The yeast proteome includes about 300 polytopic membrane proteins known or predicted to function as transporters. Such proteins ensure active or passive transport of small ions or metabolites across the plasma or internal membranes. Despite decades of research on yeast transporters, many of these remain uncharacterized in terms of substrate selectivity range, subcellular localization, and biological function. Assaying the uptake of radiolabeled compounds into whole cells or isolated organelles remains a powerful method for characterizing the function and biochemical properties of these proteins. Here we describe established protocols for measuring transporter activity in whole cells, intact vacuoles, or reconstituted vacuolar vesicles. These methods have proved particularly useful in the context of our work on yeast amino acid transporters, and can in principle be applied to assaying the uptake of other categories of compounds.
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We thank Christos Gournas, Stephan Vissers, and Kathleen Broman for critical reading of the manuscript. M.C. was the recipient of a FRIA PhD fellowship. This work was supported by a PDR grant (nr. 23655065) from the FNRS (Fédération Wallonie-Bruxelles, Belgium) and by a grant from the Cystinosis Research Foundation.
Grenson M, Mousset M, Wiame JM, Béchet J (1966) Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. I. Evidence for a specific arginine-transporting system. Biochim Biophys Acta 127:325–338CrossRefGoogle Scholar
Grenson M (1966) Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. II. Evidence for a specific lysine-transporting system. Biochim Biophys Acta 127:339–346CrossRefGoogle Scholar
Gits JJ, Grenson M (1967) Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. 3. Evidence for a specific methionine-transporting system. Biochim Biophys Acta 135:507–516CrossRefGoogle Scholar
Grenson M, Hou C, Crabeel M (1970) Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. IV. Evidence for a general amino acid permease. J Bacteriol 103:770–777PubMedPubMedCentralGoogle Scholar
Van Belle D, André B (2001) A genomic view of yeast membrane transporters. Curr Opin Cell Biol 13:389–398CrossRefGoogle Scholar
Yang Z, Huang J, Geng J, Nair U, Klionsky DJ (2006) Atg22 recycles amino acids to link the degradative and recycling functions of autophagy. Mol Biol Cell 17:5094–5104CrossRefGoogle Scholar
Nicastro R, Sardu A, Panchaud N, De Virgilio C (2017) The architecture of the rag GTPase signaling network. Biomol Ther 7:48Google Scholar
Boller T, Dürr M, Wiemken A (1975) Characterization of a specific transport system for arginine in isolated yeast vacuoles. Eur J Biochem 54:81–91CrossRefGoogle Scholar
Ohsumi Y, Anraku Y (1981) Active transport of basic amino acids driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae. J Biol Chem 256:2079–2082PubMedGoogle Scholar
Boller T, Dürr M, Wiemken A (1989) Transport in isolated yeast vacuoles: characterization of arginine permease. Methods Enzymol 174:504–518CrossRefGoogle Scholar
Kakinuma Y, Ohsumi Y, Anraku Y (1981) Properties of H+-translocating adenosine triphosphatase in vacuolar membranes of Saccharomyces cerevisiae. J Biol Chem 256:10859–10863PubMedGoogle Scholar
Sato T, Ohsumi Y, Anraku Y (1984) An arginine/histidine exchange transport system in vacuolar-membrane vesicles of Saccharomyces cerevisiae. J Biol Chem 259:11509–11511PubMedGoogle Scholar
Russnak R, Konczal D, McIntire SL (2001) A family of yeast proteins mediating bidirectional vacuolar amino acid transport. J Biol Chem 276:23849–23857CrossRefGoogle Scholar
Wiame JM, Grenson M, Arst HN (1985) Nitrogen catabolite repression in yeasts and filamentous fungi. Adv Microb Physiol 26:1–88CrossRefGoogle Scholar
Stevens T, Esmon B, Schekman R (1982) Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell 30:439–448CrossRefGoogle Scholar
Conradt B, Shaw J, Vida T, Emr S, Wickner W (1992) In vitro reactions of vacuole inheritance in Saccharomyces cerevisiae. J Cell Biol 119:1469–1479CrossRefGoogle Scholar
Gerasimaite R, Sharma S, Desfougères Y, Schmidt A, Mayer A (2014) Coupled synthesis and translocation restrains polyphosphate to acidocalcisome-like vacuoles and prevents its toxicity. J Cell Sci 127:5093–5104CrossRefGoogle Scholar
Serrano R (1983) In vivo glucose activation of the yeast plasma membrane ATPase. FEBS Lett 156:11–14CrossRefGoogle Scholar
Kane PM (1995) Disassembly and reassembly of the yeast vacuolar H(+)-ATPase in vivo. J Biol Chem 270:17025–17032PubMedGoogle Scholar