Cloning of a cDNA encoding human centrin, an EF-hand protein of centrosomes and mitotic spindle poles.
A human cDNA expression library was screened using anti-centrin antibodies to obtain a cDNA clone encoding human centrin. The cDNA clone contains an open reading frame of 516 base pairs and predicts a product of 172 amino acids with a calculated molecular mass of 19,528 and a pI of 4.61. Sequence analysis demonstrates that human centrin and centrins from higher plants, protozoa, algae, Xenopus and the yeast CDC31 gene product are closely related members of a subfamily of the EF-hand superfamily of calcium-binding proteins. The human centrin sequence has four putative calcium-binding domains as defined by the EF-hand consensus. Immunoprecipitation and western blot studies from HeLa cells confirm that human centrin is a protein of approximately 20,000 M(r) as predicted from the cDNA clone. Indirect immunofluorescence analysis of HeLa cells demonstrates that centrin is localized at the centrosome of interphase cells and that it redistributes to the region of the spindle poles during mitosis. When taken together with earlier genetic studies, these results demonstrate that centrin is a ubiquitous component of centrosomes and mitotic spindle poles of diverse organisms and suggest that centrin plays a role in centrosome separation at the time of mitosis.
Errabolu
R
R
Department of Biochemistry and Molecular Biology, Mayo Clinic Foundation, Rochester, Minnesota 55905.
Sanders
M A
MA
Salisbury
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D003001:Cloning, Molecular; D016384:Consensus Sequence; D003285:Contractile Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis; D056890:Eukaryota / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D005798:Genes, Bacterial; D006367:HeLa Cells; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010802:Phylogeny; D010944:Plants / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D008941:Spindle Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D013737:Testis / Q000378:metabolism; D014981:Xenopus
C083769:CDC31 protein, S cerevisiae; C580665:CETN1 protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D003285:Contractile Proteins; D018076:DNA, Complementary; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; C056608:caltractin