ABSTRACT. Cytosolic Hsc70/Hsp70 are known to contribute to the endoplasmic reticulum (ER)-associated degradation of membrane proteins. However, at least in mammalian cells, its partner ER-localized J-protein for this cellular event has not been identified. Here we propose that this missing protein is DNAJB12. Protease protection assay and immunofluorescence study revealed that DNAJB12 is an ER-localized single membranespanning protein carrying a J-domain facing the cytosol. Using co-immunoprecipitation assay, we found that DNAJB12 is able to bind Hsc70 and thus can recruit Hsc70 to the ER membrane. Remarkably, cellular overexpression of DNAJB12 accelerated the degradation of misfolded membrane proteins including cystic fibrosis transmembrane conductance regulator (CFTR), but not a misfolded luminal protein. The DNAJB12-dependent degradation of CFTR was compromised by a proteasome inhibitor, lactacystin, suggesting that this process requires the ubiquitin-proteasome system. Conversely, knockdown of DNAJB12 expression attenuated the degradation of CFTR. Thus, DNAJB12 is a novel mammalian ER-localized J-protein that plays a vital role in the quality control of membrane proteins.

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ABSTRACT. Misfolded proteins in the endoplasmic reticulum (ER) are dislocated out of the ER to the cytosol, polyubiquitinated, and degraded by the ubiquitin-proteasome system in a process collectively termed ERassociated degradation (ERAD). Recent studies have established that a mammalian ER-localized transmembrane J-protein, DNAJB12, cooperates with Hsc70, a cytosolic Hsp70 family member, to promote the ERAD of misfolded membrane proteins. Interestingly, mammalian genomes have another J-protein called DNAJB14 that shows a high sequence similarity to DNAJB12. Yet, very little was known about this protein. Here, we report the characterization of DNAJB14. Immunofluorescence study and protease protection assay showed that, like DNAJB12, DNAJB14 is an ER-localized, single membrane-spanning J-protein with its J-domain facing the cytosol. We used co-immunoprecipitation assay to find that DNAJB14 can also specifically bind Hsc70 via its J-domain to recruit this chaperone to ER membrane. Remarkably, the overexpression of DNAJB14 accelerated the degradation of misfolded membrane proteins including a mutant of cystic fibrosis transmembrane conductance regulator (CFTRΔF508), but not that of a misfolded luminal protein. Furthermore, the DNAJB14-dependent degradation of CFTRΔF508 was compromised by MG132, a proteasome inhibitor, indicating that DNAJB14 can enhance the degradation of a misfolded membrane protein using the ubiquitin-proteasome system. Thus, the mammalian ER possesses two analogous J-proteins (DNAJB14 and DNAJB12) that both can promote the ERAD of misfolded transmembrane proteins. Compared with DNAJB12 mRNA that was widely expressed in mouse tissues, DNAJB14 mRNA was expressed more weakly, being most abundant in testis, implying its specific role in this tissue.

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