The signal sequence of the p62 protein of Semliki Forest virus is involved in initiation but not in completing chain translocation

When microsomes were added to the C-p62-dhfr in vitro translation system a new band appeared which migrated somewhat slower than the p62-dhfr band seen in the analysis of the mixture lacking membranes (Fig. 2, lane 2) . It almost comigrated with one of the two weak C derived bands. The new band apparently corresponds to IRi2-dhfr hybrids that have been translocated into the lumen of the added microsomes and have become glycosylated. The immunoprecipitation analysis shown in Fig. 3 confirmed the identity of this material. The protease digestions in the absence (Fig. 2, lane 3) and presence of Triton X-100 (lane 4) clearly demonstrated that the slower migrating p62-dhfr molecules were indeed translocated. About half of this material remains protected in the presence of intact microsomes whereas all is digested when the membranes are solubilized with detergent. In contrast, the other translated material did not show such a pronounced membrane-dependent protease resistance. Note that protease treatment of all samples yielded a resistant protein of a small size. This most likely represents a protease-resistant C fragment.