title: Mechanisms targeting apolipoprotein B100 to proteasomal degradation: evidence that degradation is initiated by BiP binding at the N terminus and the formation of a p97 complex at the C terminus.
abstract: OBJECTIVE
  In lipid-poor states, the ubiquitin-proteasomal pathway rapidly degrades misfolded apolipoprotein B100 (apoB) cotranslationally, although the mechanism of delivery from the ER to cytosolic proteasomes is poorly understood. Here we demonstrate key roles of BiP, an ER luminal chaperone, and p97, a cytosolic ATPase anchored to the ER membrane, in the targeting of apoB for proteasomal degradation.
  
  
  RESULTS
  Using coimmunoprecipitations, we observed associations of apoB with BiP, p97, Derlin-1, VIMP, and the E3 ubiquitin ligase Hrd1 in HepG2 cells. BiP and p97 were found to bind apoB cotranslationally. Expression of C-terminal truncated apoB molecules in COS-7 cells showed an N-terminal region outside apoB15 and a C-terminal region found in apoB72 were required for BiP and p97 binding, respectively. Interestingly, overexpression of dominant negative p97 demonstrated that the ATPase activity of p97 was essential for proteasomal degradation of apoB but not for apoB binding. However, p97 activity did not appear to affect the N terminus of apoB, which may be cleaved before degradation.
  
  
  CONCLUSIONS
  These data suggest that p97 and BiP play critical roles in the cotranslational delivery of apoB to proteasomes and formation of a degradative complex. Proteasomal degradation appears to selectively target apoB molecules with large C-terminal domains.
authors.affiliation: Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: Angela C
authors.initials: AC
authors.lastname: Rutledge
authors.forename: Wei
authors.initials: W
authors.lastname: Qiu
authors.forename: Rianna
authors.initials: R
authors.lastname: Zhang
authors.forename: Rita
authors.initials: R
authors.lastname: Kohen-Avramoglu
authors.forename: Nina
authors.initials: N
authors.lastname: Nemat-Gorgani
authors.forename: Khosrow
authors.initials: K
authors.lastname: Adeli
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D053299:Apolipoprotein B-100 / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051140:Selenoproteins / Q000378:metabolism; D014161:Transduction, Genetic; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D000074405:Valosin Containing Protein
chemical_list: D053299:Apolipoprotein B-100; D018797:Cell Cycle Proteins; D015853:Cysteine Proteinase Inhibitors; C487709:DERL1 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D007976:Leupeptins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D061988:Proteasome Inhibitors; D011500:Protein Synthesis Inhibitors; C487710:SELENOS protein, human; D051140:Selenoproteins; D003513:Cycloheximide; C482345:SYVN1 protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; D000074405:Valosin Containing Protein; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde