Protein Export from Endoplasmic Reticulum to the Cytosol: In Vitro Methods

Yoshihiro Matsumura, Oregon Health and Science University, Portland, Oregon, USA
William R Skach, Oregon Health and Science University, Portland, Oregon, USA

Published online: December 2009

DOI: 10.1002/9780470015902.a0003430.pub2

Newly synthesized proteins that fail to achieve their proper conformation in the endoplasmic reticulum (ER) are recognized by quality control machinery and degraded by the ubiquitin–proteasome pathway. This process is termed ER-associated degradation (ERAD) and is implicated in a large number of inherited and acquired human diseases characterized by aberrant protein folding. Because ubiquitin, ubiquitination machinery and proteasomes are restricted to the cytosol and nucleus, ERAD substrates must be exported from the ER lumen and/or extracted from the ER membrane before or during degradation. In vitro systems that reconstitute ERAD allow detailed mechanisms of ERAD to be studied independently of ongoing protein synthesis and compensatory feedback mechanisms present in cells. Evidence indicates that retrotranslocation (or dislocation) is tightly regulated via parallel pathways that are adapted to various substrates, molecular lesions and cellular needs and stresses.

Key concepts:

  • The endoplasmic reticulum (ER) utilizes a stringent quality control machinery for secretory and transmembrane proteins.
  • Misfolded proteins are recognized by the ER quality control components and degraded by the ubiquitin–proteasome pathway.
  • ER-associated degradation (ERAD) is a multistep process.
  • Molecular chaperones and lectin-like proteins play key roles in recognizing misfolded proteins.
  • Ubiquitin ligases modify ERAD substrates by covalent attachment of ubiquitin.
  • Two ATPase associated with various cellular activities, p97 and the 19S proteasome subunit, facilitate retrotranslocation of ERAD substrates and delivery to the 20S proteasome.
  • In vitro systems reconstitute complex ERAD processes.
  • Mechanistic details of ERAD are readily amenable to in vitro manipulation and analysis.

Keywords: ERAD; cystic fibrosis; CFTR; protein folding disorders; transmembrane protein; ubiquitin-proteasome system

Figure 1. Components involved in endoplasmic reticulum (ER)-associated degradation (ERAD) and their cellular locations. In the ER lumen, molecular chaperones, Grp94, BiP and PDI, the lectin-like proteins, calnexin (CNX) and calreticulin (CRT), EDEM, OS9 and valosin-containing protein (VCP/p97)-interacting membrane protein (VIMP), play major roles in the substrate recognition. In the cytosol, molecular chaperones, Hsp90 and Hsp70, and their cochaperones Hsp40 are involved in the substrate recognition. Hrd1 and RMA1 are transmembrane E3 ubiquitin ligases, whereas CHIP is a cytosolic ligase. The Sec61 complex and Derlin proteins are proposed to be a component of retrotranslocation channel. The 19S proteasome regulatory complex and p97 mediate substrate unfolding and extraction from the membrane. The PA28 complex, an alternate proteasome cap, also binds to the degradation intermediates. Substrates are finally degraded into peptide fragment by the 20S proteasome catalytic complex.
close
 References
    Bercovich B, Stancovski I, Mayer A et al. (1997) Ubiquitin-dependent degradation of certain protein substrates in vitro requires the molecular chaperone Hsc70. Journal of Biological Chemistry 272: 9002–9010.
    Carlson E, Bays N, David L and Skach WR (2005) Reticulocyte lysate as a model system to study endoplasmic reticulum membrane protein degradation. Methods in Molecular Biology 301: 185–205.
    Carlson EJ, Pitonzo D and Skach WR (2006) p97 functions as an auxiliary factor to facilitate TM domain extraction during CFTR ER-associated degradation. EMBO Journal 25: 4557–4566.
    Carvalho P, Goder V and Rapoport TA (2006) Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins. Cell 126: 361–373.
    Connell P, Ballinger CA, Jiang J et al. (2001) The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins. Nature Cell Biology 3: 93–96.
    Denic V, Quan EM and Weissman JS (2006) A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation. Cell 126: 571–582.
    Gusarova V, Caplan AJ, Brodsky JL and Fisher EA (2001) Apoprotein B degradation is promoted by the molecular chaperones hsp90 and hsp70. Journal of Biological Chemistry 276: 24891–24900.
    Hrizo SL, Gusarova V, Habiel DM et al. (2007) The Hsp110 molecular chaperone stabilizes apolipoprotein B from endoplasmic reticulum-associated degradation (ERAD). Journal of Biological Chemistry 282: 32665–32675.
    Lee RJ, Liu CW, Harty C et al. (2004) Uncoupling retro-translocation and degradation in the ER-associated degradation of a soluble protein. EMBO Journal 23: 2206–2215.
    Lilley BN and Ploegh HL (2004) A membrane protein required for dislocation of misfolded proteins from the ER. Nature 429: 834–840.
    Mayer TU, Braun T and Jentsch S (1998) Role of the proteasome in membrane extraction of a short-lived ER-transmembrane protein. EMBO Journal 17: 3251–3257.
    McCracken AA and Brodsky JL (1996) Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP. Journal of Cell Biology 132: 291–298.
    Meacham GC, Patterson C, Zhang W, Younger JM and Cyr DM (2001) The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation. Nature Cell Biology 3: 100–105.
    Meusser B, Hirsch C, Jarosch E and Sommer T (2005) ERAD: the long road to destruction. Nature Cell Biology 7: 766–772.
    Oberdorf J, Carlson EJ and Skach WR (2001) Redundancy of mammalian proteasome subunit function during endoplasmic reticulum associated degradation. Biochemistry 40: 13397–13405.
    Oberdorf J, Carlson EJ and Skach WR (2006) Uncoupling proteasome peptidase and ATPase activities results in cytosolic release of an ER polytopic protein. Journal Cell Science 119: 303–313.
    Pickart CM and Cohen RE (2004) Proteasomes and their kin: proteases in the machine age. Nature Reviews. Molecular Cell Biology 5: 177–187.
    Shibatani T, Carlson EJ, Larabee F et al. (2006) Global organization and function of mammalian cytosolic proteasome pools: implications for PA28 and 19S regulatory complexes. Molecular Biology of the Cell 17: 4962–4971.
    Vashist S and Ng DT (2004) Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control. Journal of Cell Biology 165: 41–52.
    Vembar SS and Brodsky JL (2008) One step at a time: endoplasmic reticulum-associated degradation. Nature Reviews. Molecular Cell Biology 9: 944–957.
    Wahlman J, DeMartino GN, Skach WR et al. (2007) Real-time fluorescence detection of ERAD substrate retrotranslocation in a mammalian in vitro system. Cell 129: 943–955.
    Wiertz EJ, Tortorella D, Bogyo M et al. (1996) Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction. Nature 384: 432–438.
    Xiong X, Chong E and Skach WR (1999) Evidence that endoplasmic reticulum (ER)-associated degradation of cystic fibrosis transmembrane conductance regulator is linked to retrograde translocation from the ER membrane. Journal of Biological Chemistry 274: 2616–2624.
    Ye Y, Meyer HH and Rapoport TA (2001) The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature 414: 652–656.
    Ye Y, Shibata Y, Yun C, Ron D and Rapoport TA (2004) A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol. Nature 429: 841–847.
    Youker RT, Walsh P, Beilharz T, Lithgow T and Brodsky JL (2004) Distinct roles for the Hsp40 and Hsp90 molecular chaperones during cystic fibrosis transmembrane conductance regulator degradation in yeast. Molecular Biology of the Cell 15: 4787–4797.
    Younger JM, Chen L, Ren HY et al. (2006) Sequential quality-control checkpoints triage misfolded cystic fibrosis transmembrane conductance regulator. Cell 126: 571–582.
    Zhang Y, Nijbroek G, Sullivan ML et al. (2001) Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast. Molecular Biology of the Cell 12: 1303–1314.
 Further Reading
    Elsasser S and Finley D (2005) Delivery of ubiquitinated substrates to protein-unfolding machines. Nature Cell Biology 7: 742–749.
    Hirsch C, Gauss R, Horn SC, Neuber O and Sommer T (2009) The ubiquitylation machinery of the endoplasmic reticulum. Nature 458: 453–460.
    Nakatsukasa K and Brodsky JL (2008) The recognition and retrotranslocation of misfolded proteins from the endoplasmic reticulum. Traffic 9: 861–870.
    Nakatsukasa K, Huyer G, Michaelis S and Brodsky JL (2008) Dissecting the ER-associated degradation of a misfolded polytopic membrane protein. Cell 132: 101–112.
    Raasi S and Wolf DH (2007) Ubiquitin receptors and ERAD: a network of pathways to the proteasome. Seminars in Cell & Developmental Biology 18: 780–791.
    Ravid T and Hochstrasser M (2008) Diversity of degradation signals in the ubiquitin-proteasome system. Nature Reviews. Molecular Cell Biology 9: 679–690.

Related Sub-Topics:

Cellular Transport | Basic Cell Biology, Protein, RNA and DNA | Cell Compartments and Cellular Organelles | Techniques in Cell Biology | Protein Degradation | Biochemical and Biophysical Techniques | Proteins: Structure, Function, Metabolism | Molecular Transport
Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

Article Title: Protein Export from Endoplasmic Reticulum to the Cytosol: In Vitro Methods
 
How to Cite close
Matsumura, Yoshihiro, and Skach, William R(Dec 2009) Protein Export from Endoplasmic Reticulum to the Cytosol: In Vitro Methods. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003430.pub2]