Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTR F508

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 CFTRΔF508, by specifically promoting ubiquitylation of CFTRΔF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRΔF508 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 CFTRΔF508 and catalyzes further polyubiquitylation of CFTRΔF508 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 CFTRΔF508

SEL1L degradation intermediates stimulate cytosolic aggregation of polyglutamine‐expanded protein

Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). In mammalian cells, the HRD1–SEL1L membrane ubiquitin ligase complex plays a central role in this process. However, SEL1L is inherently unstable, and excess SEL1L is also degraded by ERAD. Accordingly, when proteasome activity is inhibited, multiple degradation intermediates of SEL1L appear in the cytosol. In this study, we searched for factors that inhibit SEL1L degradation and identified OS-9 and XTP3-B, two ER lectins that regulate glycoprotein ERAD. SEL1L degradation was characterized by a ladder of degradation products, and the C-terminal Pro-rich region of SEL1L was responsible for generation of this pattern. In the cytosol, these degradation intermediates stimulated aggregation of polyglutamine-expanded Huntingtin protein (Htt-polyQ-GFP) by interacting with aggregation-prone proteins, including Htt-polyQ-GFP. Collectively, our findings indicate that peptide fragments of ER proteins generated during ERAD may affect protein aggregation in the cytosol, revealing the interconnection of protein homeostasis across subcellular compartments

Insufficient Folding of Type IV Collagen and Formation of Abnormal Basement Membrane-like Structure in Embryoid Bodies Derived from Hsp47-Null Embryonic Stem Cells

Hsp47 is a molecular chaperone that specifically recognizes procollagen in the endoplasmic reticulum. Hsp47-null mouse embryos produce immature type I collagen and form discontinuous basement membranes. We established Hsp47(-/-) embryonic stem cell lines and examined formation of basement membrane and production of type IV collagen in embryoid bodies, a model for postimplantation egg-cylinder stage embryos. The visceral endodermal cell layers surrounding Hsp47(-/-) embryoid bodies were often disorganized, a result that suggested abnormal function of the basement membrane under the visceral endoderm. Rate of type IV collagen secretion by Hsp47(-/-) cells was fourfold lower than that of Hsp47(+/+) cells. Furthermore, type IV collagen secreted from Hsp47(-/-) cells was much more sensitive to protease digestion than was type IV collagen secreted from Hsp47(+/+) cells, which suggested insufficient or incorrect triple helix formation in type IV collagen in the absence of Hsp47. These results indicate for the first time that Hsp47 is required for the molecular maturation of type IV collagen and suggest that misfolded type IV collagen causes abnormal morphology of embryoid bodies

Simultaneous induction of the four subunits of the TRAP complex by ER stress accelerates ER degradation

The mammalian translocon-associated protein (TRAP) complex comprises four transmembrane protein subunits in the endoplasmic reticulum. The complex associates with the Sec61 translocon, although its function in vivo remains unknown. Here, we show the involvement of the TRAP complex in endoplasmic reticulum-associated degradation (ERAD). All four subunits are induced simultaneously by endoplasmic reticulum stresses from the X-box-binding protein 1/inositol-requiring 1α pathway. RNA interference knockdown of each subunit causes disruption of the native complex and significant delay in the degradation of various ERAD substrates, including the α1-antitrypsin null Hong Kong variant (NHK). In a pulse-chase experiment, the TRAP complex associated with NHK at a late stage, indicating its involvement in the ERAD pathway rather than in biosynthesis of nascent polypeptides in the endoplasmic reticulum. In addition, the TRAP complex bound preferentially to misfolded proteins rather than correctly folded wild-type substrates. Thus, the TRAP complex induced by the unfolded protein response pathway might discriminate ERAD substrates from correctly folded substrates, accelerating degradation