RbAp48 is essential for viability of vertebrate cells and plays a role in chromosome stability.

RbAp46/48, histone chaperone, is a family of evolutionarily conserved WD40 repeat-containing proteins, which are involved in various chromatin-metabolizing processes, but their in vivo functional relevance is yet unclear. In order to examine the biological role of pRbAp48 in chicken DT40 cells, we generated a tetracycline-inducible system for conditional RbAp48-knockout cells. Depletion of RbAp48 led to delayed S phase progression associated with slow DNA synthesis and nascent nucleosome formation, followed by accumulation in G2/M phase, finally leading to cell death. Prior to cell death, these cells exhibited aberrant mitosis such as highly condensed and abnormal chromosome alignment on the metaphase plate, leading to chromosome missegregation. Depletion of RbAp48 also caused dissociation of heterochromatin protein 1 (HP1) from pericentromeric heterochromatin. Furthermore, depletion of RbAp48 from cells led to elevated levels of acetylation and slightly decreased levels of methylation, specifically at Lys-9 residue of histone H3. These results suggest that RbAp48 plays an important role in chromosome stability for proper organization of heterochromatin structure through the regulation of epigenetic mark.

Molecular mechanism of ER stress-induced pre-emptive quality control involving association of the translocon, Derlin-1, and HRD1.

The maintenance of endoplasmic reticulum (ER) homeostasis is essential for cell function. ER stress-induced pre-emptive quality control (ERpQC) helps alleviate the burden to a stressed ER by limiting further protein loading. We have previously reported the mechanisms of ERpQC, which includes a rerouting step and a degradation step. Under ER stress conditions, Derlin family proteins (Derlins), which are components of ER-associated degradation, reroute specific ER-targeting proteins to the cytosol. Newly synthesized rerouted polypeptides are degraded via the cytosolic chaperone Bag6 and the AAA-ATPase p97 in the ubiquitin-proteasome system. However, the mechanisms by which ER-targeting proteins are rerouted from the ER translocation pathway to the cytosolic degradation pathway and how the E3 ligase ubiquitinates ERpQC substrates remain unclear. Here, we show that ERpQC substrates are captured by the carboxyl-terminus region of Derlin-1 and ubiquitinated by the HRD1 E3 ubiquitin ligase prior to degradation. Moreover, HRD1 forms a large ERpQC-related complex composed of Sec61α and Derlin-1 during ER stress. These findings indicate that the association of the degradation factor HRD1 with the translocon and the rerouting factor Derlin-1 may be necessary for the smooth and effective clearance of ERpQC substrates.

Pre-emptive Quality Control Protects the ER from Protein Overload via the Proximity of ERAD Components and SRP.

Cells possess ER quality control systems to adapt to ER stress and maintain their function. ER-stress-induced pre-emptive quality control (ER pQC) selectively degrades ER proteins via translocational attenuation during ER stress. However, the molecular mechanism underlying this process remains unclear. Here, we find that most newly synthesized endogenous transthyretin proteins are rerouted to the cytosol without cleavage of the signal peptide, resulting in proteasomal degradation in hepatocytes during ER stress. Derlin family proteins (Derlins), which are ER-associated degradation components, reroute specific ER proteins, but not ER chaperones, from the translocon to the proteasome through interactions with the signal recognition particle (SRP). Moreover, the cytosolic chaperone Bag6 and the AAA-ATPase p97 contribute to the degradation of ER pQC substrates. These findings demonstrate that Derlins-mediated substrate-specific rerouting and Bag6- and p97-mediated effective degradation contribute to the maintenance of ER homeostasis without the need for translocation.