Isolation of Subcellular Fractions from the Yeast Saccharomyces cerevisiae

Stephanie E. Rieder1, Scott D. Emr2

1 The Scripps Research Institute, La Jolla, California, 2 University of California San Diego, School of Medicine, La Jolla, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.8
DOI:  10.1002/0471143030.cb0308s08
Online Posting Date:  May, 2001
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Abstract

This unit presents detailed protocols for a range of centrifugation‐based subcellular fractionation procedures for the yeast Saccharomyces cerevisiae. Techniques include spheroplast preparation, glass‐bead lysis, differential centrifugation, and several density gradient procedures using a variety of gradient media. There are analytical procedures that are primarily designed to evaluate the association of proteins with organelles in the exocytic and endocytic pathways. Additionally, there are preparative protocols for isolation of yeast nuclei, vacuoles, mitochondria, peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. The unit also contains a table, with references, for alternative approaches to isolation of these organelles and fractions.

     
 
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Table of Contents

  • Basic Protocol 1: Fractionation of Spheroplasts by Differential Centrifugation
  • Support Protocol 1: Preparation of Yeast Spheroplasts Using Zymolyase
  • Basic Protocol 2: Equilibrium Density Gradient Fractionation Using Nycodenz
  • Alternate Protocol 1: Equilibrium Density Gradient Fractionation using Sucrose
  • Basic Protocol 3: Fractionation of P13,000 Membranes on Sucrose Step Gradients
  • Basic Protocol 4: Isolation of Intact Vacuoles Using Ficoll Step Gradients
  • Basic Protocol 5: Isolation of Intact Nuclei with Ficoll Step Gradients
  • Basic Protocol 6: Isolation of Lactate‐Induced Mitochondria Using Nycodenz Step Gradients
  • Basic Protocol 7: Isolation of Oleate‐Induced Peroxisomes Using Sucrose Step Gradients
  • Basic Protocol 8: Isolation of Endoplasmic Reticulum Using Sucrose Step Gradients
  • Basic Protocol 9: Isolation of Plasma Membranes from Whole Yeast Cells Using Sucrose Step Gradients
  • Basic Protocol 10: Preparation of Cytosol from Whole Yeast Cells
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Fractionation of Spheroplasts by Differential Centrifugation

  Materials
  • Desired yeast cultures in their exponential growth
  • Stop solution or spheroplast medium B (see recipe; optional), ice cold
  • HEPES/potassium acetate (HEPES/KAc) lysis buffer (see recipe), ice cold
  • 500× protease inhibitor cocktails A and B (500× PIC‐A and 500× PIC‐B; see reciperecipes)
  • Protease inhibitor stock solutions A (see recipe)
  • Dounce glass homogenizer, tight‐fitting pestle, prechilled to 4°C
  • 1.6‐ml microcentrifuge tubes, prechilled to 4°C
  • Microcentrifuge prechilled to 4°C
  • 1.6‐ml polycarbonate tubes for ultracentrifugation at 100,000 × g, prechilled on ice
  • Beckman refrigerated tabletop ultracentrifuge (or equivalent) and rotor (e.g., Beckman TLA100.3 or equivalent)
  • Additional reagents and equipment for growing yeast cells (see unit 1.6) and preparation of spheroplasts (see protocol 2)

Support Protocol 1: Preparation of Yeast Spheroplasts Using Zymolyase

  • Exponentially growing yeast cell cultures in yeast extract/peptone/dextrose (YPD) medium (see unit 1.6) or appropriate medium
  • TSD reduction buffer (see recipe)
  • Spheroplast medium A (see recipe)
  • 5 mg/ml Zymolyase 100T (ICN Immunobiochemicals) in spheroplast medium A, (store in aliquots at −20°C)
  • Spheroplast medium B (see recipe), ice cold
  • 50‐ml glass culture tubes or 100‐ml Erlenmeyer flasks
  • Shaker platform at 30°C (or appropriate growth temperature of the strains being used)
  • 250‐ml centrifuge bottles
  • 50‐ml centrifuge tubes
  • Centrifuge and rotor (e.g., Sorvall GS‐3 rotor)
  • 30°C water bath
  • Additional reagents and equipment for culturing yeast cells (unit 1.6)

Basic Protocol 2: Equilibrium Density Gradient Fractionation Using Nycodenz

  Materials
  • 20 OD 600 units of cells in mid‐logarithmic growth phase per gradient
  • HEPES/potassium acetate (HEPES/KAc) lysis buffer (see recipe), ice cold
  • 50% (w/v) Nycodenz/sorbitol stock solution (see recipe)
  • 37%, 31%, 27%, 23%, 20%, 17%, 13%, and 9% (w/v) Nycodenz in HEPES/KAc lysis buffer (see recipe), 4°C
  • 500× protease inhibitor cocktails A and B (500× PIC‐A and 500× PIC‐B; see reciperecipes)
  • Protease inhibitor stock solution A (see recipe)
  • 100 mg/ml bovine serum albumin (BSA; optional)
  • Stop solution (see recipe) or another appropriate buffer, ice cold
  • Trimmed 1‐ml pipet tips (cut ∼5 to 10 mm from the tips to increase the size of the opening)
  • Clear 14 × 89–mm ultracentrifuge tubes (e.g., Ultraclear 14 × 89–mm tubes or equivalent) and rack
  • Centrifuge and microcentrifuge at 4°C
  • Dounce homogenizer with a tight‐fitting pestle, prechilled
  • 1.6‐ml microcentrifuge tubes, prechilled
  • Refrigerated ultracentrifuge with swinging bucket rotor (e.g., Beckman SW41 Ti or equivalent), 4°C
  • Refractometer
  • Additional reagents and equipment for growing yeast cells (unit 1.6) and preparation of spheroplasts ( protocol 2)

Alternate Protocol 1: Equilibrium Density Gradient Fractionation using Sucrose

  • 60% (w/v) sucrose stock solution, pH 6.8 (see recipe)
  • Sucrose solutions for equilibrium gradients (see recipe)
  • Refractometer

Basic Protocol 3: Fractionation of P13,000 Membranes on Sucrose Step Gradients

  Materials
  • Desired yeast strains (e.g., TVY614, a pep4 prb1 prc1 mutant yeast strain)
  • Stop solution or spheroplast medium B, ice cold (see recipe; or another buffer with the appropriate osmotic support)
  • HEPES/potassium acetate (HEPES/KAc) lysis buffer, ice cold (see recipe)
  • 500× protease inhibitor cocktails A and B (500× PIC‐A and 500× PIC‐B, see reciperecipes)
  • Protease inhibitor stock solution A (see recipe)
  • 1.2 and 1.5 M sucrose gradient solutions in sucrose gradient buffer (see recipe)
  • Ultra‐clear 11 × 34–mm centrifuge tubes (e.g., Beckman or equivalent)
  • Benchtop refrigerated centrifuge and swinging bucket rotor, 4°C (e.g., Beckman TLS55 or equivalent)
  • Dounce homogenizer with a tight‐fitting pestle, prechilled
  • 1.6‐ml microcentrifuge tubes
  • Microcentrifuge, 4°C
  • Trimmed 1‐ml and 200‐µl pipet tips (∼5 to 10 mm cut from the tips)
  • Additional reagents and equipment for growing yeast cells (unit 1.6) and preparation of spheroplasts ( protocol 2)

Basic Protocol 4: Isolation of Intact Vacuoles Using Ficoll Step Gradients

  Materials
  • Desired yeast strain
  • 0.2× YPD (unit 1.6)
  • PIPES/DTT buffer (see recipe)
  • Oxalyticase buffer (see recipe)
  • Oxalyticase (45,000 U/mg, Enzogenetics; 0.5 to 1.0 mg/gradient) or an alternate β‐glucanase enzyme (e.g., Zymolyase 100T; protocol 2)
  • 15%, 8%, 4%, and 0% (w/v) Ficoll solutions, 4°C (see recipe)
  • 50× protease inhibitor cocktail (PIC, see recipe)
  • 0.4 mg/ml dextran solution, freshly made (see recipe)
  • Protein assay reagent kit (e.g., BioRad; or an alternate technique, see appendix 3B)
  • 250‐ml and 2‐liter Erlenmeyer flasks
  • Shaker platform at 30°C (or appropriate growth temperature)
  • Centrifuge and rotor, room temperature and 4°C (e.g., Beckman JA‐20 and JA‐10 rotors or equivalents)
  • 500‐ml centrifuge bottles (e.g., for Beckman JA‐10 rotor or equivalent)
  • 10‐ml glass pipets
  • 30‐ml glass Corex tubes (1 tube per gradient) or equivalent glass centrifugation tubes
  • Water bath at 30°C
  • Adapters for 30‐ml glass Corex tubes (e.g., Beckman JA‐20 rotor or equivalent)
  • Polyallomer centrifuge tubes, prechilled (e.g., 14 × 89–mm for SW41 Ti rotor)
  • Ultracentrifuge and rotor, 4°C (e.g., Beckman SW41 Ti rotor or equivalent)
  • 200‐µl pipet tips with ∼5 to 8 mm trimmed off each tip
  • 1.6‐ml microcentrifuge tubes
  • Additional reagents and equipment for growing yeast cells (unit 1.6) and determining protein concentration ( appendix 3B)

Basic Protocol 5: Isolation of Intact Nuclei with Ficoll Step Gradients

  Materials
  • Desired yeast strain
  • YPD medium (unit 1.6; or appropriate growth medium)
  • Sterile H 2O, ice cold
  • Pretreatment buffer (see recipe)
  • 1.1 M sorbitol solution (see recipe), ice cold
  • Glusulase (NEN Life Science Products)
  • 10 mg/ml Zymolyase 100 T (or 100,000 U/g yeast lytic enzyme can also be used; ICN)
  • Spheroplast recovery medium (optional; see recipe)
  • 1000× protease inhibitor cocktail‐D (1000× PIC‐D; see recipe)
  • 1000× protease inhibitor cocktail‐W (1000× PIC‐W; see recipe)
  • Ficoll cushion solution (see recipe), 4°C
  • 20%, 30%, 40%, and 50% (w/v) Ficoll lysis solutions with 1× PIC (see recipe)
  • 1× and 2× PM buffer (see recipe), 4°C
  • PSM 1 solution (see recipe)
  • TE/SDS solution (see recipe; optional)
  • Shaker platform in incubator at 30°C (or appropriate permissive temperature)
  • 250‐ml and 2‐liter Erlenmeyer flasks
  • 250‐ml centrifuge bottles
  • Hemacytometer
  • Sorvall GSA rotor or equivalent; prechill to 4°C
  • 1.6‐ml microcentrifuge tubes
  • 30°C water bath
  • 50‐ml polycarbonate centrifuge tubes (e.g., Oakridge or equivalent)
  • Swinging bucket rotor (e.g., Sorvall HB‐4 rotor or equivalent), prechill to 4°C
  • Homogenizer (e.g., Potter‐Elvehjem or a 40‐ml glass Dounce homogenizer with a loose‐fitting pestle), prechilled
  • Beckman Ultra‐Clear 25 × 89–mm centrifuge tubes (or equivalent), prechill to 4°C
  • Beckman SW28 rotor (or equivalent), 2°C
  • 20‐ml syringes with 16‐G needles
  • Additional reagents and equipment for growing yeast cells (unit 1.6) and determining protein concentration ( appendix 3B)

Basic Protocol 6: Isolation of Lactate‐Induced Mitochondria Using Nycodenz Step Gradients

  Materials
  • D273‐10B cells (e.g., or desired yeast strain)
  • Semi‐synthetic lactate medium (see recipe)
  • TSD reduction buffer (see recipe)
  • Zymolyase 20T (e.g., ∼75 mg)
  • Buffer A: 1.2 M sorbitol/20 mM potassium phosphate, pH 7.4 (see appendix 2A for phosphate buffer), room temperature
  • Semi‐synthetic lactate medium (see recipe) supplemented with 1.2 M sorbitol (optional)
  • 200 mM phenylmethylsulfonyl fluoride (PMSF; 34.5 mg/ml) in absolute ethanol; prepare fresh
  • Buffer B: 0.6 M sorbitol/20 mM potassium MES (pH 6.0), ice cold
  • 0.6% (w/v) SDS solution
  • 2× Buffer B (see recipe)
  • Buffer C: 0.6 M sorbitol/20 mM potassium HEPES (pH 7.4), ice cold
  • 18% and 14.5 % (w/v) Nycodenz solution (see recipe), prechilled
  • 100 mg/ml fatty‐acid free bovine serum albumin (BSA)
  • 200‐ml Erlenmeyer flask
  • Platform shaker, 30°C (or permissive growth temperature)
  • Sorvall GS‐3 rotor (or equivalent)
  • 250‐ml centrifuge bottles (e.g., Sorvall GS‐3 rotor or equivalent)
  • 30°C water bath
  • 1.6‐ml microcentrifuge tubes
  • 40‐ and 1‐ml glass Dounce homogenizers with a tight‐fitting pestles (or Teflon homogenizer), 4°C
  • 40‐ml centrifuge tubes (e.g., for Sorvall SS‐34 rotor or equivalent)
  • Sorvall SS‐34 rotor (or equivalent)
  • Clear ultracentrifuge tubes (e.g., Beckman SW‐41 14 × 89–mm Ultra‐Clear centrifuge tubes)
  • Beckman ultracentrifuge with a SW‐41 rotor (or equivalent)
  • Cut 1‐ml pipet tips
  • Additional reagents and equipment for growing yeast cells (unit 1.6) and determining protein concentration ( appendix 3B)

Basic Protocol 7: Isolation of Oleate‐Induced Peroxisomes Using Sucrose Step Gradients

  Materials
  • Rich growth medium (see recipe)
  • Yeast strain D273‐10B (or another strain of interest)
  • Peroxisome induction medium (see recipe)
  • TSD reduction buffer (see recipe)
  • 1.2 M sorbitol/phosphate solution (see recipe)
  • 100,000 U/g Zymolyase 100T (ICN Biochemicals)
  • 1.2 M sorbitol in MES buffer (see recipe for buffer), 4°C
  • MES buffer (see recipe), 4°C
  • 0.65 M sorbitol/MES solution (see recipe), ice cold
  • 20%, 30%, 40%, 44%, 46%, and 60% (w/w) sucrose/MES solution: ultrapure sucrose in MES buffer (see recipe), 4°C
  • 100‐ml and 2‐liter flasks
  • 50‐ml centrifuge tubes
  • 250‐ml centrifuge bottles
  • 1.6‐ml microcentrifuge tubes
  • Sorvall GS‐3 rotor
  • Centrifuge and Sorvall SS‐34 rotor (or equivalent)
  • Dounce homogenizer with a loose‐fitting pestle (optional), chilled
  • Beckman polyallomer Quick‐seal tubes (25 × 89–mm) or equivalent
  • Vertical rotor (Beckman VTi 50 or equivalent), 4°C
  • 30°C water bath
  • Syringe and wide‐gauge needle
  • Additional reagents and equipment for growing yeast cells (unit 1.6) and determining protein concentration ( appendix 3B)

Basic Protocol 8: Isolation of Endoplasmic Reticulum Using Sucrose Step Gradients

  Materials
  • Desired yeast strain (∼5000 OD 600 units of cells in exponential growth phase)
  • HEPES lysis buffer (see recipe), 4°C
  • 1 M DTT
  • Protease inhibitor stock solutions B (see recipe)
  • 1.5 M and 1.2 M sucrose/HEPES solution (see recipe)
  • 250‐ml centrifugation bottles
  • Motor‐driven Potter‐Elvehjem homogenizer (or a glass Dounce homogenizer)
  • 1.6‐ml centrifuge tubes
  • 1.6‐ml polycarbonate ultracentrifuge microcentrifuge tubes, prechilled
  • Sorvall GS‐3 rotor
  • Trimmed 1‐ml and 200‐µl pipet tips (i.e., ∼5 to 10 mm cut from the tips)
  • 4‐ml Dounce homogenizer, prechilled
  • Beckman Ultraclear 11 × 34–mm centrifuge tubes (or equivalent), prechilled
  • Swinging bucket rotor (e.g., Beckman SW 50.1 or equivalent), 4°C
  • Additional reagents and equipment for growing yeast cells (unit 1.6), spheroplast preparation (see protocol 2), and Lowry assay for proteins ( appendix 3B)

Basic Protocol 9: Isolation of Plasma Membranes from Whole Yeast Cells Using Sucrose Step Gradients

  Materials
  • 1‐liter culture of desired yeast cells in exponential growth phase
  • 0.4 M, 1.1 M, 1.65 M, 2.25 M sucrose/imidazole solutions (see recipe), 4°C
  • Protease inhibitor stock solutions C (see recipe)
  • Breakage buffer (see recipe), 4°C
  • Plasma membrane storage buffer (optional; see recipe)
  • 250‐ml centrifuge bottles
  • 50‐ml polycarbonate centrifuge tubes
  • Acid‐washed glass beads (0.45‐mm diameter; see recipe), prechilled to 4°C
  • Sorvall GS‐3 rotor
  • Sorvall SS‐34 rotor (or equivalent) and appropriate centrifugation tubes, 4°C
  • 14 × 89–mm Beckman Ultra‐clear centrifugation tubes
  • Beckman SW41 or SW40Ti rotor (or equivalent), 4°C
  • Beckman 50 Ti rotor (or equivalent)
  • Additional reagents and equipment for growing yeast cells (unit 1.6) and culture preparation ( protocol 2)

Basic Protocol 10: Preparation of Cytosol from Whole Yeast Cells

  Materials
  • Desired yeast strains
  • YPD (unit 1.6 or appropriate growth medium)
  • PIPES lysis buffer (see recipe), prechilled
  • 50× protease inhibitor cocktail (50× PIC; see recipe) or equivalent
  • Acid‐washed glass beads (∼0.45‐mm diameter; BDH or Sigma; see recipe), prechilled to 4°C
  • Methylene blue
  • BioRad protein assay reagent kit (or equivalent)
  • 250‐ml and 2‐liter Erlenmeyer flasks
  • Platform shaker, 30°C (or appropriate permissive temperature)
  • Centrifuge rotors (e.g., Beckman JA‐10 and JA‐20 rotors or equivalents)
  • 500‐ml centrifuge bottles
  • 30‐ml glass Corex tubes (1 tube per cytosol preparation), prechilled
  • Ice‐water bath
  • Adapters for 30‐ml glass Corex tubes (for Beckman JA‐20 rotor or equivalent)
  • Ultracentrifuge tubes, 4°C
  • Ultracentrifuge rotor (Beckman TLA 100.2 rotor or equivalent)
  • Tabletop ultracentrifuge, 4°C
  • Microcentrifuge tubes, prechilled
  • Additional reagents and equipment for growing yeast cells (unit 1.6) and determining protein concentration ( appendix 3B)
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Literature Cited

Literature Cited
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   Aris, J.P. and Blobel, G. 1991. Isolation of yeast nuclei. Methods Enzymol. 194:735‐749.
   Becherer, K.A., Rieder, S.E., Emr, S.D., and Jones, E.W. 1996. Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome‐like vacuole in yeast. Mol. Biol. Cell 7:579‐594.
   Bowser, R. and Novick, P. 1991. Sec15 protein, an essential component of the exocytotic apparatus, is associated with the plasma membrane and with a soluble 19.5S particle. J. Cell Biol. 112:1117‐1131.
   Daum, G., Bohni, P.C., and Schatz, G. 1982. 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.
   Distel, B., van der Leij, I., and Kos, W. 1996. Peroxisome isolation. Methods Mol. Biol. 53:133‐138.
   Dobrota, M. and Hinton, R. 1992. Conditions for density gradient separations. In Preparative Centrifugation: A Practical Approach (D. Rickwood, ed.) pp. 77‐137. Oxford‐IRL Press, New York.
   Dove, J.E., Brockenbrough, J.S., and Aris, J.P. 1998. Isolation of nuclei and nucleoli from the yeast Saccharomyces cerevisiae. Methods Cell Biol. 53:33‐46.
   Erdmann, R. and Blobel, G. 1995. Giant peroxisomes in oleic acid‐induced Saccharomyces cerevisiae lacking the peroxisomal membrane protein Pmp27p. J. Cell Biol. 128:509‐523.
   Evans, W.H. 1992. Isolation and characterization of membranes and cell organelles. In Preparative Centrifugation: A Practical Approach (D. Rickwood, ed.) pp. 233‐270. Oxford‐IRL Press, New York.
   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. 260:213‐223.
   Haas, A. 1995. A quantitative assay to measure homotypic vacuole fusion in vitro. Methods Cell Sci. 17:283‐294.
   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. Oxford: IRL Press, New York.
   Kalinich, J.F. and Douglas, M.G. 1989. In vitro translocation through the yeast nuclear envelope. Signal‐dependent transport requires ATP and calcium. J. Biol. Chem. 264:17979‐17989.
   Kunau, W.H., Beyer, A., Franken, T., Gotte, K., Marzioch, M., Saidowsky, J., Skaletz‐Rorowski, A., and Wiebel, F.F. 1993. Two complementary approaches to study peroxisome biogenesis in Saccharomyces cerevisiae: Forward and reversed genetics. Biochimie 75:209‐224.
   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.
   Panaretou, B. and Piper, P. 1996. Isolation of yeast plasma membranes. Methods Mol. Biol. 53:117‐121.
   Pon, L., Moll, T., Vestweber, D., Marshallsay, B., and Schatz, G. 1989. Protein import into mitochondria: ATP‐dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins. J. Cell Biol. 109:2603‐2016.
   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.
   Roberts, C.J., Raymond, C.K., Yamashiro, C.T., and Stevens, T.H. 1991. Methods for studying the yeast vacuole. Methods Enzymol. 194:644‐661.
   Sanderson, C.M. and Meyer, D.I. 1991. Purification and functional characterization of membranes derived from the rough endoplasmic reticulum of Saccharomyces cerevisiae. J. Biol. Chem. 266:13423‐13430.
   Scott, J.H. and Schekman, R. 1980. Lyticase: Endoglucanase and protease activities that act together in yeast cell lysis. J.Bacteriol. 142:414‐423.
   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.
   Thieringer, R. and Kunau, W.H. 1991. The beta‐oxidation system in catalase‐free microbodies of the filamentous fungus Neurospora crassa. Purification of a multifunctional protein possessing 2‐enoyl‐CoA hydratase, L‐3‐hydroxyacyl‐CoA dehydrogenase, and 3‐hydroxyacyl‐CoA epimerase activities. J. Biol. Chem. 266:13110‐13117.
   Uchida, E., Ohsumi, Y., and Anraku, Y. 1988. Purification of yeast vacuolar membrane H+‐ATPase and enzymological discrimination of three ATP‐driven proton pumps in Saccharomyces cerevisiae. Methods Enzymol. 157:544‐562.
   Veenhuis, M., Mateblowski, M., Kunau, W.H., and Harder, W. 1987. Proliferation of microbodies in Saccharomyces cerevisiae. Yeast 3:77‐84.
   Vida, T.A. and Emr, S.D. 1995. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128:779‐792.
   Walworth, N.C., Goud, B., Ruohola, H., and Novick, P.J. 1989. Fractionation of yeast organelles. Methods Cell Biol. 31:335‐356.
   Wuestehube, L.J. and Schekman, R.W. 1992. Reconstitution of transport from endoplasmic reticulum to Golgi complex using endoplasmic reticulum‐enriched membrane fraction from yeast. Methods Enzymol. 219:124‐136.
   Yaffe, M.P. 1991. Analysis of mitochondrial function and assembly. Methods Enzymol. 194:627‐643.
   Ziman, M., Chuang, J.S., and Schekman, R.W. 1996. Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway. Mol. Biol. Cell 7:1909‐1919.
   Zinser, E. and Daum, G. 1995. Isolation and biochemical characterization of organelles from the yeast, Saccharomyces cerevisiae. Yeast 11:493‐536.
Key References
   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://www.proteome.com/
  The Yeast Protein Database (Proteome) 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, size).
   http://genome-www.stanford.edu/Saccharomyces/
  Saccharomyces Genome Database (SGD) provides access to the complete sequence of the S. cerevisiae genome, the confirmed and predicted open reading frames (ORFs), protein information, and a number of useful internet links.
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