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PMID: 10637302 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

The Rib43a protein is associated with forming the specialized protofilament ribbons of flagellar microtubules in Chlamydomonas.

Molecular biology of the cell ·Vol. 11 ·No. 1 ·2000-01-00 ·Pages 201-15

Norrander JM, deCathelineau AM, Brown JA, Porter ME, Linck RW

Abstract

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.

MeSH Terms
Algal Proteins/genetics,metabolism Amino Acid Sequence Animals Base Sequence Chlamydomonas reinhardtii/genetics,metabolism,ultrastructure Chromosome Mapping Cloning, Molecular Cross Reactions Humans Microscopy, Electron Microscopy, Fluorescence Microtubules/metabolism Molecular Sequence Data Mutation Protozoan Proteins Rabbits Sequence Analysis
Chemicals
Algal Proteins Protozoan Proteins
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Norrander J M
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455, USA.
deCathelineau A M
Brown J A
Porter M E
Linck R W
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Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1059-1524
DOI
10.1091/mbc.11.1.201
Published
2000-01-00
Pages
201-15
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC14768
Subset
IM
Grants
NIGMS NIH HHS · GM-55667 · United States
NIGMS NIH HHS · GM-35648 · United States
NIGMS NIH HHS · R01 GM035648 · United States
NIGMS NIH HHS · R01 GM055667 · United States
NIGMS NIH HHS · R37 GM055667 · United States
Databases
GENBANK
AF196576, AF196577
External Links
PubMed source DOI: 10.1091/mbc.11.1.201 PMC Full Text View journal details
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