Subcellular Localization of Simian Virus 40 Large Tumor Antigen

Abstract

The distribution of SV-40 large tumor [T] antigen in subcellular fractions from SV-40-transformed hamster (H-50) and mouse (VLM) cells and from SV-40-infected monkey cells was determined. Solubilized [35S]-methionine- or 32Pi-labeled surface membrane and nuclear fractions were prepared, immunoprecipitated with hamster anti-T serum, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. T antigen with an apparent molecular weight of .apprx. 96,000 was detected in both subcellular fractions. Minor components of .apprx. 68,000 and .apprx. 56,000 with anti-T reactivity which labeled with [35S]methionine were also detected in both fractions from H-50 cells, as were components of .apprx. 140,000 and .apprx. 56,000 from VLM cells. The 56,000 component appeared to be greatly reduced in 32Pi-labeled surface membrane fractions. Normal cells or cells transformed with a heterologous agent, e.g., polyoma virus or a chemical carcinogen, lacked immunoprecipitable tumor antigen. Cell fractionation was monitored by [3H]thymidine labeling, NADH-diaphorase activity and Na+-K+-dependent ATPase activity. These analyses revealed only trace contamination of surface membranes by nuclei, extremely low levels of nuclear rupture during homogenization and an approximate 10-fold enrichment of surface membrane. Reconstruction experiments demonstrated that soluble tumor antigen failed to associate or copurify with surface membranes during fractionation procedures. A protein is apparently present in the plasma membrane of cells transformed or infected by SV-40 that is immunologically indistinguishable from nuclear tumor antigen.