Purification and characterization of a basal body-associated Ca2+-binding protein.
Isolated basal body complexes from the unicellular alga, Chlamydomonas reinhardtii were found to contain a low molecular mass acidic polypeptide, distinct from calmodulin, but with biochemical features in common with members of the calmodulin family of calcium-binding proteins. These common characteristics included a relative low molecular mass of 20 kD, an experimentally determined acidic pI of 5.3, an altered electrophoretic mobility in SDS-polyacrylamide gels in the presence of added calcium, and a calcium-dependent binding to the hydrophobic ligand phenyl-Sepharose which allowed its purification by affinity chromatography. The relatedness of the basal body-associated 20-kD calcium-binding protein (CaBP) to calmodulin was confirmed by amino acid compositional analysis and partial peptide sequencing of the isolated protein. A rabbit antibody specific for the 20-kD CaBP was raised and used to determine by indirect immunofluorescence the cellular localization of the protein in Chlamydomonas cells. In interphase cells the antibody stained intensely the region between the paired basal bodies, two fibers extending between the basal bodies and the underlying nucleus, and an array of longitudinal filaments surrounding the nucleus. The two basal body-nuclear connecting fibers were identified in thin-section electron micrographs to be narrow striated fiber roots. In mitotic cells the 20-kD CaBP was specifically associated with the poles of the mitotic spindle at the sites of the duplicated basal body complexes.
Huang
B
B
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Watterson
D M
DM
Lee
V D
VD
Schibler
M J
MJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001345:Autoradiography; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000302:isolation & purification*; D002147:Calmodulin / Q000032:analysis; D002696:Chlamydomonas / Q000032:analysis* / Q000648:ultrastructure; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D007118:Immunoassay; D007150:Immunohistochemistry; D007399:Interphase; D008854:Microscopy, Electron; D008938:Mitosis; D008969:Molecular Sequence Data
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D002147:Calmodulin