Subcellular Localization of Simian Virus 40 Large Tumor Antigen

Abstract

The distribution of simian virus 40 large tumor antigen in subcellular fractions from simian virus 40-transformed hamster (H-50) and mouse (VLM) cells and from simian virus 40-infected monkey cells was determined. Solubilized [ 35 S]-methionine- or 32 P i -labeled surface membrane and nuclear fractions were prepared, immunoprecipitated with hamster anti-T serum, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tumor antigen with an apparent molecular weight of ∼96,000 was detected in both subcellular fractions. Minor components of ∼68,000 and ∼56,000 with anti-T reactivity which labeled with [ 35 S]methionine were also detected in both fractions from H-50 cells, as were components of ∼140,000 and ∼56,000 from VLM cells. The 56,000 component appeared to be greatly reduced in 32 P i -labeled surface membrane fractions. Normal cells or cells transformed with a heterologous agent, such as polyoma virus or a chemical carcinogen, lacked immunoprecipitable tumor antigen. Cell fractionation was monitored by [ 3 H]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. These results indicate the presence of a protein in the plasma membrane of cells transformed or infected by simian virus 40 that is immunologically indistinguishable from nuclear tumor antigen.