Ciliary and flagellar microtubules contain a specialized set of three protofilaments, termed ribbons, that are composed of
tubulin and several associated
proteins. Previous studies of sea urchin sperm flagella identified three of the ribbon
proteins as
tektins, which form coiled-coil filaments in doublet microtubules and which are associated with basal bodies and centrioles. To study the function of
tektins and other ribbon
proteins in the assembly of flagella and basal bodies, we have begun an analysis of ribbons from the unicellular biflagellate, Chlamydomonas reinhardtii, and report here the molecular characterization of the ribbon
protein rib43a. Using
antibodies against rib43a to screen an expression library, we recovered a full-length
cDNA clone that encodes a 42,657-Da
polypeptide. On Northern blots, the rib43a
cDNA hybridized to a 1. 7-kb transcript, which was up-regulated upon deflagellation, consistent with a role for rib43a in flagellar assembly. The
cDNA was used to isolate RIB43a, an approximately 4.6-kb genomic clone containing the complete rib43a coding region, and restriction fragment length polymorphism analysis placed the RIB43a gene on linkage group III. Sequence analysis of the RIB43a gene indicates that the substantially coiled-coil rib43a
protein shares a high degree of sequence identity with clones from Trypanosoma cruzi and Homo sapiens (genomic, normal fetal kidney, and endometrial and
germ cell tumors) but little sequence similarity to other
proteins including
tektins. Affinity-purified
antibodies against native and bacterially expressed rib43a stained both flagella and basal bodies by immunofluorescence microscopy and stained isolated flagellar ribbons by immuno-electron microscopy. The structure of rib43a and its association with the specialized protofilament ribbons and with basal bodies is relevant to the proposed role of ribbons in forming and stabilizing doublet and triplet microtubules and in organizing their three-dimensional structure.