Figure 5.

Site-specific photocross-linking of the signal sequence of nascent polypeptides to translocation components. (A) To analyze a preinsertion stage of translocation, RNCs containing 59 amino acids of preprolactin (pPL) or 50 amino acids of prepro-α-factor (αF) were synthesized with photoreactive probes at various positions of the signal sequence. To this end, truncated mRNAs with stop codons at different positions in the signal sequence-coding region were translated in vitro in the presence of a modified suppressor Phe-tRNA containing a diazirino group in the side chain of the amino acid. The RNCs were incubated with canine microsomes and subjected to irradiation. Cross-linked products were analyzed by immunoprecipitation with antibodies against Sec61α or TRAM, followed by SDS-PAGE and autoradiography. To analyze stages at which the nascent chains were inserted into the channel, RNCs with chains of 86 amino acids containing photoreactive probes at the various positions were employed in similar cross-linking experiments. (B) RNCs carrying prepro-α-factor chains of different length were incubated with or without microsomal membranes and treated with proteinase K. The percentage of nascent chains protected is given.