Figure 7. Hsp104 acts prior to substrate retrotranslocation.

(A) A schematic of the in vitro ubiquitination/retrotranslocation assay. (B) Ubiquitinated Q394X was immunoprecipitated from the retrotranslocated supernatant (S) fractions and the membrane bound pellet (P) fractions after immunoprecipitation. Reactions were run in the presence of either wild type (HSP104) or hsp104Δ cytosol, and in the presence or absence of ATP, as indicated. The native molecular weight (arrow) and high molecular weight species (asterisk) of Q394X are marked. (C) The in vitro assay was scaled up 8-fold with cytosol purified from BY4742 wild type yeast and ER-enriched microsomes purified from BY4742 yeast containing either an empty vector (E) or the Q394X expression vector (Q). The substrate was immunoprecipitated from the pellet (membrane) or supernatant (cytosol, retrotranslocated) fractions and the indicated components were subsequently examined by immunoblot. (D) The membrane bound (P) fraction in a ubiquitination/retrotranslocation assay was collected and washed in 6 M urea to differentiate between membrane-integrated (P) and retrotranslocated, membrane-associated Q394X (US). (E) ER-enriched microsomes were washed with 6 M urea and collected by centrifugation. Samples were also either diluted in buffer or incubated in the presence of SDS. The solubilized fractions (S) and the re-suspended pelleted fractions (P) were TCA precipitated and resolved by SDS-PAGE. Blots were incubated with antibodies against the HA tag in Q394X, the ER resident integral membrane protein, Sec61, and the ER-associated cytosolic proteins, Cdc48 and Hsp104. (F) The stability of Q394X was determined in mutant cdc48-2 yeast containing either an empty vector (EV) or a vector to over-express Hsp104 (OE104). Cultures were shifted to 39°C for 2.5 hrs and chased for 1 hr at 39°C. N = 3 independent experiments. +/− SEM. **P < 0.01, ***P<0.001.