Proteasomes: Isolation and Activity Assays

Yanjie Li1, Robert J. Tomko2, Mark Hochstrasser1

1 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, 2 Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.43
DOI:  10.1002/0471143030.cb0343s67
Online Posting Date:  June, 2015
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Abstract

In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin‐proteasome system. In this system, the protein substrate is often first covalently modified with a chain of ubiquitin polypeptides. This chain serves as a signal for delivery to the 26S proteasome, a 2.5‐MDa, ATP‐dependent multisubunit protease complex. The proteasome consists of a barrel‐shaped 20S core particle (CP) that is capped on one or both of its ends by a 19S regulatory particle (RP). The RP is responsible for recognizing the substrate, unfolding it, and translocating it into the CP for destruction. Here we describe simple, one‐step purifications scheme for isolating the 26S proteasome and its 19S RP and 20S CP subcomplexes from the yeast Saccharomyces cerevisiae, as well as assays for measuring ubiquitin‐dependent and ubiquitin‐independent proteolytic activity in vitro. © 2015 by John Wiley & Sons, Inc.

Keywords: proteasome; ubiquitin; ATPase; purification; proteolytic activity

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Purification of Active 26S Proteasomes
  • Support Protocol 1: Growth of Yeast Strains and Production of Yeast Cell Powder
  • Support Protocol 2: Regeneration of Anti‐FLAG M2 Affinity Gel
  • Basic Protocol 2: Purification of the 19S Regulatory Particle (RP)
  • Basic Protocol 3: Purification of Active 20S CP
  • Basic Protocol 4: In‐Gel Peptidase Activity Assay for 20S CP and 26S Proteasomes
  • Basic Protocol 5: In‐Solution Peptidase Activity Assay for 20S and 26S Proteasomes
  • Basic Protocol 6: Measuring Degradation of Polyubiquitinated Sic1PY
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Purification of Active 26S Proteasomes

  Materials
  • Cell powder from Saccharomyces cerevisiae strain MHY5841 (RPN11‐6 × Gly‐3 × FLAG:kanMX6; Hochstrasser lab strain) or similar
  • Buffer A (see recipe)
  • 10× ATP regenerating system (see recipe)
  • 500 mM ATP stock (see recipe)
  • BCA Assay Kit (Pierce, cat. no. 23227)
  • Anti‐FLAG M2 affinity gel (Sigma, cat. no. A2220)
  • 3 × FLAG peptide (Sigma, cat. no. F4799)
  • Bovine serum albumin to prepare SDS‐PAGE standards
  • Gelcode Blue stain (Thermo Scientific, cat. no. 24592)
  • Liquid nitrogen
  • 40‐ml high‐speed centrifuge tubes, pre‐cooled on ice
  • High‐speed refrigerated centrifuge and rotor
  • 50‐ml disposable polypropylene conical centrifuge tubes (e.g., BD Falcon)
  • End‐over‐end rotator or Nutator
  • Refrigerated benchtop centrifuge
  • Vivaspin 500 centrifugal concentrator (MWCO 100‐kDa; Sartorius, cat. no. VS0141)
  • G‐Box (Syngene) or similar gel documentation system
  • Additional reagents and equipment for SDS‐PAGE [see unit 6.1; Gallagher ( ) and Table 3.43.1]
Table 3.3.1   MaterialsRecipe to Make Two 12% 1‐mm SDS‐PAGE Gels

Resolving gel, 12% acrylamide Stacking gel, 4% acrylamide
Solution [Stock] For 15 ml Solution [Stock] For 5 ml
H 2O 4.23 ml H 2O 3.541 ml
Tris·Cl, pH 8.8 1.5 M 3.75 ml Tris·Cl, pH 6.8 1 M 625 μl
Acrylamide 40% 4.38 ml Acrylamide 40% 487 μl
Bis‐acrylamide 2% 2.4 ml Bis‐acrylamide 2% 267 μl
SDS 20% 75 μl SDS 20% 25 μl
APS 10% 150 μl APS 10% 50 μl
TEMED 15 μl TEMED 5 μl

Support Protocol 1: Growth of Yeast Strains and Production of Yeast Cell Powder

  Materials
  • Saccharomyces cerevisiae strain MHY5841 (RPN11‐6 × Gly‐3 × FLAG:kanMX6; Hochstrasser lab strain), MHY6952 (PRE1‐6 × Gly‐3 × FLAG:kanMX6; Hochstrasser lab strain) or similar
  • YPD agar plate (see recipe)
  • YPD liquid medium (see recipe)
  • Liquid nitrogen
  • 30°C incubator with shaker
  • 50‐ml disposable polypropylene conical centrifuge tubes (e.g., BD Falcon)
  • 4‐liter Erlenmeyer flasks
  • Large (∼500 ml) and small (∼40 ml) high‐speed centrifuge tubes
  • High‐speed refrigerated centrifuge and rotor
  • Bench‐top centrifuge, 4°C
  • Ceramic mortar, 80 mm × 130 mm (VWR, cat. no. 89038‐152 or equivalent)
  • Ceramic pestle, 194 mm (VWR, cat. no. 89038‐168 or equivalent)
  • Hammer
  • Scoopula or spoon

Support Protocol 2: Regeneration of Anti‐FLAG M2 Affinity Gel

  Materials
  • Used anti‐FLAG M2 affinity gel (Basic Protocol protocol 11, protocol 42, or protocol 53)
  • Buffer A (see recipe)
  • 0.1 M glycine, pH 3.5
  • 1% (w/v) NaN 3 in deionized H 2O
  • 50‐ml disposable polypropylene conical centrifuge tubes (e.g., BD Falcon)
  • pH strips

Basic Protocol 2: Purification of the 19S Regulatory Particle (RP)

  Materials
  • Cell powder from Saccharomyces cerevisiae strain MHY5841 (RPN11‐6 × Gly‐3 × FLAG:kanMX6; Hochstrasser lab strain) or similar
  • Buffer A500 (see recipe)
  • Buffer A (see recipe)
  • 10× ATP regenerating system (see recipe)
  • 500 mM ATP stock (see recipe)
  • BCA Assay Kit (Pierce, cat. no. 23227)
  • Anti‐FLAG M2 affinity gel (Sigma, cat. no. A2220)
  • 3 × FLAG peptide (Sigma, cat. no. F4799)
  • Bovine serum albumin to prepare SDS‐PAGE standards
  • Gelcode Blue stain (Thermo Scientific, cat. no. 24592)
  • Liquid nitrogen
  • 40‐ml high‐speed centrifuge tubes
  • High‐speed refrigerated centrifuge and rotor
  • 50‐ml disposable polypropylene conical centrifuge tubes
  • End‐over‐end rotator or Nutator
  • Refrigerated benchtop centrifuge
  • Vivaspin 500 centrifugal concentrator (100 kDa molecular weight cutoff; Sartorius, cat. no. VS0141)
  • G‐Box (Syngene) or similar gel documentation system
  • Additional reagents and equipment for SDS‐PAGE [see unit 6.1; Gallagher ( ) and Table 3.43.1]

Basic Protocol 3: Purification of Active 20S CP

  Materials
  • Cell powder from Saccharomyces cerevisiae strain MHY6952 (PRE1‐6 × Gly‐3 × FLAG:kanMX6; Hochstrasser lab strain) or similar
  • 20S lysis buffer (see recipe)
  • 20S washing buffer (see recipe)
  • Anti‐FLAG M2 affinity gel (Sigma, cat. no. A2220)
  • BCA Assay Kit (Pierce, cat. no. 23227)
  • Buffer A (see recipe)
  • 3 × FLAG peptide (Sigma, cat. no. F4799)
  • Gelcode Blue stain (Thermo Scientific, cat. no. 24592)
  • Liquid nitrogen
  • 40‐ml high‐speed centrifuge tubes
  • High‐speed refrigerated centrifuge and rotor
  • 50‐ml disposable polypropylene conical centrifuge tubes
  • End‐over‐end rotator or Nutator
  • Refrigerated benchtop centrifuge
  • Vivaspin 500 centrifugal concentrator (MWCO 100‐kDa; Sartorius, cat. no. VS0141)
  • G‐Box (Syngene) or similar gel documentation system
  • Additional reagents and equipment for SDS‐PAGE [see unit 6.1; Gallagher ( ) and Table 3.43.1]

Basic Protocol 4: In‐Gel Peptidase Activity Assay for 20S CP and 26S Proteasomes

  Materials
  • 0.9 M Tris‐Boric acid buffer, pH 8.3 (see recipe)
  • 1 M MgCl 2 (see recipe)
  • 500 mM ATP stock (see recipe)
  • 25% (w/v) sucrose
  • 40% (w/v) acrylamide (BioRad, cat. no. 161‐0140)
  • 2% bis‐acrylamide (BioRad, cat. no. 161‐0142)
  • 20% (w/v) sodium dodecyl sulfate (SDS) in deionized H 2O
  • 10% (w/v) ammonium persulfate (APS) in deionized H 2O
  • N,N,N’,N’‐tetramethylethylenediamine (TEMED; Sigma, cat. no. T7024)
  • 1 μM purified 20S CP (see protocol 5) and/or 26S (see protocol 1) proteasomes
  • Buffer A (see recipe)
  • 5× native gel loading buffer (see recipe)
  • Developing buffer (see recipe)
  • Mini gel electrophoresis system (BioRad) or similar
  • Gel releasers (BioRad, cat. no. 165‐3320), or similar tools for the system of choice
  • Gel tray (GenHunter, cat. no. B107 or similar)
  • 30°C shaker
  • G‐Box (Syngene) or similar gel‐documentation system

Basic Protocol 5: In‐Solution Peptidase Activity Assay for 20S and 26S Proteasomes

  Materials
  • 1 μM purified 20S CP (see protocol 5) and/or 26S (see protocol 1) proteasomes
  • Developing buffer (see recipe)
  • 1% (w/v) SDS in deionized H 2O

Basic Protocol 6: Measuring Degradation of Polyubiquitinated Sic1PY

  Materials
  • Buffer A (see recipe)
  • 10 × ATP regenerating system (see recipe)
  • 10 mM dithiothreitol (DTT) in deionized H 2O
  • 20 mM ATP (prepare from 500 mM ATP stock; see recipe)
  • 0.5 μM polyubiquitinated T7‐Sic1PY
  • 1 μM purified 26S proteasomes ( protocol 1)
  • Anti‐T7 antibody (EMD Millipore, cat. no. 69522)
  • Tris‐buffered saline (TBS; appendix 2A)/0.1% Tween‐20 (TBS‐T) with or without blocking protein (e.g., 5% nonfat milk)
  • 5× SDS loading buffer (see recipe)
  • Temperature‐controlled heat block or water bath set to 30°C
  • Temperature‐controlled heat block or water bath, set to 100°C
  • Mini gel electrophoresis system (BioRad) or similar (also see unit 6.1; Gallagher, )
  • Electroblotting apparatus (BioRad) or a similar electroblotting system (also see unit 6.2; Gallagher et al., )
  • Chemiluminescence imaging equipment (G‐Box, Syngene) or a similar imaging system
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1; Gallagher, ) and immunoblotting (unit 6.2; Gallagher et al., )
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Figures

Videos

Literature Cited

Literature Cited
  Ben‐Nissan, G. and Sharon, M. 2014. Regulating the 20S proteasome ubiquitin‐independent degradation pathway. Biomolecules 4:862‐884.
  Erales, J. and Coffino, P. 2014. Ubiquitin‐independent proteasomal degradation. BBA‐Mol. Cell Res. 1843:216‐221.
  Gallagher, S.R. 2007. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Cell Biol. 37:6.1.1‐6.1.38.
  Gallagher, S., Winston, S.E., Fuller, S A., and Hurrell, J.G. 2011. Immunoblotting and immunodetection. Curr. Protoc. Cell Biol. 52:6.2.1‐6.2.28.
  Glickman, M. and Coux, O. 2001. Purification and characterization of proteasomes from Saccharomyces cerevisiae. Curr. Protoc. Protein Sci. 24:21.5.1‐21.5.17.
  Goldberg, A.L. 2003. Protein degradation and protection against misfolded or damaged proteins. Nature 426:895‐899.
  Leggett, D.S., Glickman, M.H., and Finley, D. 2005. Purification of proteasomes, proteasome subcomplexes, and proteasome‐associated proteins from budding yeast. Methods Mol. Biol. 301:57‐70.
  Saeki, Y., Isono, E., and Toh, E.A. 2005. Preparation of ubiquitinated substrates by the PY motif‐insertion method for monitoring 26S proteasome activity. Methods Enzymol. 399:215‐227.
  Schmidt, M. and Finley, D. 2014. Regulation of proteasome activity in health and disease. BBA‐Mol. Cell Res. 1843:13‐25.
  Tomko, R.J. and Hochstrasser, M. 2013. Molecular architecture and assembly of the eukaryotic proteasome. Annu. Rev. Biochem. 82:415‐445.
  Zhang, J., Wu, P., and Hu, Y. 2013. Clinical and marketed proteasome inhibitors for cancer treatment. Curr. Med. Chem. 20:2537‐2551.
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