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 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 approximately 15:1 (alpha:beta) in the dynactin complex.
Clark
S W
SW
Department of Biological Chemistry, UCLA School of Medicine, USA.
Staub
O
O
Clark
I B
IB
Holzbaur
E L
EL
Paschal
B M
BM
Vallee
R B
RB
Meyer
D I
DI
D000199:Actins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary; D004285:Dogs; D000072159:Dynactin Complex; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells; D006801:Humans; D008868:Microtubule Proteins / Q000737:chemistry; D008869:Microtubule-Associated Proteins; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D014018:Tissue Distribution
D000199:Actins; D018076:DNA, Complementary; D000072159:Dynactin Complex; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; C076834:centractin