Novel localization of pp60 src in rous sarcoma virus-transformed rat and goat cells and in chicken cells transformed by viruses rescued from these mammalian cells

Abstract

We have investigated by indirect immunofluorescence and subcellular fractionation the intracellular location of pp60 src in RSV-transformed mammalian cells and in CEF cells transformed by virus rescued from these cells. Two independently derived cell lines were examined: RR1022 cells isolated from an in vivo sarcoma induced in an Amsterdam rat by infection with SR-RSV-D; and Pcl, cells isolated from a soft agar colony of normal goat skin fibroblasts transformed in vitro by SR-RSV-D. Transforming viruses (RSV-RR and RSV-Pcl) were rescued from RR1022 and Pcl cells by fusion with CEF cells. Immunofluorescence microscopy showed association of pp60 src with the nuclear envelope and the juxtanuclear reticular membranes in the transformed mammalian cells and in CEF cells transformed by the rescued viruses, in contrast to the plasma membrane localization of pp60 src seen in SR-RSV-transformed CEF cells. Results of subcellular fractionation by differential centrifugation and fractionation of particulate fractions by equilibrium centrifugation in discontinuous sucrose gradients were in agreement with the differences in pp60 src distribution observed by immunofluorescence microscopy. Although the mammalian cell lines were independently derived, pp60 srcs isolated from RR1022 and Pcl cells both lacked amino-terminal 21- and 18-kilodalton [ 35S]methionine S. aureus V8 protease peptides found in SR-RSV-D pp60 src. Proteolytic peptides identical to those of pp60 src from the mammalian cells were obtained from pp60 src proteins isolated from rescued virus-transformed CEF cells, suggesting that the alteration in the amino-terminal half of the src protein represents a stable change, and that an alteration in the primary structure of pp60 src is responsible for the altered intracellular membrane localization of pp60 src in these cells.