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

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.

Role of Hsc70 binding cycle in CFTR folding and endoplasmic reticulum–associated degradation

“…Under normal physiological conditions, CFTR dislocation and proteolysis are tightly coupled, and CFTR is released from the ER membrane as TCA-soluble fragments ( Xiong et al ., 1999 ; Oberdorf et al ., 2006 ), because proteolytic capacity of the proteasome exceeds the ability of AAA-ATPases, p97 (VCP/CDC48) and the 19S proteasome regulatory subunit, to unfold and extract CFTR from the lipid bilayer ( Ye et al ., 2001 ; Lee et al ., 2004 ; Carlson et al ., 2006 ; Oberdorf et al ., 2006 ; Matsumura and Skach, 2009 ). Inhibition of β-subunit activity, however, uncouples extraction from proteolysis and allows one to measure retrotranslocation independently of peptide cleavage.…”

“…RRL lacks endogenous transcriptional mechanisms but maintains a native-like proteostatic environment with a complete complement of biosynthetic machinery ( Nimmesgern and Hartl, 1993 ; Frydman et al ., 1994 ) and a robust ubiquitin–proteasome system that reflects the terminally differentiated state of reticulocytes ( Xiong et al ., 1999 ; Oberdorf and Skach, 2002; Matsumura and Skach, 2009 ). Thus it provides the unique ability to 1) manipulate (co)chaperones without inducing compensatory feedback mechanisms; 2) analyze stoichiometry of (co)chaperones; 3) monitor specific steps of membrane integration, ubiquitination, dislocation, and proteolysis; and 4) directly evaluate substrate fate by both SDS–PAGE and conversion to TCA-soluble peptides ( Frydman et al ., 1994 ; Terada et al ., 1997 ; Carlson et al ., 2006 ; Matsumura and Skach, 2009 ). Most importantly, our conditions allow the effects of chaperone function during synthesis to be analyzed independently of degradation processes.…”

Endoplasmic Reticulum Protein Quality Control Is Determined by Cooperative Interactions between Hsp/c70 Protein and the CHIP E3 Ligase

“…Importantly, this system is readily amenable to biochemical manipulation and lacks compensatory transcriptional and translational mechanisms. It is thus possible to perturb chaperone levels and activities without secondary consequences that typically complicate similar maneuvers in intact cells (43)(44)(45)(46). Moreover, because CFTR is the only radiolabeled client protein, it is relatively simple to characterize CHIP, Hsp/c70, and CFTR interactions that are responsible for ubiquitination and degradation.…”