Related Experiment Videos
Cloning and sequencing of rat plectin indicates a 466-kD polypeptide chain with a three-domain structure based on a
Abstract:
We have determined the complete cDNA sequence of rat plectin from a number of well-characterized overlapping lambda gt11 clones. The 4,140-residue predicted amino acid sequence (466,481 D) is consistent with a three-domain structural model in which a long central rod domain, having mainly an alpha-helical coiled coil conformation, is flanked by globular NH2- and COOH-terminal domains. The plectin sequence has a number of repeating motifs. The rod domain has five subregions approximately 200-residues long in which there is a strong repeat in the charged amino acids at 10.4 residues that may be involved in association between plectin molecules. The globular COOH-terminal domain has a prominent six-fold tandem repeat, with each repeat having a strongly conserved central region based on nine tandem repeats of a 19-residue motif. The plectin sequence has several marked similarities to that of desmoplakin (Green, K. J., D. A. D. Parry, P. M. Steinert, M. L. A. Virata, R. M. Wagner, B. D. Angst, and L.A. Nilles. 1990. J. Biol. Chem. 265:2,603-2,612), which has a shorter coiled-coil rod domain with a similar 10.4 residue charge periodicity and a COOH-terminal globular domain with three tandem repeats homologous to the six found in plectin. The plectin sequence also has homologies to that of the bullous pemphigoid antigen. Northern blot analysis indicated that there is a significant degree of conservation of plectin genes between rat, human, and chicken and that, as shown previously at the protein level, plectin has a wide tissue distribution. There appeared to be a single rat plectin gene that gave rise to a 15-kb message. Expression of polypeptides encoded by defined fragments of plectin cDNA in E. coli has also been used to localize the epitopes of a range of monoclonal and serum antibodies. This enabled us to tentatively map a sequence involved in plectin-vimentin and plectin-lamin B interactions to a restricted region of the rod domain.