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

Surface staining of nonpermeabilized cells was readily detectable for cells expressing sis-El(ins), sis-El(Ql), and sis-TGN38A derivatives (see Fig. 4 , B, D, and H), indicating that these fusion proteins are efficiently transported to the cell surface, as expected. The sis-TGN38 construct (F) displays decreased but detectable surface staining, indicating that a portion of the population of this protein reaches the cell surface. This is consistent with the behavior of native TGN38, which has been shown to recycle between the TGN and the cell surface (Reaves et al., 1993) . Since a small amount of this sis-q'GN38 fusion protein reaches the cell surface, this may explain the transforming activity of this construct in the focus formation assays (see Table I ). However, at this time we have no way of determining if the TGNretained population of the fusion protein contributes in any way to the transforming activity. While Fig. 4 clearly demonstrates that the sis-E1 mutants and the sis-TGN38 derivatives reach the cell surface, it does not answer the question of whether the sis-E1 or sis-E1-G fusions do not reach the surface. To address this question, double-label immunofluorescence was performed using the constructs bearing the VSV-G cytoplasmic tail. This allowed for simultaneous examination of both intracellular and cell surface populations of the various fusion proteins within the same cell. Cell surface protein was detected with an antibody against v-sis. The cells were then permeabilized, and intracellular fusion proteins were detected with a monoclonal antibody to the VSV-G protein. As positive controls, both the sis-El(ins)-G and the sis-El(Ql)-G fusion proteins were included in this assay. As in Fig. 4, Fig. 5 shows that these proteins were readily detected within the cell (E and G) as well as on the cell surface (F and H). As another control, sis-G was included. This fusion protein was created in our lab for previous experiments, and it localizes to the cell surface (Hannink and Donoghue, 1986a) . A and B of Fig. 5 clearly demonstrate both intracellular and surface staining for this construct. C demonstrates the reticular and perinuclear intracellular staining consistently seen for sis-El-G, indicating presence of this protein within the ER and Golgi complex.