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


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.



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Ravid T and Hochstrasser M (2008) Diversity of degradation signals in the ubiquitin‐proteasome system. Nature Reviews. Molecular Cell Biology 9: 679–690.

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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. [doi: 10.1002/9780470015902.a0003430.pub2]