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 Stefan Geimer, Judith Clees, Michael Melkonian, and Karl-Ferdinand Lechtreck Botanisches Institut, Universität zu Köln, D-50931 Köln, Germany Abstract. The flagellar basal apparatus comprises the 95-kD protein exclusively decorated the striated, basal bodies and the attached fibrous structures, which wedge-shaped fibers, termed sinister fibers (sf-fibers), together form the organizing center for the cytoskele- attached to the basal bodies of S. similis. Striated fibers ton in many flagellated cells. Basal apparatus were iso- with a periodicity of 98 nm were assembled in vitro lated from the naked green flagellate Spermatozopsis from the purified protein expressed from the cloned Downloaded from similis and shown to be composed of several dozens of cDNA indicating that the 95-kD protein could be a ma- different polypeptides including a protein band of 95 kD. jor component of the sf-fibers. This structure intercon- Screening of a cDNA library of S. similis with a poly- nects specific triplets of the basal bodies with the micro- clonal antibody raised against the 95-kD band resulted tubular bundles that emerge from the basal apparatus. in a full-length clone coding for a novel protein of 834 The sf-fibers and similar structures, e.g., basal feet or amino acids (90.3 kD). Sequence analysis identified satellites, described in various eukaryotes including jcb.rupress.org nonhelical NH2- and COOH-terminal domains flanking vertebrates, may be representative for cytoskeletal el- a central domain of z650 residues, which was predicted ements involved in positioning of basal bodies/centri- to form a series of coiled-coils interrupted by short oles with respect to cytoskeletal microtubules and spacer segments. Immunogold labeling using a poly- vice versa. clonal antibody raised against the bacterially expressed on September 9, 2017 he base of eukaryotic flagella is formed by special- central structures of the basal apparatus—a functional ho- ized microtubular structures, basal bodies. The struc- mologue of the centrosomal complex of animal cells Tture of the basal bodies, consisting of nine microtu- (Baron and Salisbury, 1992). The basal apparatus consists bular triplets, is highly conserved and also characteristic for of the basal bodies and a diverse array of filaments at- centrioles, which are part of the centrosome in many eu- tached to them. Sets of flagellar root microtubules usually karyotic cells. During sexual and asexual reproduction, basal radiate from the basal apparatus and form the major part bodies are often converted into centrioles, indicating their of the microtubular cytoskeleton (which is involved in the functional similarity (Kellogg et al., 1994). It is generally determination of polarity, shape, and internal organization accepted that basal bodies/centrioles function as a template of flagellated cells) (Moestrup, 1982; Melkonian, 1984; for the structure of the axoneme (Lange and Gull, 1996b). Holmes and Dutcher, 1989). Thus, analysis of the flagellar An open question is whether or not centrioles contribute basal apparatus could provide information about the basal to the centrosome of animal cells, for example, by organiz- bodies/centrioles as centrosomal organizers. ing its microtubule nucleation sites or by controlling its du- In green flagellates like Chlamydomonas reinhardtii, two plication. types of the filaments present in the basal apparatus have The basal bodies of many flagellated cells not only serve been analyzed in detail (Lechtreck and Melkonian, 1991a). as assembly sites for the axoneme but also represent the One is characterized by the presence of centrin, a 20-kD protein belonging to the EF-hand family of calcium-bind- ing proteins (Huang et al., 1988). Centrin-based filaments Address all correspondence to Karl-F. Lechtreck, Botanisches Institut, (system II fibers; Melkonian, 1980) form a connection be- Gyrhofstrasse 15, Universität zu Köln, D-50931 Köln, Germany. Tel.: 49-221- tween the basal bodies, interconnect the basal bodies and 470-3795. Fax: 49-221-470-5181. E-mail: [email protected] J. Clees’ present address is Universitätskrankenhaus Eppendorf, Zen- the cell nucleus, and are part of the flagellar transitional trum für Molekulare Neurobiologie, Institut für Biosynthese Neuraler region; they undergo calcium-induced contractions and Strukturen, Martinistrasse 85, D-20251 Hamburg, Germany. are involved in various motile functions of the basal appa- The Rockefeller University Press, 0021-9525/98/03/1149/10 $2.00 The Journal of Cell Biology, Volume 140, Number 5, March 9, 1998 1149–1158 http://www.jcb.org 1149 This document was created with FrameMaker 4.0.4 ratus (Melkonian et al., 1992; Salisbury, 1995). The other Tris-HCl, pH 7.4, 2 mM EDTA), disintegrated by homogenization, well-characterized filaments of the basal apparatus are washed with TE buffer/0.5 % Triton X-100 (800 g, 30 min), and then the suspension was loaded onto a discontinuous sucrose gradient (6 ml 60%, the noncontractile, striated microtubule-associated fibers 9 ml 50%, 9 ml 40%, and 6 ml 20% in TE buffer/0.1% Triton X-100) and 1 (SMAFs, or system I fibers) (Melkonian, 1980) that are centrifuged for 1 h at 12,500 g in a swing-out rotor (HB-4; Sorvall, Bad composed of striated fiber (SF)-assemblin, a highly a-heli- Homburg, Germany). Highly enriched basal apparatus were collected cal 30-kD protein (Lechtreck and Melkonian, 1991b; We- from a milky band below the 20–40% sucrose interphase, and the proteins ber et al., 1993). These are attached to the four bundles of were separated by preparative SDS-PAGE. A band of 95 kD was excised from Coomassie blue–stained gels, concentrated by electrophoresis, and flagellar root microtubules characteristic for Chlamydo- then blotted onto nitrocellulose membrane. The membrane with z10 mg monas-like flagellates and are thought to function as stabi- of the 95-kD band was dissolved in dimethyl sulfoxide, mixed with com- lizing elements in the basal apparatus (Lechtreck and Sil- plete Freunds adjuvant, and subcutanously injected at several sites into a flow, 1997). Electron microscopical studies have revealed young, male rabbit. For subsequent booster injections on day 15 and 53, we used acrylamide gel pieces with z10 mg antigen, homogenized in PBS, additional filamentous structures attached to the basal and then mixed with incomplete Freunds adjuvant. An IgG fraction (anti- bodies in the green algal basal apparatus (Ringo, 1967; p95) was purified from the serum taken 10 d after the second injection us- Beech and Melkonian, 1993; Geimer et al., 1997a): transi- ing a fast protein liquid chromatography (FPLC) system (Pharmacia LKB, tional fibers (connectives between the basal bodies and Uppsala, Sweden) and a protein A column (Pharmacia LKB). the plasma membrane), proximal plates or connecting fi- bers, and linkers between basal bodies and flagellar root Cloning and Sequencing of a cDNA Coding for the microtubules. The latter may establish the spatial relation- 95-kD Protein ship between the basal bodies and the flagellar root micro- Total RNA from S. similis was isolated by the phenol/SDS method basi- tubules, as well as direct the microtubular roots to specific cally following the protocol of Palmiter (1974). To purify mRNA, a self- locations in the cell. In contrast to centrin- and SF-assemb- packed oligo-dT column (Boehringer Mannheim GmbH, Mannheim, lin–based fibers, these other fibers and linkers are rela- Germany) was used. A Uni-Zap XR cDNA library was constructed using a cDNA synthesis and cloning kit (Stratagene, Heidelberg, Germany) ac- Downloaded from tively small, making it more difficult to identify their bio- cording to the manufacturer’s instructions with the following modifica- chemical composition. tions: first-strand synthesis was done for 15 min, 458C; 20 min, 508C; and We have recently developed a method to obtain homo- 20 min, 558C, with reverse transcriptase from GIBCO BRL (Eggenstein, geneous preparations of basal apparatus from the naked, Germany). The library was screened with anti-p95, and positive clones were analyzed by restriction mapping and partial sequencing after in vivo biflagellate green alga Spermatozopsis similis (Geimer et al., excision of the pBluescript phagemid. In addition, bacterial lysates ob- 1997b). The complex, filamentous system surrounding the tained from isopropyl-b-d-thiogalactopyranoside (IPTG)-induced cul- two basal bodies in the isolated state has been studied in tures were analyzed with anti-p95 in Western blots. A 3.1-kb clone (clone jcb.rupress.org detail by serial thin section electron microscopy (Geimer 7/1) produced an immunoreactive band of 95 kD. The insert was se- a quenced completely in both directions (Abiprism, 310 Gene Analyzer; et al., 1997 ). Besides the major proteins of the basal appa- Perkin Elmer Applied Biosystems, Weiterstadt, Germany). The AutoAs- ratus (tubulin, centrin, and SF-assemblin) more than two sembler 1.4.0 (Perkin Elmer Applied Biosystems) program was used for dozen different polypeptides were present in our prepara- gene assembly and the sequence was analyzed using programs (COILS, tions. Here we identify a novel 95-kD protein as a compo- FASTA, BLAST) provided by European Bioinformatics Institute (EBI; nent of the sinister fibers (sf-fibers), which are linkers be- http://www.ebi.ac.uk/services/services.html) and Baylor College of Medi-