The v-sis oncoprotein loses transforming activity when targeted to the early Golgi complex

To examine proteolytic processing of our fusion proteins, NIH3T3 cells expressing the desired proteins were metabolically labeled with [35S]Cys and [35S]Met, and labeled proteins were immunoprecipitated and separated by SDS-PAGE. As shown in Fig. 3 , the sis-E1-G protein showed no detectable processed forms (Fig. 3, lane 2) , implying retention in an early Golgi compartment. On the other hand, the mutant derivatives sis-El(Ql)-G and sis-El(ins)-G both exhibited processing, which appears as a doublet of lower mo- lecular mass bands (Fig. 3, lanes 3 and 4, indicated by arrows). Similar results were obtained using the constructs lacking the G tail (data not shown). The diffuse signal above the major bands most likely represents heterogeneity of O-linked oligosaccharides which, although previously observed , have not been extensively characterized. In summary, these results are consistent with localization of sis-E1 and sis-E1-G to the early Golgi complex, whereas the proteins encoded by the other constructs have clearly progressed through the secretory pathway beyond the trans-Golgi complex.