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

To verify that the constructs used in this study were capable of encoding the desired proteins, the coding regions from each construct were swapped into a pSP64(polyA) vector (Promega Biotec, Madison, WI) for in vitro transcription and translation experiments. Messenger RNA was synthesized (Melton, 1987) and translation reactions were performed in rabbit reticulate lysate in the presence of [35S]Cys. SDS-PAGE analysis of the products of these reactions demonstrated proteins with molecular weights that correlated with the expected sizes of each of the fusion proteins (data not shown). This indicates that our constructs encode the desired fusion proteins. The results summarized in Table I indicate that sis-E1 and sis-E1-G chimeric proteins are not able to cause transformation. The most likely explanation is that these proteins are anchored in a cellular compartment where they are unable to functionally interact with PDGF receptors. However, one might postulate other explanations for the lack of transforming activity exhibited by these proteins. For example, a critical post-translational event may be altered by forcing v-sis to remain intracellular, thereby abolishing its activity. N-linked glycosylation of v-sis is not required for its biological activity (Sauer et al., 1986) , and bacteriaUy-expressed PDGF-BB (which has no N-or O-linked glycosylation) is biologically active (Hoppe et al., 1989) . Therefore, glycosylation of v-sis, although altered in these fusion proteins, does not constitute a critical processing event. However, v-sis must dimerize in order to function , so perhaps these fusion proteins do not dimerize correctly. To address this, we infected NIH3T3 cells with viral supernatants collected from cells transfected with the indicated v-sis fusion constructs (see legend to Fig. 2) . The fusion proteins were immunoprecipitated using the a-sis antibody, and one half of the sample was treated with nonreducing sample buffer to examine dimeric proteins, while the other half was treated with reducing sample buffer to examine monomeric forms. Fig. 2 , lanes 3 and 9 shows that the sis-E1 chimera indeed dimerizes, yielding a dimer band of •62-64 kD under nonreducing conditions, and a 32-kD monomer band un-der reducing conditions. Comparison of the left and right panels of Fig. 2 further illustrates that all of the chimeric proteins dimerize as expected, indicating that this critical processing event is not altered in the fusion proteins. Thus, these data suggest that the transforming activity of the v-sis fusion proteins described here most closely correlates with their subcellular localization within the cell, as verified further below, rather than resulting from differences in posttranslational processing events.