α-32P]ATP was before the of that not we observed labeling of Vps33p we did not the 8-azido-[α-32P]ATP, that the on of the was the only unlabeled min before the of the the with unlabeled a of the and was and the two the was protein Vps33p, was to be a soluble protein subcellular Biol. PubMed Scopus Google Scholar). the function of Vps33p, we an in that intercompartmental transport of to the yeast vacuole in permeabilized cells J. Cell Biol. PubMed Scopus Google Scholar). The a and function for protein Biol. PubMed Scopus Google Scholar, T. J. 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The also exists in soluble and particulate forms J. Cell Google and is to Vps33p J. J. PubMed Scopus (45) Google Scholar). However, similarity to proteins that bind or ATP J. Cell Google Scholar, Stevens T.H. J. Cell Biol. Google Scholar). fractionated after in glucose of Vps33p and and of cofractionated with the cytosolic marker after min of and In with and for min caused of Vps33p and to with permeabilized cell but the of that was did not After the cells in to the and with and for of Vps33p and of fractionated with the and that the of Although not the of Vps33p was the same was to the cells during that protein was not to the of Vps33p. 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Nature. 1993; PubMed Scopus Google Scholar). rise to an that the in Vps33p with protein in a as to an ATP binding The of a specific binding unique specificity to a vesicle docking or transport Although the molecular details of ATP binding to Vps33p are the on specificity or not be in these The of is an where was not to bind ATP after photoaffinity and T. S. J. J. J. Biol. PubMed Scopus Google Scholar). However, the of an with ATP these C. J.E. 1997; PubMed Scopus Google Scholar). the amino of for the not a or and to bind ATP, and of these proteins also the M. T.C. J. Biol. PubMed Scopus Google Scholar). that many different ATP binding and that amino acid data are or J. PubMed Scopus Google Scholar). The interaction of Vps33p with ATP not to characteristics of a binding Molecular details of the interaction may be protein of Vps33p are The energy-dependent localization of Vps33p an in the in protein are in proteins that bind or ATP as S. J. Biol. PubMed Google and the fusion S.W. K. Brunner M. Rothman J.E. J. Cell Biol. PubMed Scopus Google H. J.E. 1997; PubMed Scopus Google Scholar). The nature of Vps33p is The of Vps33p and the Sec18p, but not an that continually these proteins between soluble and particulate forms with ATP The of Vps33p in living yeast cells and for of cellular permeabilized cell of Vps33p is with the remaining to fluorescent which appeared localization is of the prevacuolar compartment in which is in K. Biol. PubMed Scopus Google Scholar, Stevens T.H. Biol. PubMed Scopus Google Scholar, H. Stevens T.H. J. Cell Biol. 1995; PubMed Scopus Google Scholar). However, data with that the of Vps33p that is particulate not with the prevacuolar compartment as the of J. Cell Biol. 1993; PubMed Scopus Google Scholar). the fluorescent spots may be a or subcellular a and interactions the yeast C and Biol. 1997; PubMed Scopus Google Scholar). and a with of associated and Vps33p. as a protein and as a of the vacuole Biol. 1997; PubMed Scopus Google Scholar). The sedimentation characteristics of the are very to we have observed with Vps33p after that ATP is to Vps33p the The many SEC1 that these proteins function vesicle-mediated in eukaryotic cells J. J. PubMed Scopus (45) Google Scholar, J. PubMed Scopus Google Scholar). However, the molecular which proteins their role in vesicle is The that proteins play a role in SNARE homologue, A. M. PubMed Scopus Google and yeast interactions in to transport 1997; PubMed Scopus Google Scholar). the a homologue, transport between the and C. J. Biol. PubMed Scopus Google that proteins not a function different vesicle-mediated the secretory and endocytic this Vps33p may a unique not of the SEC1 they are not to bind or on Vps33p on the details of ATP binding in the molecular of transport between the prevacuolar compartment and lysosome/vacuole. In we Vps33p ATP after of a The multimeric nature of Vps33p be with and sedimentation The interaction between and the that their protein may directly T. J. Cell Biol. 1997; PubMed Scopus Google Scholar). We this with protein to is associated with Vps33p after of the full-length Vps33p-GFP fusion protein in a with the or not Vps33p to the prevacuolar of intercompartmental transport to the vacuole be the system to the molecular details of ATP binding and Vps33p We are to this system with that show a in protein transport of in or ATP binding or hydrolysis to Vps33p the to a membrane or membrane to the yeast vacuole. We and for the and for for and and for the We also for on photoaffinity labeling with Whiteheart for to and for of