Figure 5. Orm2 is retro‐translocated by the Dsc complex and Cdc48 for proteasomal degradation.

• A
  WT (pdr5∆) cells expressing FLAG‐Orm2 and Sec61‐GFP (integral ER membrane protein) treated with MG‐132 were fractionated into insoluble membrane (P100) and soluble cytoplasmic (S100) fractions and analyzed by SDS–PAGE and Western blot with the indicated antibodies.
• B, C
  WT cells, tul1Δ, and cdc48‐3 mutants (all in a pdr5∆ strain background) were left untreated or treated with the proteasome inhibitor (MG‐132) as indicated and subsequently subjected to subcellular fractionation as in (A). S100 and P100 fractions were subjected to denaturing FLAG‐Orm2 immunoprecipitations (IP). Input and eluted fractions (with FLAG peptide) were analyzed by SDS–PAGE and Western blotting with the indicated antibodies.
• D
  P100 fractions from MG‐132‐treated WT (pdr5∆) cells expressing FLAG‐Orm2 and Sec61‐GFP were extracted with Na₂CO₃ solution at the indicated pH and subsequently fractionated by 100,000 × g centrifugation into soluble (S) and insoluble membrane (M) fractions, subjected to denaturing FLAG‐immunoprecipitations and analyzed by Western blotting with the indicated antibodies. The asterisk indicates reactivity of the secondary antibody with IgG heavy chains.
• E
  Schematic representation of Orm2 degradation by the EGAD pathway.