Terminally misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytoplasm and degraded by proteasomes through a mechanism known as ER-associated degradation (ERAD). EDEM, a postulated Man8B-binding protein, accelerates the degradation of misfolded proteins in the ER. Here, EDEM was shown to interact with calnexin, but not with calreticulin, through its transmembrane region. Both binding of substrates to calnexin and their release from calnexin were required for ERAD to occur. Overexpression of EDEM accelerated ERAD by promoting the release of terminally misfolded proteins from calnexin. Thus, EDEM appeared to function in the ERAD pathway by accepting substrates from calnexin.

Proteins that are unfolded or misfolded in the endoplasmic reticulum (ER) must be refolded or degraded to maintain the homeostasis of the ER. Components of both productive folding and ER-associated degradation (ERAD) mechanisms are known to be up-regulated by the unfolded protein response (UPR). We describe two novel components of mammalian ERAD, Derlin-2 and -3, which show weak homology to Der1p, a transmembrane protein involved in yeast ERAD. Both Derlin-2 and -3 are up-regulated by the UPR, and at least Derlin-2 is a target of the IRE1 branch of the response, which is known to up-regulate ER degradation enhancing α-mannosidase–like protein (EDEM) and EDEM2, receptor-like molecules for misfolded glycoprotein. Overexpression of Derlin-2 or -3 accelerated degradation of misfolded glycoprotein, whereas their knockdown blocked degradation. Derlin-2 and -3 are associated with EDEM and p97, a cytosolic ATPase responsible for extraction of ERAD substrates. These findings indicate that Derlin-2 and -3 provide the missing link between EDEM and p97 in the process of degrading misfolded glycoproteins.

SummaryEpithelial cell contacts consist of not only bicellular contacts but also tricellular contacts, where the corners of three cells meet. At tricellular contacts, tight junctions (TJs) generate specialized structures termed tricellular TJs (tTJs) to seal the intercellular space. Tricellulin is the only known molecular component of tTJs and is involved in the formation of tTJs, as well as in the normal epithelial barrier function. However, the detailed molecular mechanism of how tTJs are formed and maintained remains elusive. Using a localization-based expression cloning method, we identified a novel tTJ-associated protein known as lipolysis-stimulated lipoprotein receptor (LSR). Upon LSR knockdown in epithelial cells, tTJ formation was affected and the epithelial barrier function was diminished. Tricellulin accumulation at the tricellular contacts was also diminished in these cells. By contrast, LSR still accumulated at the tricellular contacts upon tricellulin knockdown. Analyses of deletion mutants revealed that the cytoplasmic domain of LSR was responsible for the recruitment of tricellulin. On the basis of these observations, we propose that LSR defines tricellular contacts in epithelial cellular sheets by acting as a landmark to recruit tricellulin for tTJ formation.

SummaryTricellular tight junctions (tTJs) seal the extracellular space at tricellular contacts (TCs), where the corners of three epithelial cells meet. To date, the transmembrane proteins tricellulin and lipolysis-stimulated lipoprotein receptor (LSR) are known to be molecular components of tTJs. LSR recruits tricellulin to tTJs, and both proteins are required for the full barrier function of epithelial cellular sheets. In the present study, we show that two LSR-related proteins, immunoglobulin-like domain-containing receptor (ILDR) 1 and ILDR2, are also localized at TCs and recruit tricellulin. At least one of LSR, ILDR1 and ILDR2 was expressed in most of the epithelial tissues in mice. The expressions of LSR, ILDR1 and ILDR2 were generally complementary to each other, although LSR and ILDR1 were co-expressed in some epithelia. ILDR1 was required for the establishment of a strong barrier of the epithelium, similar to LSR, when introduced into cultured epithelial cells, whereas ILDR2 provided a much weaker barrier. We further analyzed human ILDR1, mutations in which cause a familial deafness, DFNB42, and found that most DFNB42-associated ILDR1 mutant proteins were defective in recruitment of tricellulin. We also found that tricellulin mutant proteins associated with another familial deafness, DFNB49, were not recruited to TCs by ILDR1. These findings show the heterogeneity of the molecular organization of tTJs in terms of the content of LSR, ILDR1 or ILDR2, and suggest that ILDR1-mediated recruitment of tricellulin to TCs is required for hearing. Given their common localization at epithelial cell corners and recruitment of tricellulin, we propose to designate LSR, ILDR1 and ILDR2 as angulin family proteins.

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin-proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTR⌬F508, by specifically promoting ubiquitylation of CFTR⌬F508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTR⌬F508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTR⌬F508 and catalyzes further polyubiquitylation of CFTR⌬F508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTR⌬F508.