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

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 centrin-containing fibrous structures associated with the Chlamydomonas centrosome.