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.

Human protein-disulfide isomerase (hPDI)-related protein (hPDIR), which we previously cloned from a human placental cDNA library (Hayano, T., and Kikuchi, M. (1995) FEBS Lett. 372, 210 -214), and its mutants were expressed in the Escherichia coli pET system and purified by sequential nickel affinity resin chromatography. Three thioredoxin motifs (CXXC) of purified hPDIR were found to contribute to its isomerase activity with a rank order of CGHC > CPHC > CSMC, although both the isomerase and chaperone activities of this protein were lower than those of hPDI. Screening for hPDIR-binding proteins using a T7 phage display system revealed that ␣ 1 -antitrypsin binds to hPDIR. Surface plasmon resonance experiments demonstrated that ␣ 1 -antitrypsin interacts with hPDIR, but not with hPDI or human P5 (hP5). Interestingly, the rate of oxidative refolding of ␣ 1 -antitrypsin with hPDIR was much higher than with hPDI or hP5. Thus, the substrate specificity of hPDIR differed from that associated with isomerase activity, and the contribution of the CSMC motif to the oxidative refolding of ␣ 1 -antitrypsin was the most definite of the three (CSMC, CGHC, CPHC). Substitution of SM and PH in the CXXC motifs with GH increased isomerase activity and decreased oxidative refolding. In contrast, substitution of GH and PH with SM decreased isomerase activity and increased oxidative refolding. Because CXXC motif mutants lacking isomerase activity retain chaperone activity for the substrate rhodanese, it is clear that, similar to PDI and hP5, the isomerase and chaperone activities of hPDIR are independent. These results suggest that the central dipeptide of the CXXC motif is critical for both redox activity and substrate specificity.Protein-disulfide isomerase (PDI) 1 catalyzes the oxidation, reduction, and isomerization of protein disulfide bonds (1). It is believed to accelerate the folding of disulfide-bonded proteins by catalyzing the disulfide interchange reaction, which is the rate-limiting step during protein folding that occurs within the luminal space of the endoplasmic reticulum (ER) (2-4). Recently, many PDI homologs have been identified (5-7), and elucidation of their functions, most of which are unknown, may explain the observed diversity of these proteins. PDI has two distinct regions that contain the CGHC sequence. This sequence is similar to a motif found in thioredoxin, CGPC (also called the TX or CXXC motif). CGHC sequences serve as the active sites in PDI for thiol-disulfide bond exchange reactions (8). PDI and its homologs form a diverse protein superfamily whose members are characterized by two or three CXXC motifs (5).We have identified the cDNA of a novel human PDI (hPDI)-related protein (hPDIR) from a human placental cDNA library (9). The deduced amino acid sequence of hPDIR indicates that it contains three CXXC motifs (Cys-Ser-Met-Cys, Cys-Gly-HisCys, and Cys-Pro-His-Cys) and a putative ER retention signal (Lys-Glu-Glu-Leu) at its carboxyl terminus (9). Based on the number and relative positions of CXXC motifs...

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This report demonstrates for the first time that P5, a member of the protein disulphide isomerase (PDI) family, is present in the mitochondria. Various organelles were screened for proteins bearing the CGHC motif using an affinity column conjugated with the phage antibody 5E, which cross-reacts with PDI family proteins. P5 was found in bovine liver mitochondrial extract and identified by Western blot analysis using anti-P5 antibody and by mass spectrometric analysis. Results of cell fractionation, proteinase sensitivity experiments and immuno-electron microscopy supported the mitochondrial localization of P5 and also indicated the presence of ERp57, another PDI family protein, in mitochondria. Our findings will be useful for the elucidation of the translocation mechanism of PDI family proteins and their roles in mitochondria.

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