Characterization of a 50-kDa polypeptide in cytoplasmic dynein preparations reveals a complex with p150GLUED and a novel actin.
Earlier work identified a series of accessory polypeptides of 150, 74, 59, 57, 55, 53, 50, and 45 kDa copurifying with cytoplasmic dynein. In the present study immunoprecipitation of the 50-kDa polypeptide from bovine brain cytosol with a specific monoclonal antibody revealed coprecipitating components of 150, 135, 62, and 45 kDa, which were completely distinct from the polypeptides immunoprecipitated using an antibody to the well established 74-kDa cytoplasmic dynein subunit. The 150- and 135-kDa polypeptides reacted with an antibody to p150Glued, the mammalian homologue of the Drosophila Glued gene. N-terminal microsequencing of tryptic peptides of the major 45-kDa component of the complex revealed it to be the alpha-isoform of centractin, a novel form of actin. Immunoblotting of sucrose gradient-fractionated brain cytosol revealed p150Glued, p50, and centractin to cosediment exclusively at 20 S. Immunofluorescence microscopy using antibody to p150Glued revealed centrosomal staining, which was abolished by microtubule depolymerization. Together these results reveal the 50-kDa polypeptide to be part of a cytosolic complex distinct from cytoplasmic dynein. However, the immunolocalization data indicate an association with microtubule minus ends, suggesting a possible interaction with cytoplasmic dynein in the cell.
Paschal
B M
BM
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Holzbaur
E L
EL
Pfister
K K
KK
Clark
S
S
Meyer
D I
DI
Vallee
R B
RB
D000199:Actins / Q000032:analysis*; D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry*; D004398:Dyneins / Q000032:analysis*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000032:analysis*; D051381:Rats; D016415:Sequence Alignment
D000199:Actins; D010455:Peptides; D011506:Proteins; D004398:Dyneins