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

To define at what time point during p62-dhfr chain synthesis the Asn13 becomes glycosylated we performed a time-course experiment essentially as described by Rothman and Lodish (1977) (Fig. 5) . In this experiment a 150-#1 translation was initiated. After 1.5 rain ATA was added (0.075 raM) to block additional starting of chain synthesis. Then, at 0.5-rain intervals, two 7.5/~1 aliquots were removed; one for mixing with 40 #1 of hot PAGE sample buffer (2 % SDS) and the other one for further incubation after mixing with 0.75/~1 of 20% TX-100. The first sample from each time point was used for the determination of the time needed for chain completion, which is a function of the translation rate, and the other one allowed determination of the time course of glycosylation of the translocated chain. Triton X-100 solubilizes the microsomal membranes and thereby inactivates glycosylation (but not chain elongation). Therefore, only those p62-dhfr chains that have presented Asnl3 tO the glycosylation machinery before TX-100 addition have had the possibility to become glycosylated. In Fig. 5, lanes 1-10, one can see that completed p62dhfr chains (197 residues with initiator Met) appear after a 3-min incubation from the time point of ATA addition. If one assumes constant chain initiation during the Figure 5 . Time course of p62 dhfr glycosylation. A 150-#1 translation was initiated. After a preincubation time of 1.5 min ATA was added to inhibit further initiation of chain synthesis. Then, at intervals of 0.5 min (indicated by 0.5', 1.0', 1.5', 2.0', 2.5', 3.0', 3.5', 4.0', 4.5', and 5.0') two 7.5-/zl samples were removed, one for mixing with PAGE sample buffer and another one for mixing with TX-100 (final concentration 1%) and further incubation at 30°C (for a total time of 20 min after ATA addition as indicated by the lower row of time points in the figure) . Lanes 1-10 show the samples removed for mixing with the PAGE sample buffer. From these results the approximate rate of translation can be derived. Completed chains appear in the 3-min sample. Lanes 11-20 show the samples in which the membranes have been solubilized with Triton X-100 for inactivation of the glycosylation machinery. From these analyses it is possible to estimate when Asn13 is modified during p62-dhfr synthesis. The first glycosylated forms are clearly visible in the 1.5-min sample, a time point where only about half of the p62-dhfr chain has been synthesized. The nature of the material in the two weak bands seen in lanes 1-5 is unclear. Their transient appearance before the completion of the p62-dhfr chain suggests that they represent complexes of nascent p62-dhfr chains. Figure 6 . Time course of p62 d-4 glycosylation. Seven translations in the presence of microsomal membranes were started in parallel. After a 3-rain initial incubation at 30°C, ATA was added in order to inhibit further initiation of chain synthesis. Incubation was then continued for 35 rain. At the indicated time points (5, 10, 15, 20, 25, 30 , and 35 rain) TX-100 (TX) was added to stop further chain glycosylation. At the same time one half of each sample was removed and put on ice in order to measure the extent of chain elongation at each time point. All samples were analyzed by SDS-PAGE (10%) and autoradiography. Lanes 3-9 show the analysis of the samples incubatedwith TX-100 and lanes 10--16 the analysis of the portions put on ice at the different time points. The complete sequence of treatments for each sa