A Systematic Search for Endoplasmic Reticulum (ER) Membrane-associated RING Finger Proteins Identifies Nixin/ZNRF4 as a Regulator of Calnexin Stability and ER Homeostasis

Abstract

To identify novel regulators of endoplasmic reticulum (ER)-linked protein degradation and ER function, we determined the entire inventory of membrane-spanning RING finger E3 ubiquitin ligases localized to the ER. We identified 24 ER membraneanchored ubiquitin ligases and found Nixin/ZNRF4 to be central for the regulation of calnexin turnover. Ectopic expression of wild type Nixin induced a dramatic down-regulation of the ER-localized chaperone calnexin that was prevented by inactivation of the Nixin RING domain. Importantly, Nixin physically interacts with calnexin in a glycosylation-independent manner, induces calnexin ubiquitination, and p97-dependent degradation, indicating an ER-associated degradation-like mechanism of calnexin turnover.The endoplasmic reticulum (ER) 3 is a major cellular site for production, folding, quality control, and distribution of proteins. Many regulatory mechanisms are in place to keep these processes in balance and therefore to ensure cellular fitness and survival, with ubiquitin-dependent protein degradation playing an important part (1). One major challenge the ER faces is an overload with unfolded or folding proteins. An excess of folding proteins in the ER triggers a cellular response called the unfolded protein response (UPR) (2, 3). UPR entails lowering of the protein load by the attenuation of protein translation and the up-regulation of chaperones thereby increasing the protein folding capacity of the cells. If the capacity of UPR is exceeded, the cell utilizes ER-associated degradation (ERAD), a system for the recognition of terminally misfolded proteins and their disposal (4). Misfolded proteins destined for ERAD are ubiquitinated by the RING domain containing ubiquitin ligases (5), Hrd1 (6), and Doa1 (7, 8), retrotranslocated across the ER membrane into the cytosol by the AAA-ATPase p97 (9), and then degraded by the 26 S proteasome.Upon entry into the ER, most nascent polypeptides are recognized by glycosidases and modified on specific asparagine residues (Asn-Xaa-(Thr/Ser)) with the N-glycan GlcNAc 2 Man 9 Gluc 3 (10). Core glycosylation of nascent polypeptides decreases their overall hydrophobicity. Trimming of the terminal two glucose residues by glucosidase I allows for binding of the lectins/chaperones calreticulin and calnexin thereby facilitating the proper folding of the newly synthesized protein (10,11).Although the importance of regulated degradation of ER resident proteins is firmly established, only a small number of RING finger-containing ubiquitin ligases are known to be involved in such processes to date, namely SYVN1/hHrd1 (6, 12), AMFR/gp78 (13), TEB4/MARCH6 (14), RNF5/Rma1 (15), RNF77/TRIM13 (16), and RNF13 (17). Given the importance of protein metabolism and degradation in the ER and the vast number of ubiquitin ligases encoded in the human genome, we asked whether other ubiquitin ligases are involved in the regulation of ER-related degradation processes.Based on the assumption that the ER lumen is devoid of E1 and E2 ubiquitination activity and on the...

Protein Folding and Quality Control in the ER

“…In contrast to yeast, mammalian ERAD E3 ligases are more diverse (HRD1, RMA1, Parkin, CHIP, gp78, TEB4, TRC8, etc.) and additional ERAD E3 ligases continue to be identified, which indicates that various complexes are needed to monitor different classes of substrates Tsai and Weissman 2010;Neutzner et al 2011). In mammalian ERAD, the degradation of N-glycosylated proteins is well-characterized.…”

RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation

“…This was confirmed by experiments showing that RNF170 is an integral membrane protein and that it has an ER-like distribution pattern. RNF170 can thus be added to the list of known mammalian ER membrane E3 ligases (13,20). These include Hrd1 and gp78 (the mammalian homologs of yeast Hrd1p) (11,13), TEB4 (the mammalian homolog of yeast Doa10) (11,13), RNF5 (21), Kf-1 (22), TRC8 (23), RNF121 (17), RNF122 (24), and RNF180 (25).…”

ERAD‐dependent control of the Wnt secretory factor Evi

“…However, only a few regulatory ERAD substrates have been described so far. Given the number of ubiquitin ligases present in the ER (Neutzner et al , 2011), the physiological role of regulatory ERAD is likely underestimated at present.…”