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Fig. 3. Sec61β associates with cytosolic chaperones. (A) Sec61β was synthesised as in described for Fig. 2 and polypeptide chains were released from the ribosome by puromycin treatment. Samples were treated with apyrase to deplete nucleotide triphosphates, crosslinking reagents were added as shown and the resulting products resolved by SDS-PAGE. The location of Sec61β chains and the approximate molecular mass of major adducts are indicated (in kDa). (B) Products of SMCC cross-linking were subjected to immunoprecipitation with antisera recognising specific cytosolic components or a non-related serum (NRS). Adducts with SRP54 (filled circle) and Hsp40 (star) are shown. (C) Sec61β (lanes 1 to 5) or a version without the hydrophobic TM region, Sec61β-TM (lanes 6 to 10), were synthesised as for A) and total products analysed either before (lanes 1 and 6) or after (lanes 2 and 7) SMCC mediated cross-linking. Adducts were identified by immunoprecipitation carried out in the absence of prior SDS denaturation and using antisera specific for either Sec61β (lanes 4 and 9) or Hsc70 (lanes 5 and 10). A non-related serum was used as a control (lanes 3 and 8), adducts with Hsc70 are identified (filled square).
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