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

  1. Yoshihiro Matsumura,
  2. William R Skach

Published Online: 15 DEC 2009

DOI: 10.1002/9780470015902.a0003430.pub2

eLS

eLS

How to Cite

Matsumura, Y. and Skach, W. R. 2009. Protein Export from Endoplasmic Reticulum to the Cytosol: In Vitro Methods. eLS. .

Author Information

  1. Oregon Health and Science University, Portland, Oregon, USA

Publication History

  1. Published Online: 15 DEC 2009

Abstract

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