Heterotypic tetramer (A 2D 2) complexes of non-epidermal keratins isolated from cytoskeletons of rat hepatocytes and hepatoma cells

Abstract

Cytoskeletal residues obtained after extraction of rat liver and cultured rat hepatoma cells (line MH 1C 1) were used to isolate eytokeratin subunit complexes by solubilization in low salt buffer containing 4 m-urea. Alternatively, the complexes were prepared by solubilixation of total cytoskeletal proteins in 9.5 m-urea or 6 m-guanidinium hydrochloride (Gu · HCl), followed by separation using reversed phase high pressure liquid chromatography and dialysis first against either 9.5 m-urea or 6 m-Gu · HCl and then against buffers containing either 4 m-urea or 2 m-Gu · HCl, respectively. The complexes contained only two cytokeratin polypeptides in a 1:1 ratio as demonstrated by electrophoresis and isoelectric focusing, i.e. components A ( M r 55,000; isoelectric point in 9.5 m-urea, pH 6.4) and D ( M r 49,000; isoelectric point, pH 5.38) which were separated from each other at urea concentrations higher than 7 m. The complex had a sedimentation coefficient s 25. w of 4.96 S in 2 m-Gu · HCl. Sedimentation equilibrium analysis gave an average M r value of 207,000 which was interpreted as a tetramer containing two chains each of A and D. This complex was also directly demonstrated by gel electrophoresis under non-dissociating conditions. Using dimethyl suberimidate to cross-link the complex in solution of 4 m-urea or 2 m-Gu · HCl, we identified covalently linked heterodimers of A and D, and a tetrameric unit containing equal amounts of A and D which was the largest cross-link product obtained. This complex was similar to the tetrameric complex of rat and human vimentin formed under the same conditions. The constituents of the cross-linked products were identified by two-dimensional (“diagonal”) gel electrophoresis, involving the cleavage of the bis(amidine) cross-links after the initial separation in the first dimension. Identical cross-link products were recognized when eytokeratin filaments were used. By electron microscopy the complexes appeared as threads of 2 to 3 nm diameter with a mean length of approximately 48 nm. On dialysis to low salt buffer, the complexes formed 2 to 3 nm protofilaments, intertwisted 3 to 4 nm protofilaments and typical 7 to 11 nm intermediate-sized filaments. Complexes formed from equivalent cytokeratins of other species such as man and cow, as well as heterologous recombinations such as human component A mixed with bovine component D and vice versa, showed the same characteristics. The data are not compatible with a three-stranded coiled-coil organization. We conclude that hepatic cytokeratin occurs in subunit complexes that are heterotypic tetramers containing two molecules each of polypeptides A and D. We suggest that these complexes represent two-stranded coiled-coils containing one molecule of A and D, which in turn associate into pairs of heterodimers, i.e. tetramers. We propose that these heterodimer pairs represent the basic subunits of polymerization of cytokeratin filaments. The results are discussed in relation to current concepts of molecular arrangements in other types of cytokeratin and in non-keratinous intermediate-sized filaments.