Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like domain-dependent manner.

Accumulation of aberrant proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response pathway that helps the cell to survive under these stress conditions. Herp is a mammalian ubiquitin domain protein, which is strongly induced by the unfolded protein response. It is involved in ER-associated protein degradation (ERAD) and interacts directly with the ubiquitin ligase Hrd1, which is found in high molecular mass complexes of the ER membrane. Here we present the first evidence that Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like (UBL) domain-dependent manner. We found that upon exposure of cells to ER stress, elevation of Herp steady state levels is accompanied by an enhanced association of Herp with pre-existing Hrd1. Hrd1-associated Herp is rapidly degraded and substituted by de novo synthesized Herp, suggesting a continuous turnover of the protein at Hrd1 complexes. Further analysis revealed the presence of multiple Hrd1 copies in a single complex enabling binding of a variable number of Herp molecules. Efficient ubiquitylation of the Hrd1-specific ERAD substrate α1-antitrypsin null Hong Kong (NHK) required the presence of the Herp UBL domain, which was also necessary for NHK degradation. In summary, we propose that binding of Herp to Hrd1-containing ERAD complexes positively regulates the ubiquitylation activity of these complexes, thus permitting survival of the cell during ER stress.

The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway.

To eliminate misfolded proteins that accumulate in the endoplasmic reticulum (ER) the cell mainly relies on ubiquitin-proteasome dependent ER-associated protein degradation (ERAD). Proteolysis of ERAD substrates by the proteasome requires their ubiquitylation and retro-translocation from the ER to the cytoplasm. Here we describe a high molecular mass protein complex associated with the ER membrane, which facilitates ERAD. It contains the ubiquitin domain protein (UDP) HERP, the ubiquitin protein ligase HRD1, as well as the retro-translocation factors p97, Derlin-1 and VIMP. Our data on the structural arrangement of these ERAD proteins suggest that p97 interacts directly with membrane-resident components of the complex including Derlin-1 and HRD1, while HERP binds directly to HRD1. We propose that ubiquitylation, as well as retro-translocation of proteins from the ER are performed by this modular protein complex, which permits the close coordination of these consecutive steps within ERAD.

PI31 is a modulator of proteasome formation and antigen processing.

Regulation of the proteasome system, which is responsible for the generation of most MHC class I-bound peptides, occurs through the interaction of the 20S proteasome with several regulatory proteins. One of these is PI31, which acts in vitro as an inhibitor of proteasome activity. Here, we demonstrate that, rather than inhibiting proteasome function, PI31 acts as a selective modulator of the proteasome-mediated steps in MHC class I antigen processing. Overexpression of PI31 in mouse embryonic cells has no impact on proteasome-mediated proteolysis. Instead, PI31, which localizes at the nuclear envelope/endoplasmic reticulum membrane, selectively interferes with the maturation of immunoproteasome precursor complexes. Consequently, overexpression of PI31 abrogates MHC class I presentation of an immunoproteasome-dependent cytotoxic T lymphocyte epitope and reduces the surface MHC class I levels on IFN-gamma-treated mouse embryonic cells. Thus, PI31 represents a cellular regulator of proteasome formation and of proteasome-mediated antigen processing.