| dc.description | Apolipoprotein (apo) B-100 is an atypical secretory protein in that its translocation across the ER membrane can be inefficient. Analysis of truncated apoB variants revealed that sequences in the beta1 domain between apoB-39 and apoB-42 and predicted to be composed of amphipathic beta-strands, governed the secretion efficiency and intracellular stability of apoB. Unlike smaller truncated apoB proteins, apoB-42 and apoB-100 were secreted poorly and were degraded by an ALLN-sensitive pathway. To test the hypothesis that sequences from the beta1 domain are involved in apoB degradation, fusion proteins were created that contained apoB-29 linked to fragments derived from the beta1 domain of apoB or to liver FABP, a known beta-sheet protein. Fusion proteins containing the segments apoB-34-42 or apoB-37-42 were degraded rapidly, mostly by the proteasome, whereas other fusion proteins were stable and secreted efficiently. The apoB-34-42 segment increased the susceptibility to polyubiquitination and the affinity of apoB to the microsomal membrane by decreasing translocation efficiency. The presence of specific sequences in the beta1 domain of human apoB may increase degradation by promoting the cytosolic exposure and polyubiquitination of the protein. Polyubiquitinated apoB proteins can be recognized by cytosolic ER-associated degradation (ERAD) factors involved in the delivery of degradation proteins target to the proteasome. Of these factors, the AAA-ATPase p97 has been shown to have a role in ERAD by recognizing and retrotranslocating protein from the ER to the cytosol for delivery to the 26S proteasome. To investigate the potential role of p97 in the retro-translocation and proteasomal degradation of apoB-100, cytosolic proteins were analyzed using a digitonin-permeabilised cell system. Accumulation of cytosolic ubiquitinated apoB proteins was observed when cells were treated with the proteasome inhibitor ALLN or MG132. Using crosslinking agents, we showed that p97 associates with apoB-100. Reducing intracellular levels of p97 by transfecting p97-targeted siRNA increased the intracellular levels of apoB-100 by impairing proteasomal degradation. Reduced levels of p97 also decreased the accumulation of cytosolic apoB-100, supporting the role of p97 in retro-translocation. Taken together, these experiments suggested that p97 is a key player in the recognition and retro-translocation of apoB-100 for proteasomal degradation. | en_US |
