Int. zyxwvutsrqp J. Cancer: zyxwvuts 25, zyxwvutsr 517-527 (1980) SUBCELLULAR DISTRIBUTION OF SIMIAN VIRUS 40 T ANTIGEN SPECIES IN VARIOUS CELL LINES: THE56K PROTEIN Samuel W. LUBORSKY and K. CHANDRASEKARAN Macromolecular Biology Section, Immunology Program, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205, USA. zyxwvu The subcellular distribution of SV40 anti-T serum- specific species was examined in SV40-transformed, T- antigen-positive tissue culture cell lines of rat and of A U N and BALB/c mouse origin. Cells were labelled with [’*S]methionine. The cytoplasm, nuclear and mem- brane fractions were obtained, and their radioim- munoprecipitates analyzed by gel electrophoresis. Tests were performed to determine the purity of these sub- cellular fractions, and negligible cross-contamination was found. The cytoplasm fractions lacked detectable anti-T serum reactivity. Large amounts of both large T antigen and a 56K protein were always present together both in the nuclear fractions and, in a somewhat lesser amount, in the plasma membrane fractions of all cell lines examined. Analysis of density gradient sedimenta- tion profiles of the immunoprecipitates of whole-cell ex- tracts indicated these species were associated in some fashion, probably with each other. The activity of the 56K protein may be associated with its presence on the cell surface where, either alone or acting together with the large T antigen, it might provide the surface activity responsible for tumor-specific surface and/or traneplan- tation antigen activities. Immunoprecipitates from simian virus 40 (SV40) transformed cells, obtained following treatment with hamster SV40 anti-T serum, generally contain three polypeptides: the tumor antigens (T Ag), large T Ag (Crawford et a[., 1978; Prives et al., 1977; Carroll and Smith, 1976; Tegtmeyer, 1974) and small t Ag (Crawford et al., 1978; Sleigh et al., 1978; Prives et al., 1977; Tabuchi et al., 1976) and the intermediate size, 56,000 molecular weight protein (56K protein) (Chang et al., 19796; Edwards et al., 1979; Kress et al., 1979; Lane and Crawford, 1979; Melero et al., 1979; Gaudray et al., 1978). Such immunoprecipi- tates, commonly analyzed by sodium dodecylsulfate- polyacrylamide gel electrophoresis (SDS-PAGE), contain antigens of molecular weights near 94K and 19K, for the large T Ag and the small t Ag, respec- tively. These values are used to denote these pro- teins operationally. While it is known that large T antigen is located predominantly in the nucleus of Sv40-infected or transformed cells (Chang et al., 1979a; Crawford et al., 1978; Paucha et al., 1977; Prives et al., 1977; Tenen et al., 1977; Tabuchi et al., 1976; Livingston et al., 1974; Leduc et al., 1969; Pope and Rowe, 1964), and the small t antigen is found in the cytoplasm (Sleigh et at., 1978; Prives et al., 1977; Tabuchi et al., 1976), until recently little was known about the properties and distribution of 56K protein. There has been a recent report of the presence of large T antigen and some 56K protein in the membrane fractions of various SV40-trans- formed cell lines (Soule and Butel, 1979). Our re- sults confirm and extend this work, and deal in more detail with the subcellular distribution of the 56K protein. The designation, “56K protein” is an operational description of a class of proteins with molecular weights (by SDS-PAGE) in the range -52K-63K which precipitate with SV40 large T antigen follow- ing treatment with hamster anti-T serum. The 56K protein is important as it has been consistently ob- served in most SV40-transformed cells and recent evidence suggests that its origin is cellular (Chang et al., 19796; Kress et al., 1979; Lane and Crawford, 1979; Linzer et al., 1979; Linzer and Levine, 1979; Melero et al., 1979; Smith et al., 1979). It is impor- tant to know its subcellular distribution vis-Ci-vis the other SV40 early antigens, in order better to under- stand first, the binding to or coordinate biosynthesis with these antigens, and ultimately, its function. Re- ports have appeared recently which include informa- tion on subcellular distribution of anti-T reactive species (Soule and Butel, 1979). We have examined distributions of the three antigen species, especially with regard to the species on the cell surface, in various transformed murine tissue culture lines with well-characterized biological (especially immunolog- ical) characteristics. We have included certain con- trol experiments to provide a measure of the efficacy of subcellular separations. We found both 94K and 56K molecular weight species present in both nuc- lear and surface membrane cell fractions, but absent from the cytoplasm of the transformed cells ex- amined. In order to survey fractions from many cell lines repetitively for anti-T serum reactive material, we chose a fractionation procedure which entailed minimum manipulation and rapidly provided cell fractions of reasonable purity. Cell homogenates were separated into nuclear (N), cytoplasmic (C), and surface membrane (M) fractions, which were immunoprecipitated with hamster anti-T serum and analyzed by SDS-polyacrylamide gel electro- phoresis. MATERIAL AND METHODS Cells All work was performed with T-Ag-positive, con- tinous culture SV40-transformed cell lines. Mouse lines used were zyxw SV AL/N, a high passage number virus-transformed mass culture previously described (Coll et al., 1977; Smith and Mora, 1972), and 215, derived from an early passage, cloned, normal AL/ N mouse embryo line transformed by SV40 and iso- lated by cloning single cells (Winterbourne and Mora, 1978). Another mouse line, 315, was derived Received: November 14, 1979, and in revised form Ja- nuary 29, 1980.