The Yeast Vacuolar Membrane Proteome

Abstract

Transport of solutes between the cytosol and the vacuolar lumen is of crucial importance for various functions of vacuoles, including ion homeostasis; detoxification; storage of different molecules such as amino acids, phosphate, and calcium ions; and proteolysis. To identify proteins that catalyze solute transport across the vacuolar membrane, the membrane proteome of purified Saccharomyces cerevisiae vacuoles was analyzed. Subtractive proteomics was used to distinguish contaminants from true vacuolar proteins by comparing the relative abundances of proteins in pure and crude preparations. A robust statistical analysis combining enrichment ranking with the double boundary iterative group analysis revealed that 148 proteins were significantly enriched in the pure vacuolar preparations. Among these proteins were well characterized vacuolar proteins, such as the subunits of the vacuolar H ؉ -ATPase, but also proteins that had not previously been assigned to a cellular location, many of which are likely novel vacuolar membrane transporters, e.g. for nucleosides and oligopeptides. Although the majority of contaminating proteins from other organelles were depleted from the pure vacuolar membranes, some proteins annotated to reside in other cellular locations were enriched along with the vacuolar proteins. In many cases the enrichment of these proteins is biologically relevant, and we discuss that a large group is involved in membrane fusion and protein trafficking to vacuoles and may have multiple localizations. Other proteins are degraded in vacuoles, and in some cases database annotations are likely to be incomplete or incorrect. Our work provides a wealth of information on vacuolar biology and a solid basis for further characterization of vacuolar functions. The pH difference of ϳ1.7 pH units between the vacuolar lumen and the cytosol is used as the driving force for substrate-proton antiport systems in the vacuolar membrane (1). Transport processes across the vacuolar membrane are important for many crucial functions of vacuoles: storage of organic molecules such as polyphosphates, mannans, and other carbohydrates (2, 3); detoxification, i.e. removal and accumulation of harmful substances such as heavy metals and drugs; and proton and ion homeostasis. Another major function of lysosomes and vacuoles is the intracellular proteolysis of cytosolic and membrane proteins (4, 5) and turnover of organelles, e.g. lipid bodies, mitochondria, peroxisomes, and portions of nuclei (6 -9). Hydrolytic proteins in the lumen of vacuoles, including proteases, lipases, phosphatases, and nucleases, carry out these functions. Whereas vacuolar luminal proteins have been studied fairly extensively (10, 11), our knowledge about the integral membrane proteins in the yeast vacuolar membrane is limited.

Heptahelical protein PQLC2 is a lysosomal cationic amino acid exporter underlying the action of cysteamine in cystinosis therapy

“…The function of the five other PQ-loop proteins is unknown. At least two of them, Yol092p and Ydr352p (hereafter called Ypq1 and Ypq2), were reported in proteomic and genome-scale protein localization studies to be located at the membrane of the vacuole, the lysosome of yeast (23,24). By colabeling with fluorescent FM4-64 dye, we observed that Ypq1-GFP and Ypq2-GFP fusion proteins are indeed located at the vacuolar membrane and found that the same is true for another member of the family, Ybr147p, hereafter called Ypq3 (Fig.…”

Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance

“…This in vivo interaction did not require aldolase catalytic activity, as mutants lacking the catalytic site were still capable of interacting (Lu et al, 2007), although the exact mechanism of regulation remains unclear, and it has not been related to a specific stress condition. Enolase also appears to bind to yeast vacuoles through a peripheral membrane association, but the target protein on the membrane remains unknown (Decker and Wickner, 2006;Wiederhold et al, 2009). In order to determine if in M. crystallinum these enzymes are associated to the tonoplast by means of physical interaction with subunits of the V-ATPase, we performed reciprocal immunoprecipitation assays on tonoplast isolated from control and salt-treated plants using antibodies against aldolase, enolase, VHA-B, and VHA-E subunits ( Figure 5).…”

Bioengineered yeast-derived vacuoles with enhanced tissue-penetrating ability for targeted cancer therapy

“…Because of their subnanometer size, the engineered, drug-carrying vacuoles could rapidly extravasate and accumulate in the periphery of tumor tissue, where, after cancer receptor engagement, they could undergo endocytosis before releasing their payload and producing cytotoxic effects. In addition, with the available rapid protocols for vacuole preparation and methods for large-scale fermentation of yeasts, it is possible to obtain high yields of vacuoles in a cost-effective manner (26,33).…”