Purification and characterization of a basal body-associated Ca2+-binding protein.

Abstract

Abstract. 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.

Analysis of microtubule sliding patterns inChlamydomonasflagellar axonemes reveals dynein activity on specific doublet microtubules

“…However, previous studies in Chlamydomonas suggest that the effect is mediated in part by calmodulin associated with the axoneme (Smith, 2002a;Yang et al, 2001). Additional Ca 2+ binding proteins that may play a role in Ca 2+ induced changes in motility include the Ca 2+ binding protein centrin/caltractin (Guerra et al, 2003;Huang et al, 1988;Piperno et al, 1992;Salisbury et al, 1988;Yanagisawa and Kamiya, 2001), the 18 kDa light chain of the outer dynein arm in Chlamydomonas (King and Patel-King, 1995), a Ca 2+ regulated nucleotide-diphosphate kinase in Chlamydomonas flagella (Patel-King et al, 2002) and the outer dynein arm docking complex protein DC3 (Casey et al, 2003a;Casey et al, 2003b). …”

Centrin 2 Stimulates Nucleotide Excision Repair by Interacting with Xeroderma Pigmentosum Group C Protein

“…Centrin was first identified as a small acidic calcium-binding protein in the flagellar apparatus of unicellular green algae (20,21,50). In Chlamydomonas reinhardtii, centrin is localized in nucleus-basal body connectors, distal striated fibers, and a flagellar transition zone (51,65).…”

Drosophila gamma-tubulin is part of a complex containing two previously identified centrosomal MAPs.

“…A number of such proteins have already been identified (for example, Kuriyama and Borisy, 1985;Klotz et ai., 1986;Baron and Salisbury, 1988;Huang et al, 1988b;Whitfield et al, 1988;Kellogg et al, 1989;Rout and Kilmartin, 1990), and cDNAs encoding several of them have been cloned and sequenced (Huang et al, 1988a;Kuriyama et al, 1990;Ohta et al, 1990;Joswig et al, 1991). Moreover, antibodies raised against certain centrosomal proteins have been shown to inhibit aspects of centrosomal function when injected into cells (Moudjou et al, 1992;Joshi et al, 1992), and genetic studies in yeasts have identified genes whose products are involved in the function of the spindle pole body, the centrosome equivalent in yeasts (Byers, 1981;Baum et al, 1986Baum et al, , 1988Rose and Fink, 1987;Uzawa et al, 1990;Winey et al, 1991;Vallen et al, 1992).…”