Fig. 5. TM3 moves out of the ER translocon upon chain extension. (A) Membrane-associated integration intermediates of OP150 to OP259 containing a single cysteine probe in TM3 (residue 115) were treated with BMH and the resulting adducts analysed following immunoprecipitation as previously described. An adduct with a putative ribosomal protein of 21 kDa was observed with OP150[Cys115] (panel A, lanes 2 and 3, labelled R) and in some cases adducts containing both Sec61 and Sec61ß were seen (labelled ß). All other symbols as previously defined. (B) Membrane-associated integration intermediates of OP204 containing a single cysteine probe in TM4 (residue 154) were analysed as for panel A. A novel adduct was observed with the OP204[Cys154] intermediate (panel B, lanes 2 and 3, filled circle), this remains to be fully characterised. Trace levels of radiolabelled Sec61 resulting from the translation of endogenous mRNA that sometimes remained after the nuclease treatment of the semi-intact cells (data not shown) can be seen in panel B, lane 5 (X).