The cruciated microtubule-associated fibers of the green alga Dunaliella bioculata consist of a 31 kDa SF-assemblin

Karl-Ferdinand Lechtreck 2 Sandra Frins 1 Joachim Bilski 1 Annette Teltenktter 1 Klaus Weber 0 Michael Melkonian 1 0 Max Planck Institute for Biophysical Chemistry, Department of Biochemistry , PO Box 2841, D-37018 Gottingen , Germany 1 Botanisches Institut der Universitat zu Koln , Gyrhofstrasse 15, D-50931 Koln , Germany 2 University of Minnesota, Department of Genetics and Cell Biology , St Paul, MN 55108-1095 , USA SUMMARY Cytoskeletons of Dunaliella bioculata, the biflagellate wallless green alga, were isolated and analyzed using a monoclonal and a polyclonal antibody raised against SFassemblin, the major protein of the two striated microtubule-associated fibers of the alga Spermatozopsis similis. Indirect immunofluorescence showed antigenic structures associated with the four microtubular flagellar roots. SDS-PAGE followed by immunoblot analysis revealed a cross-reacting polypeptide of 31 kDa. This protein of D. bioculata was isolated using gel filtration chromatography in 8 M urea and in vitro reassembly of striated fibers. Microsequencing of the purified protein yielded various peptides, which could be aligned along the sequence of SF-assemblin from S. similis. A complete sequence of the Dunaliella protein was The basal bodies of flagellate/ciliate eukaryotic cells are commonly associated with microtubular and fibrous flagellar roots (Pitelka, 1969). The latter can be classified into at least two types, which differ in their ultrastructure and biochemical composition (Lechtreck and Melkonian, 1991a). One is contractile (system-II-fibers) and consists predominantly of Centrin/Caltractin, a calcium-binding EF-hand type protein (reviewed by Melkonian et al., 1992). In general centrinrelated proteins are associated with the centrosome of eukaryotes (Lee and Huang, 1993; Ogawa and Shimizu, 1993; Errabolu et al., 1994). The second type of fibrous flagellar roots (system I fibers) is composed of fine filaments (2-4 nm diameter), which exhibit a narrowly cross-striated banding. They are apparently noncontractile (Dingle and Larson, 1981). Here we refer to the kinetodesmal fibers of ciliates (Rubin and Cunningham, 1973; Williams et al., 1979; Hyams and King, 1985), the flagellar rootlets of the amoeboflagellate Naegleria gruberi (Larson and Dingle, 1981), the striated rootlets of the ciliate gill epithelium of molluscs (Stephens, 1975), and the striated microtubuleassociated fibers of flagellate green algae (Lechtreck and obtained by cDNA cloning. It documents the non helical head domain followed by a helical rod domain with a 29 residue repeat pattern based on four heptads followed by a skip residue. Compared to SF-assemblin of S. similis the SF-assemblin of Dunaliella has a shorter head and a slightly longer rod domain. The two algal SF-assemblins share only 57% sequence identity. We conclude that SFassemblin and related proteins in various protists are representatives of a new class of a -helical proteins characterized by the ability to form a special segmented coiled coil and to assemble into striated fibers of 2 nm protofilaments in vivo and in vitro. Melkonian, 1991b). Recently, the striated microtubule-associated fibers of the flagellate green alga Spermatozopsis similis were isolated, reassembled in vitro, and a 34 kDa protein (SFassemblin) was identified as the major constituent (Lechtreck and Melkonian, 1991b). Interestingly, the only protein found in the data banks with a significant homology to SF-assemblin is b -giardin, a 30 kDa protein from the parasitic protozoan flagellate Giardia lamblia (Holberton et al., 1988; Weber et al., 1993). b -giardin is located in striated fibers associated with the complex microtubular structure of the sucking disk (Peattie et al., 1989). SF-assemblin and b -giardin consist of a small nonhelical N-terminal head domain and a rod domain of 253 amino acids. They show a striking structural similarity over the entire rod domain i.e. a 29 residue repeat pattern based on four heptads followed by a skip residue (Weber et al., 1993). A similar structure for proteins forming microtubule-associated bundles of 2 nm filaments in an archezoan protist and a green alga indicates that related molecules may be widespread among eukaryotes. Despite their structural homology, SF-assemblin and b giardin show only low sequence identity (<20%). Therefore we were interested in the evolutionary conservation of the structure and sequence of SF-assemblin. For this purpose the cytoskeletons of the marine green flagellate Dunaliella spp., which contain striated fibers associated with all four microtubular roots in contrast to the two striated fibers described in Spermatozopsis similis, were analyzed with antibodies against SFassemblin. The immunoreactive 31 kDa protein was purified from D. bioculata, reassembled into striated fibers and the sequence was determined from cDNA clones. SF-assemblin from Spermatozopsis and the homologous protein of Dunaliella share only 57% sequence identity, indicating that although the protein structure is retained, sequence divergence has occurred. MATERIAL AND METHODS Strains and culture conditions Dunaliella bioculata (strain number SAG 19-4, Sammlung von Algenkulturen, University of Gttingen), D. primolecta (SAG 183.80), D. minuta (PLY 430, Plymouth Culture Collection, Plymouth, UK), and D. tertiolecta (CCMP MCIIIA, ProvasoliGuillard Center for Culture of Marine Phytoplankton, West Boothbay Habor, Maine, USA) were cultured in the artificial sea water medium ASP-H (McFadden and Melkonian, 1986). The culture conditions were 15C, 20 mE m- 2 second- 1, and a L/D-cycle of 14/10 hours. The procedures for culturing Spermatozopsis similis (strain number SAG B 1.85) have been described (Lechtreck and Melkonian, 1991b). Isolated basal apparatuses of S. similis were purified by sucrose gradient centrifugation, resuspended in PBS (1.5 mg/ml), sonified, and mixed with Complete Freunds Adjuvant (Sigma). Three month old female mice ((C57 BL/6 BALB/c)F1) were injected subcutaneously with 190 m g antigen and after four weeks again with 120 m g antigen without Freunds Adjuvant. Five days after the final boost the spleens were removed and the cells were fused to the myeloma cell line X63-Ag8.653 (Kearney et al., 1979). Hybridoma cells were produced following the method of Khler and Milstein (1976) and plated into microtiter plates with mouse peritoneal macrophage feeder cells. Once hybridoma colonies appeared the supernatants were analyzed against purified SF-assemblin by ELISA (Bremerich et al., 1995). Six positive clones were identified and clone BAS 6.5 was subcloned by limited dilution. Immunolocalization techniques Cells of D. bioculata and D. tertiolecta were harvested by centrifugation (100 g, 5 minutes, 15C), washed once with culture medium (60-100 g, 2-5 minutes, RT), resuspended in MT/Mg2+ (30 mM Hepes, 15 mM KCl, 5 mM Na-EGTA, 5 mM MgSO4, pH 7) and lysed by the addition of an equal volume MT/Mg2+ including 0.2 or 2% (...truncated)