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

We also studied the timing of the glycosylation of Asn~3 in its normal background, i.e., during p62 chain synthesis. For this experiment we used the pGEM SFVd-4 construct. This encodes the C and the p62 membrane protein variant, p62 d-4, in which a few residues of the cytoplasmic protein domain have been exchanged as compared to the wild type sequence (see Materials and Methods and Fig, 1) . Fig. 6 , lane 2, shows that RNA, which has been transcribed from this construct, directs the synthesis of C and p62 d-4 chains. The protein has catalyzed correct C-p62 cleavage and the p62 signal sequence has catalyzed the insertion of about half of the p62 d-4 chains across the added microsomal membranes. These migrate as glycosylated 60-58 kD proteins in contrast to the noninserted molecules which have an apparent molecular mass of 'x,52 kD. The glycosylated and translocated nature of the 60-58 kD material was clearly demon-strated in experiments similar to those described above for the p62-dhfr hybrid molecule (not shown).