BETA-CENTRACTIN - CHARACTERIZATION AND DISTRIBUTION OF A NEW MEMBER OF THE CENTRACTIN FAMILY OF ACTIN-RELATED PROTEINS

An examination of human-expressed sequence tags indicated the existenc e of an isoform of centractin, an actin-related protein localized to m icrotubule-associated structures. Using one of these tags, we isolated and determined the nucleotide sequence of a full-length cDNA clone. T he protein encoded represents the first example of multiple isoforms o f an actin-related protein in a single organism. Northern analysis usi ng centractin-specific probes revealed three species of mRNA in HeLa c ells that could encode centractin isoforms. One mRNA encodes the previ ously-identified centractin (now referred to as alpha-centractin). The full-length cDNA clone isolated using the expressed sequence tag enco des a new member of the centractin family, beta-centractin. A probe sp ecific for alpha-centractin hybridized to the third species of mRNA ob served (referred to as gamma-centractin). Comparisons of Northern blot s of human tissues indicated that alpha-centractin and beta-centractin mRNAs are equally distributed in all populations of mRNA examined, wh ereas the expression of gamma-centractin appears to be tissue specific . The amino acid sequence of beta-centractin, deduced from the cDNA, i ndicates a 91% identity with alpha-centractin, increasing to 96% simil arity when conservative amino acid changes are taken into account. As antibodies previously raised against alpha-centractin reacted only poo rly with beta-centractin, new antibodies were produced and combined wi th two-dimensional gel electrophoresis to discriminate the two isoform s. Using this system, the subcellular distribution of the alpha- and b eta-isoforms were determined. Both isoforms were found predominantly i n the cytosolic fraction as a part of a previously identified 20S comp lex (referred to as the dynactin complex) with no evidence for a free pool of either isoform. The isoforms were found in a constant ratio of similar to 15:1 (alpha:beta) in the dynactin complex.