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

To demonstrate that sis-E1 and sis-E1-G fusion proteins are indeed targeted to the early Golgi complex, we have used double-label immunofluorescence to colocalize these chimeric proteins with known Golgi markers. The Golgi markers used were (a) Lens culinaris lectin, a carbohydratebinding protein that binds to terminal o~-mannosyl and o~-Dglucosyl residues (Kornfeld et al., 1981) , and has been shown to stain primarily the Golgi complex of cells (Hsu et al., 1992; Machamer et al., 1993) ; and (b) a monoclonal antibody 10Et, described by Wood et al. (1991) , which was localized to the cis-Golgi complex of NRK cells by immunoelectron microscopy. In these studies, the sis-E1-G chimera was expressed in NIH3T3 cells by infection with retroviral supernatants, and was detected in fixed and permeabilized cells with a polyclonal rabbit antisera to v-sis. This in turn was visualized with a rhodamine-conjugated goat anti-rabbit IgG. To visualize the Golgi complex, these same cells were treated with either a fluorescein-conjugated Lens culinaris lectin, or with the mouse mAb 10E6, which was visualized with a fluorescein-conjugated goat anti-mouse IgG. The two cells shown for each condition in Fig. 6 are representative Cells expressing various fusion proteins were processed for immunofluorescence. Surface proteins were detected by a rabbit serum directed against the v-sis protein, and rhodamine-conjugated goat antirabbit antibody (B, D, F, and H). Intrac~llular proteins were detected by a mouse mAb against the COOH-terminal portion of the VSV-G protein and a biotin-conjugated goat anti-mouse antibody, followed by FITC-conjugated streptavidin (A, C, E, and G). A and B , sis-G, C and D, sis-El-G; E and F, sis-El(ins) of the sis-El-G-expressing cells generated in these immunofluorescence assays. The percentage of ceils expressing protein was higher using this infection protocol than that obtained by transient transfections. This percentage varied from •10-15 %. As Fig. 6 shows, the sis-E1-G fusion protein clearly colocalizes with both the lectin (see A and B, C and D) and the mAb 10E6 (see E and F, G and H). Thus, the E1 cis-Golgi targeting signal functions correctly and targets v-sis to the early Golgi when incorporated into a fusion protein.