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

In vitro transcription (0.3 #g supercoiled template DNA per 10 #1 vol) in the presence of SP6 RNA Polymerase (6-8 U) and the cap structure was carried out as previously described (Zerial et al., 1986) . In vitro translation reactions using a rabbit reticulocyte lysate were performed at 30°C essentially as described . 1.5 #1 of the in vitro synthesized RNA was translated in a total volume of 15 #1. Potassium, magnesium, and spermidine concentrations were 100, 1.2, and 0.375 mM, respectively. When indicated, 1 #1 of ER membranes was included. In some translocations the membranes were pretreated with 200 #M peptide for 5 min on ice. The final peptide concentration in the total translation mixture here was, after addition of the pretreated membranes, adjusted to 100/~M. To obtain partial synchronization of translation, ATA was added after a preincubation of 1.5-3.0 rain (Borgese et al., 1974) . A final ATA concentration of 0.075 mM was found to be sufficient for inhibiting initiation of chain synthesis (see control in Fig. 6 , lane/). Higher concentrations of ATA inhibited first transloeation and then also chain elongation. For protease protection experiments, proteinase K was added to a final concentration of 0.1 mg/ml and the samples were incubated at 0*C for 30 min in the presence or absence of 1% Triton X-100. Proteolysis was stopped by the addition of PMSF (final concentration 2 mg/ml) and samples were kept at 0*C for 5 min before further processing for electrophoresis (Cutler and Garoff, 1986) . Bands containing labeled protein were visualized by fluorography. Quantitation of proteins was done by cutting the bands out of the dried gel, solubilizing them with Protosol (from DuPont de Nemours, NEN) according to the instructions of the manufacturer, and finally counting the 3~S radioactivity in a liquid scintillator (Wallac LKB, Turku, Finland). The localization of the bands on the dried gel was done with the aid of the fluorograph in transillumination.