Mutational analysis of centrin: an EF-hand protein associated with three distinct contractile fibers in the basal body apparatus of Chlamydomonas.

Abstract

Abstract. Centrin, a 20-kD phosphoprotein with four calcium-binding EF-hands, is present in the centrosome/basal body apparatus of the green alga Chlamydomonas reinhardtii in three distinct locations: the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions. In each location, centrin is found in fibrous structures that display calcium-mediated contraction. The mutant vfl2 has structural defects at all of these locations and is defective for basal body localization and/or segregation. We show that the vfl2 mutation is a G-to-A transition in the centrin structural gene which converts a glutamic acid to a lysine at position 101, the first amino acid of the E-helix of the protein's third EF-hand. This proves that centrin is required to construct the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions, and it demonstrates the importance of amino acid 101 to normal centrin function. Based on immunofluorescence analysis using anti-centrin antibodies, it appears that vfl2 centrin is capable of binding to the basal body but is incapable of polymerizing into filamentous structures. 19 phenotypic revertants of vfl2 were isolated, and 10 of them, each of which had undergone further mutation at codon 101, were examined in detail. At the DNA level, 1 of the 10 was wild type, and the other 9 were pseudorevertants encoding centrins with the amino acids asparagine, threonine, methionine, or isoleucine at position 101. No ultrastructure defects were apparent in the revertants with asparagine or threonine at position 101, but in those with methionine or isoleucine at position 101, the distal striated fibers were found to be incomplete, indicating that different amino acid substitutions at position 101 can differentially affect the assembly of the three distinct centrincontaining fibrous structures associated with the Chlamydomonas centrosome.

Characterization of Centrin Genes in Paramecium

“…Yet both molecular and genetic analysis show that there is a single centrin gene (Huang et al, 1988). In the $2-220 mutant, a point mutation in the centrin gene leads to defective assembly of each of the three fibrous structures (Taillon et al, 1992;Taillon and Jarvik, 1995).…”

“…2) could be due to the hetereogeneity of the ICLl components, and to the unusual richness in Gln and Pro residues of their N-termini. lar mass, isoelectric point, Ca2+-binding capacity), and their ability to form filamentous structures with Caz '-dependent contractile properties, previously suggested that these proteins might be centrins (Garreau de Loubresse et al, 1991), which also form contractile fibrous arrays in other protists such as ChZamydomonas (Salisbury et al, 1988;Taillon et al, 1992;Sanders and Salisbury, 1994).…”

“…These filamentous arrays are varied in their morphology and function, as exemplified by the striated rootlets and nucleus-basal body connectors of unicellular algae (Salisbury et al, 1984(Salisbury et al, , 1988Schulze et al, 1987;Taillon et al, 1992;Katsaros et al, 1993 ;Sanders and Salisbury, 1994), or the myonemes of the ciliate Eudiplodinum maggii (David and Viguks, 1994).…”

The Uni2 Phosphoprotein is a Cell Cycle–regulated Component of the Basal Body Maturation Pathway inChlamydomonas reinhardtii

“…The bld2 mutation in the gene encoding -tubulin (Dutcher et al, 2002) and the uni3 mutation in the gene encoding ␦-tubulin (Dutcher and Trabuco, 1998) reveal the roles of these tubulins in assembly of B tubules and C tubules in triplet micro-tubules of the basal body wall. Mutations in the VFL1 gene encoding a basal body protein (Silflow et al, 2001) and in the VFL2 gene encoding centrin (Taillon et al, 1992) show the roles of these gene products in basal body positioning and segregation. Genomic and proteomic approaches have provided information on the protein composition of the flagella and basal bodies (Li et al, 2004;Keller et al, 2005;Pazour et al, 2005).…”

Binding of Centrins and Yeast Calmodulin to Synthetic Peptides Corresponding to Binding Sites in the Spindle Pole Body Components Kar1p and Spc110p

“…Centrin-based fiber contraction also plays an important role in flagellar excision in Chlamydomonas (21). Furthermore, green algal centrin is associated with the MTOC named the basal body in these organisms (22); however, the function of centrin at this location is not understood. Human cells contain two highly related centrin isogenes (8,9).…”