Regulation of Endoplasmic Reticulum-associated Degradation by RNF5-dependent Ubiquitination of JNK-associated Membrane Protein (JAMP)

Abstract

Clearance of misfolded proteins by endoplasmic reticulum (ER)-associated degradation (ERAD) requires concerted activity of chaperones, adaptor proteins, ubiquitin ligases, and proteasomes. RNF5 is a ubiquitin ligase anchored to the ER membrane implicated in ERAD via ubiquitination of misfolded proteins. Among RNF5-associated proteins is JNK-associated membrane protein (JAMP), a 7-transmembrane protein located within the ER membrane that facilitates degradation of misfolded proteins through recruitment of proteasomes and ERAD regulatory components. Here we demonstrate that RNF5 associates with JAMP in the ER membrane. This association results in Ubc13-dependent RNF5-mediated noncanonical ubiquitination of JAMP. This ubiquitination does not alter JAMP stability but rather inhibits its association with Rpt5 and p97. Consequently, clearance of misfolded proteins, such as CFTR⌬508 and T cell receptor ␣, is less efficient, resulting in their greater accumulation. Significantly, the RNF5 effect on JAMP is seen prior to and after ER stress response, thereby highlighting a novel mechanism to limit ERAD and proteasome assembly at the ER, to the actual ER stress response.Degradation of misfolded proteins is part of a complex quality control system that clears diverse proteins independent of sequence or functional similarity (1-3). The unfolded protein response reduces the burden caused by unfolded protein accumulation in the endoplasmic reticulum (ER) 2 (4), in part by activating its key regulatory proteins IRE1-XBP1 and ATF6, which results in transcriptional activation of genes important for unfolded protein response, including components of the ER-associated degradation system (ERAD) (5).ERAD is regulated by an ER quality control system that marks proteins that cannot fold or assemble into multiprotein complexes for ubiquitin-dependent degradation (1-3). This system consists of molecular chaperones such as BiP (1-3), which interacts with misfolded protein to enable their transfer across the ER membrane via the multispanning membrane proteins Derlin-1/2/3 and Sec61(6), and the AAA (ATPase p97 (also known as VCP or cdc48)). Subsequent to translocation, misfolded proteins are ubiquitinated via ERanchored ubiquitin ligases, such as the vertebrate gp78, Parkin, RNF5/RMA1, and Hrd1 (7-10), followed by proteasome-mediated degradation.RNF5 (RING finger domain E3 ligase; also known as RMA1) is one of the few ER-associated E3 ubiquitin ligases. Anchored to the ER membrane via its C terminus, RNF5 consists of a classic RING domain (which confers ligase activity), a single transmembrane-spanning domain located within the C-terminal region, and a formin-like homology domain. RNF5 has been shown to promote degradation of misfolded proteins, such as mutant CFTR (CFTR⌬508) (9, 10). Intriguingly, RNF5-mediated degradation of mutant CFTR requires cooperation with another ligase, as has also been shown for gp78 or CHIP, suggesting a two-stage process in which the first ligase promotes substrate mono-ubiquitination, whereas the second media...

Peripheral Endoplasmic Reticulum Localization of Gp78 Ubiquitin Ligase Activity

“…Moreover, expression of a dominant negative mutant of p97 ATPase function is sufficient to induce accumulation of p97 itself, Derlin-1 and polyubiquitylated substrates in the ERQC, identifying this site as the potential retrotranslocation site in the ER (Wakana et al, 2008). A role for central ER translocation of polyubiquitylated substrate in proteasomal targeting is consistent with the previously reported interaction of gp78 not only with p97 (Ballar et al, 2007;Li et al, 2005;Zhong et al, 2004), but also with various components of the retrotranslocation machinery, including the Derlins, Sec61 and JAMP1, which recruit proteasomes to the ER (Li et al, 2005;Tcherpakov et al, 2009;Ye et al, 2005). Localization of CLIMP-63, a transmembrane protein involved in maintaining the architecture of ER sheets, is stabilized via interaction with ribosomes (Shibata et al, 2010).…”

Regulation of Glutamine Carrier Proteins by RNF5 Determines Breast Cancer Response to ER Stress-Inducing Chemotherapies

“…Since RNF5 is implicated in the clearance of misfolded proteins as part of the ERAD response (Tcherpakov et al, 2009; Younger et al, 2006), we asked whether RNF5 interaction with Gln-carrier proteins required changes in protein folding following ERS. To do so, we subjected BCa cells to 35 S-methionine-cysteine-based labeling followed by immunopurification of 35 S-labeled SLC1A5 and limited proteolytic cleavage using BNPS-Skatole.…”

HECT and RING finger families of E3 ubiquitin ligases at a glance

“…Adding further complexity to ERAD pathways, gp78 can be targeted for degradation by autoubiquitylation and by ubiquitylation through HRD1 that -in turn -stabilizes gp78 substrates (Ballar et al, 2010;Shmueli et al, 2009). Other ER-localized E3s, including RNF5, MARCH6 and RNF139, have also been implicated in ERAD (Morito et al, 2008;Stagg et al, 2009;Tcherpakov et al, 2009;Zavacki et al, 2009).…”