Solubilization of SV40 Plasma‐Membrane‐Associated Large Tumor Antigen Using Single‐Phase Concentrations of 1‐Butanol

Abstract

The nature of the interaction of the simian virus 40 (SV40) transforming protein, large tumor antigen (T-ag), with the plasma membrane of transformed cells is not well understood. We report here that SV40 plasma-membrane-associated large tumor antigen (pmT-ag) can be solubilized by using single-phase concentrations of 1-butanol. Purified plasma membranes from SV40-transformed mouse cells yielded T-ag when treated with 2.5% butanol; solubilization of T-ag from the purified membranes in butanol was temperature dependent, with approximately 10-fold more T-ag extracted at 37 degrees C than at 22 degrees C; and application of 2.5% butanol to mKSA cells after cellular surface proteins had been radiolabeled with 125I resulted in the release of iodinated T-ag. Butanol-extracted pmT-ag coprecipitated with p53 and several cellular proteins ranging in size from 35 to 60 kDa. One cellular component migrated at a mobility similar to that of tubulin (56 kDa), and a monoclonal antibody against the alpha subunit of tubulin coprecipitated T-ag. Immunoblotting of proteins immunoprecipitated with monoclonal antibodies against T-ag or p53 from butanol extracts with a monoclonal antibody against the beta subunit of tubulin revealed specific coprecipitation of tubulin with T-ag and p53. This suggests that complexes composed of tubulin, T-ag, and p53 exist in butanol extracts. Control experiments eliminated the possibility of an artifactual association of tubulin with T-ag and p53 induced by butanol. Two-dimensional gel analyses revealed that 2.5% butanol at 37 degrees C extracted a subset of membrane-associated proteins and some cytosolic proteins, as well as a number of proteins that were not soluble in either high salt or detergent. Thus, the butanol extraction conditions employed in this study recovered a species of pmT-ag that appears to complex with tubulin. As butanol reportedly is less deleterious to native protein structures than other agents, including high salts and detergents, this extraction procedure may be useful for studying the structure and function of other membrane-associated proteins.

Formation of the tetraploid intermediate is associated with the development of cells with more than four centrioles in the elastase-simian virus 40 tumor antigen transgenic mouse model of pancreatic cancer.

“…Therefore, additional experiments will be necessary to determine whether cells with more than 4 of 2N, 4N, 6N, 8N, 10N, and greater (31)(32)(33) (34)(35)(36)(37), coprecipitates with p53 and tubulin (38), and localizes with p53 to cytoplasmic, mitotic, and centriolar microtubules in situ (39). p53 also interacts with p34cdc2, a gene product that has been implicated in cell cycle checkpoints in yeast (40) (Fig.…”