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© The Rockefeller University Press,
0021-9525/1998//1149 $5.00
The Journal of Cell Biology, Volume 140, Number 5,
, 1998 1149-1158
Article |
A Novel 95-kD Protein Is Located in a Linker between Cytoplasmic Microtubules and Basal Bodies in a Green Flagellate and Forms Striated Filaments In Vitro
The flagellar basal apparatus comprises the basal bodies and the attached fibrous structures, which together form the organizing center for the cytoskeleton in many flagellated cells. Basal apparatus were isolated from the naked green flagellate Spermatozopsis similis and shown to be composed of several dozens of different polypeptides including a protein band of 95 kD. Screening of a cDNA library of S. similis with a polyclonal antibody raised against the 95-kD band resulted in a full-length clone coding for a novel protein of 834 amino acids (90.3 kD). Sequence analysis identified nonhelical NH2- and COOH-terminal domains flanking a central domain of
650 residues, which was predicted to form a series of coiled-coils interrupted by short spacer segments. Immunogold labeling using a polyclonal antibody raised against the bacterially expressed 95-kD protein exclusively decorated the striated, wedge-shaped fibers, termed sinister fibers (sf-fibers), attached to the basal bodies of S. similis. Striated fibers with a periodicity of 98 nm were assembled in vitro from the purified protein expressed from the cloned cDNA indicating that the 95-kD protein could be a major component of the sf-fibers. This structure interconnects specific triplets of the basal bodies with the microtubular bundles that emerge from the basal apparatus. The sf-fibers and similar structures, e.g., basal feet or satellites, described in various eukaryotes including vertebrates, may be representative for cytoskeletal elements involved in positioning of basal bodies/centrioles with respect to cytoskeletal microtubules and vice versa.
Abbreviations used in this paper: IPTG, isopropyl-β-D-thiogalactopyranoside; sf-fibers, sinister fibers; SMAF, striated microtubule-associated fibers.
This work is dedicated to the memory of Annette Teltenkötter (2 May 1997).
Address all correspondence to Karl-F. Lechtreck, Botanisches Institut, Gyrhofstrasse 15, Universität zu Köln, D-50931 Köln, Germany. Tel.: 49-221-470-3795. Fax: 49-221-470-5181. E-mail: kflecht{at}novell.biolan.uni-koeln.de
J. Clees' present address is Universitätskrankenhaus Eppendorf, Zentrum für Molekulare Neurobiologie, Institut für Biosynthese Neuraler Strukturen, Martinistrasse 85, D-20251 Hamburg, Germany.
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