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

被引:38
作者
CLARK, SW
STAUB, O
CLARK, IB
HOLZBAUR, ELF
PASCHAL, BM
VALLEE, RB
MEYER, DI
机构
[1] UNIV CALIF LOS ANGELES, INST MOLEC BIOL, LOS ANGELES, CA 90024 USA
[2] UNIV PENN, SCH VET MED, PHILADELPHIA, PA 19104 USA
[3] WORCESTER FDN EXPTL BIOL INC, CELL BIOL GRP, SHREWSBURY, MA 01545 USA
[4] Scripps Res Inst, DEPT CELL BIOL, LA JOLLA, CA 92037 USA
关键词
D O I
10.1091/mbc.5.12.1301
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
An examination of human-expressed sequence tags indicated the existence of an isoform of centractin, an actin-related protein localized to microtubule-associated structures. Using one of these tags, we isolated and determined the nucleotide sequence of a full-length cDNA clone. The protein encoded represents the first example of multiple isoforms of an actin-related protein in a single organism. Northern analysis using centractin-specific probes revealed three species of mRNA in HeLa cells that could encode centractin isoforms. One mRNA encodes the previously-identified centractin (now referred to as alpha-centractin). The full-length cDNA clone isolated using the expressed sequence tag encodes a new member of the centractin family, beta-centractin. A probe specific for alpha-centractin hybridized to the third species of mRNA observed (referred to as gamma-centractin). Comparisons of Northern blots of human tissues indicated that alpha-centractin and beta-centractin mRNAs are equally distributed in all populations of mRNA examined, whereas the expression of gamma-centractin appears to be tissue specific. The amino acid sequence of beta-centractin, deduced from the cDNA, indicates a 91% identity with alpha-centractin, increasing to 96% similarity when conservative amino acid changes are taken into account. As antibodies previously raised against alpha-centractin reacted only poorly with beta-centractin, new antibodies were produced and combined with two-dimensional gel electrophoresis to discriminate the two isoforms. Using this system, the subcellular distribution of the alpha- and beta-isoforms were determined. Both isoforms were found predominantly in the cytosolic fraction as a part of a previously identified 20S complex (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.
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页码:1301 / 1310
页数:10
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