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 approximately 40 min. Depletion of cellular ATP increased the association rate to a half-time of approximately 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. 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-32P]ATP, could bind directly to Vps33p with low affinity. Interestingly, excess unlabeled ATP could enhance photoaffinity labeling of 8-azido-[alpha-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.
Highlights
The compartmentalized nature of eukaryotic cells demands high fidelity mechanisms to ensure maintenance of each organelle’s unique content
Spheroplasts from a vps33– 4 mutant strain were able to mature 35– 40% of carboxypeptidase Y (CPY) in vivo, while the majority was secreted as the Golgi-modified p2 precursor
Depleting cellular ATP results in a rapid change of Vps33p from a cytosolic protein to one that fractionates as a particulate protein
Summary
The compartmentalized nature of eukaryotic cells demands high fidelity mechanisms to ensure maintenance of each organelle’s unique content. The SNARE hypothesis describes fundamental molecular mechanisms that give rise to the specificity of vesicle-mediated protein transport in eukaryotic cells [1]. 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 [5, 6]. The VPS33 gene was identified in a selection for yeast mutants with defects in vacuolar protein sorting [17]. Loss of VPS33 function results in three prominent phenotypes; 1) temperature-sensitive growth (restrictive temperature is 38 °C), 2) severe missorting of both soluble and membrane vacuolar proteins, and 3) abnormal vacuole morphology. Vps33p reversibly changed its localization from the cytosol to a particulate fraction when ATP was depleted in yeast cells, which was observed for the NSF1 homologue, Sec18p
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