The cellular secretory pathway is not utilized for biosynthesis, modification, or intracellular transport of the simian virus 40 large tumor antigen.

Abstract

Unlike most proteins, which are localized within a single subcellular compartment in the eucaryotic cell, the simian virus 40 (SV40) large tumor antigen (T-ag) is associated with both the nucleus and the plasma membrane. Current knowledge of protein processing would predict a role for the secretory pathway in the biosynthesis and transport of at least a subpopulation of T-ag to account for certain of its chemical modifications and for its ability to reach the cell surface. We have examined this prediction by using in vitro translation and translocation experiments. Preliminary experiments established that translation of T-ag was detectable with as little as 0.1 microgram of the total cytoplasmic RNA from SV40-infected cells. Therefore, by using a 100-fold excess of this RNA, the sensitivity of the assays was above the limits necessary to detect the theoretical fraction of RNA equivalent to the subpopulation of plasma-membrane-associated T-ag (2 to 5% of total T-ag). In contrast to a control rotavirus glycoprotein, the electrophoretic mobility of T-ag was not changed by the addition of microsomal vesicles to the in vitro translation mixture. Furthermore, T-ag did not undergo translocation in the presence of microsomal vesicles, as evidenced by its sensitivity to trypsin treatment and its absence in the purified vesicles. Identical results were obtained with either cytoplasmic RNA from SV40-infected cells or SV40 early RNA transcribed in vitro from a recombinant plasmid containing the SP6 promoter. SV40 early mRNA in infected cells was detected in association with free, but not with membrane-bound, polyribosomes. Finally, monensin, an inhibitor of Golgi function, failed to specifically prevent either glycosylation or cell surface expression of T-ag, although it did depress overall protein synthesis in TC-7 cells. We conclude from these observations that the constituent organelles of the secretory pathway are not involved in the biosynthesis, modification, or intracellular transport of T-ag. The initial step in the pathway of T-ag biosynthesis appears to be translation on free cytoplasmic polyribosomes. With the exclusion of the secretory pathway, we suggest that T-ag glycosylation, palmitylation, and transport to the plasma membrane are accomplished by previously unrecognized cellular mechanisms.