Overview of Subcellular Fractionation Procedures for the Yeast Saccharomyces cerevisiae

Stephanie E. Rieder1, Scott D. Emr1

1 University of California, San Diego, La Jolla, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.7
DOI:  10.1002/0471143030.cb0307s07
Online Posting Date:  May, 2001
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Abstract

This unit provides an overview of centrifugation‐based fractionation procedures adapted for the yeast Saccharomyces cerevisiae. The goals, merits, limitations, and critical parameters of are discussed in order to facilitate the development of subcellular fractionation strategies. Topics include yeast cell lysate preparation, differential velocity centrifugation, density gradient centrifugation, and the analysis of subcellular fractions.

     
 
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Table of Contents

  • Yeast Strain Selection and Growth Conditions
  • Preparation of Yeast Cell Lysates
  • Isolation of Subcellular Fractions: Procedural Options
  • Analysis of Subcellular Fractions
  • Figures
  • Tables
     
 
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Materials

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Figures

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Literature Cited

Literature Cited
   Aris, J.P. and Blobel, G. 1991. Isolation of yeast nuclei. Methods Enzymol. 194:735‐749.
   Babst, M., Wendland, B., Estepa, E.J., and Emr, S.D. 1998. The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function. EMBO J. 17:2982‐2993.
   Baker, D., Hicke, L., Rexach, M., Schleyer, M., and Schekman, R. 1988. Reconstitution of SEC gene product‐dependent intercompartmental protein transport. Cell 54:335‐344.
   Botstein, D., Chervitz, S.A., and Cherry, J.M. 1997. Yeast as a model organism. Science 277:1259‐1260.
   Brodsky, J.L., Hamamoto, S., Feldheim, D., and Schekman, R. 1993. Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70. J Cell Biol. 120:95‐102.
   Byers, B. and Goetsch, L. 1991. Preparation of yeast cells for thin‐section electron microscopy. Methods Enzymol. 194:602‐608.
   Cereghino, J.L., Marcusson, E.G., and Emr, S.D. 1995. The cytoplasmic tail domain of the vacuolar protein sorting receptor Vps10p and a subset of VPS gene products regulate receptor stability, function, and localization. Mol. Biol. Cell 6:1089‐1102.
   Cherry, J.M., Ball, C., Weng, S., Juvik, G., Schmidt, R., Adler, C., Dunn, B., Dwight, S., Riles, L., Mortimer, R.K., and Botstein, D. 1997. Genetic and physical maps of Saccharomyces cerevisiae. Nature 387:67‐73.
   Clark, M.W. 1991. Immunogold labeling of yeast ultrathin sections. Methods Enzymol. 194:608‐626.
   Clayton, R.A., White, O., Ketchum, K.A., and Venter, J.C. 1997. The yeast genome directory: The first genome from the third domain of life. Nature 387:459‐462.
   Daum, G., Bohni, P.C., and Schatz, G. 1982a. Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J. Biol. Chem. 257:13028‐13033.
   Daum, G., Gasser, S.M., and Schatz, G. 1982b. Import of proteins into mitochondria. Energy‐dependent, two‐step processing of the intermembrane space enzyme cytochrome b2 by isolated yeast mitochondria. J. Biol. Chem. 257:13075‐13080.
   Dobrota, M. and Hinton, R. 1992. Conditions for density gradient separations. In Preparative Centrifugation: A Practical Approach (D. Rickwood, ed.) pp. 77‐137. IRL Press, New York.
   Dulic, V., Egerton, M., Elguindi, I., Raths, S., Singer, B., and Riezman, H. 1991. Yeast endocytosis assays. Methods Enzymol. 194:697‐710.
   Evans, W.H. 1992. Isolation and characterization of membranes and cell organelles. In Preparative Centrifugation: A Practical Approach (D. Rickwood, ed.) pp. 233‐270. IRL Press, New York.
   Franzusoff, A., Rothblatt, J., and Schekman, R. 1991. Analysis of polypeptide transit through yeast secretory pathway. Methods Enzymol. 194:662‐674.
   Garrels, J.I., McLaughlin, C.S., Warner, J.R., Futcher, B., Latter, G.I., Kobayashi, R., Schwender, B., Volpe, T., Anderson, D.S., Mesquita‐Fuentes, R., and Payne, W.E. 1997. Proteome studies of Saccharomyces cerevisiae: Identification and characterization of abundant proteins. Electrophoresis 18:1347‐1360.
   Gaynor, E.C., te Heesen, S., Graham, T.R., Aebi, M., and Emr, S.D. 1994. Signal‐mediated retrieval of a membrane protein from the Golgi to the ER in yeast. J. Cell Biol. 127:653‐665.
   Glick, B.S. and Pon, L.A. 1995. Isolation of highly purified mitochondria from Saccharomyces cerevisiae. Methods Enzymol. 260:213‐23.
   Hasek, J. and Streiblova, E. 1996. Fluorescence microscopy methods. Methods Mol. Biol. 53:391‐405.
   Hicke, L., Zanolari, B., Pypaert, M., Rohrer, J., and Riezman, H. 1997. Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N‐ethylmaleimide‐sensitive fusion protein. Mol. Biol. Cell 8:13‐31.
   Hinton, R.H. and Mullock, B.M. 1997. Isolation of subcellular fractions. In Subcellular Fractionation: A Practical Approach (J. M. Graham and D. Rickwood, eds.) pp. 31‐69. IRL Press, New York.
   Hodges, P.E., Payne, W.E., and Garrels, J.I. 1998. The Yeast Protein Database (YPD): A curated proteome database for Saccharomyces cerevisiae. Nucl. Acids Res. 26:68‐72.
   Horazdovsky, B.F., DeWald, D.B., and Emr, S.D. 1995. Protein transport to the yeast vacuole. Curr. Opin. Cell Biol. 7:544‐551.
   Jones, E.W. 1990. Vacuolar proteases in yeast Saccharomyces cerevisiae. Methods Enzymol. 185:372‐386.
   Jones, E.W. 1991a. Tackling the protease problem in Saccharomyces cerevisiae. Methods Enzymol. 194:428‐453.
   Jones, E.W. 1991b. Three proteolytic systems in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 266:7963‐7966.
   Klionsky, D.J. and Emr, S.D. 1989. Membrane protein sorting: Biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase. EMBO J. 8:2241‐2250.
   Lazarow, P.B., Thieringer, R., Cohen, G., Imanaka, T., and Small, G. 1991. Protein import into peroxisomes in vitro. Methods Cell Biol. 34:303‐326.
   Luck, H. 1963. Catalase. In Methods of Enzymatic Analysis (H.U. Bergmeyer, ed.) pp. 885‐894. Academic Press, New York.
   Marcusson, E.G., Horazdovsky, B.F., Cereghino, J.L., Gharakhanian, E., and Emr, S.D. 1994. The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene. Cell 77:579‐586.
   Mewes, H.W., Albermann, K., Bahr, M., Frishman, D., Gleissner, A., Hani, J., Heumann, K., Kleine, K., Maierl, A., Oliver, S.G., Pfeiffer, F., and Zollner, A. 1997. Overview of the yeast genome. Nature 387:7‐65.
   Perrot, M., Sagliocco, F., Mini, T., Monribot, C., Schneider, U., Shevchenko, A., Mann, M., Jeno, P., and Boucherie, H. 1999. Two‐dimensional gel protein database of Saccharomyces cerevisiae(update 1999). Electrophoresis 20:2280‐2298.
   Prescianotto‐Baschong, C. and Riezman, H. 1998. Morphology of the yeast endocytic pathway. Mol. Biol. Cell 9:173‐189.
   Pringle, J.R., Adams, A.E., Drubin, D.G., and Haarer, B.K. 1991. Immunofluorescence methods for yeast. Methods Enzymol. 194:565‐602.
   Raths, S., Rohrer, J., Crausaz, F., and Riezman, H. 1993. end3 and end4: Two mutants defective in receptor‐mediated and fluid‐phase endocytosis in Saccharomyces cerevisiae. J. Cell Biol. 120:55‐65.
   Rieder, S.E. and Emr, S.D. 1997. A novel RING finger protein complex essential for a late step in protein transport to the yeast vacuole. Mol. Biol. Cell 8:2307‐2327.
   Rieder, S.E., Banta, L.M., Kohrer, K., McCaffery, J.M., and Emr, S.D. 1996. Multilamellar endosome‐like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant. Mol. Biol. Cell 7:985‐999.
   Romanos, M.A., Scorer, C.A., and Clare, J.J. 1992. Foreign gene expression in yeast: A review. Yeast 8:423‐488.
   Rothblatt, J. and Schekman, R. 1989. A hitchhiker's guide to analysis of the secretory pathway in yeast. Methods Cell Biol. 32:3‐36.
   Schekman, R. 1985. Protein localization and membrane traffic in yeast. Annu. Rev. Cell Biol. 1:115‐143.
   Scott, J.H. and Schekman, R. 1980. Lyticase: Endoglucanase and protease activities that act together in yeast cell lysis. J. Bacteriol. 142:414‐423.
   Sherman, F. 1991. Getting started with yeast. Methods Enzymol. 194:3‐21.
   Shevchenko, A., Jensen, O.N., Podtelejnikov, A.V., Sagliocco, F., Wilm, M., Vorm, O., Mortensen, P., Boucherie, H., and Mann, M. 1996. Linking genome and proteome by mass spectrometry: Large‐scale identification of yeast proteins from two dimensional gels. Proc. Natl. Acad. Sci. U.S.A. 93:14440‐14445.
   Singer, B. and Riezman, H. 1990. Detection of an intermediate compartment involved in transport of alpha‐factor from the plasma membrane to the vacuole in yeast. J. Cell Biol. 110:1911‐1922.
   Singer‐Kruger, B., Frank, R., Crausaz, F., and Riezman, H. 1993. Partial purification and characterization of early and late endosomes from yeast. Identification of four novel proteins. J. Biol. Chem. 268:14376‐14386.
   Van Den Hazel, H.B., Kielland‐Brandt, M.C., and Winther, J.R. 1996. Review: Biosynthesis and function of yeast vacuolar proteases. Yeast 12:1‐16.
   Vida, T.A., Graham, T.R., and Emr, S.D. 1990. In vitro reconstitution of intercompartmental protein transport to the yeast vacuole. J. Cell Biol. 111:2871‐2884.
   Walworth, N.C., Goud, B., Ruohola, H., and Novick, P.J. 1989. Fractionation of yeast organelles. Methods Cell Biol. 31:335‐356.
   Westphal, V., Marcusson, E.G., Winther, J.R., Emr, S.D., and van den Hazel, H.B. 1996. Multiple pathways for vacuolar sorting of yeast proteinase A. J. Biol. Chem. 271:11865‐11870.
   Winzeler, E.A., Shoemaker, D.D., Astromoff, A., Liang, H., Anderson, K., Andre, B., Bangham, R., Benito, R., Boeke, J.D., Bussey, H., Chu, A.M., Connelly, C., Davis, K., Dietrich, F., Dow, S.W., El Bakkoury, M., Foury, F., Friend, S.H., Gentalen, E., Giaever, G., Hegemann, J.H., Jones, T., Laub, M., Liao, H., Davis, R.W. et al. 1999. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901‐906.
   Wuestehube, L.J., Duden, R., Eun, A., Hamamoto, S., Korn, P., Ram, R., and Schekman, R. 1996. New mutants of Saccharomyces cerevisiae affected in the transport of proteins from the endoplasmic reticulum to the Golgi complex. Genetics 142:393‐406.
   Zinser, E. and Daum, G. 1995. Isolation and biochemical characterization of organelles from the yeast, Saccharomyes cerevisiae. Yeast 11:493‐536.
   Zubenko, G.S., Park, F.J., and Jones, E.W. 1983. Mutations in PEP4 locus of Saccharomyces cerevisiae block final step in maturation of two vacuolar hydrolases. Proc. Natl. Acad. Sci. U.S.A. 80:510‐514.
Key References
   Dobrota and Hinton, 1992. See above.
  This review provides an excellent overview of the theory and design of subcellular fractionation experiments. Other helpful reviews include Evans () and Hinton and Mullock (; see above).
   Sikorski, R. and Peters, R. 1998. Yeast sites on the net. Science 279:1968.
  This review provides information on multiple Internet resources available for S. cerevisiae.
   Walworth et al., 1989. See above.
  This older review provides an excellent summary of the subcellular fractionation techniques applied to S. cerevisiae, with a focus on organelles along the secretory pathway.
   Zinser and Daum, 1995. See above.
  This review provides an excellent summary of the subcellular fractionation techniques applied to S. cerevisiae and the corresponding references.
Internet Resources
  http://genome-www.stanford.edu/Saccharomyces/
  Saccharomyces Genome Database (SGD) provides access to the complete sequence of the S. cerevisiae genome, confirmed and predicted open reading frames (ORFs), protein information, and a number of useful Internet links.
  http://www.atcc.org/searchengine/ygsc.html
  Yeast Genetic Stock Center at the ATCC, which can provide many published yeast strains.
  http://www.proteome.com/
  The Yeast Protein Database (Proteome, Inc.) is an excellent source of comprehensive and up‐to‐date information on S. cerevisiae proteins. The information is derived from the published literature and DNA sequence databases. The protein information provided, including summaries of the protein and gene characteristics and the corresponding literature references, can be searched and categorized in several convenient ways (e.g., according to subcellular location, gene name, or size).
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