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First published online 28 February 2006
doi: 10.1242/jcs.02811

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Radial spoke proteins of Chlamydomonas flagella

¹ Department of Biological Sciences, Marquette University, Milwaukee, WI 53233, USA
² Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA
³ Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, 113-0033, Japan
⁴ Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA
⁵ Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA
⁶ Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030, USA
⁷ Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA

View larger version (37K): [in a new window]   Fig. 1. 2D maps of RSPs in 20S radial spoke fractions. Newly identified proteins whose sequences were completely determined are labeled in red. RSPs 15 and 18, marked in green, yielded peptides that matched regions of the genome where no gene model currently exists. Proteins identified previously are in blue. Those that remain to be identified are in black. RSPs 20, 22 and 23, which also were identified previously, are not visible because they are poorly stained by the non-formaldehyde silver stain (RSPs 20 and 23) or are too small to be retained in the gel (RSP22). The proteins were resolved in 12% (A) and 6% (B) gels following non-equilibrium isoelectric focusing. Figures only show the relevant area of pH 4.6-7.3 (A) and 5-6.5 (B). The acidic end is to the right. Horizontal bars indicate molecular markers of 130, 100, 83, 54, 40 and 24 kDa, from top to bottom.

View larger version (123K): [in a new window]   Fig. 2. Predicted sequences of the 10 newly identified RSPs. The GenBank accession numbers for the nucleotides are listed in parentheses. Peptide sequences obtained from MS are in bold font. The underlined polypeptide sequences in RSP17 (asterisks) were described previously (Pazour et al., 2005). The N-terminal end of the RSP14 sequence may not be complete.

View larger version (22K): [in a new window]   Fig. 3. Confirmation that the MS analysis identified the correct RSPs. (A) Western blot showing that antibodies based on the sequences predicted for RSPs 7, 8, 9, 10, 11 and 12 recognize proteins present in wild-type (WT) axonemes but absent in axonemes from the spokeless mutant pf14. (B) Western blot of sucrose density gradient fractions showing that an antibody based on the sequence predicted for RSP11 recognizes a protein that co-sediments with solubilized 20S WT radial spokes (upper panel) or 15S spoke stalks from the spoke-head-less mutant pf17 (lower panel). The blot was also probed with an antibody to RSP16 (Yang et al., 2005) as a marker for the 20S spokes and 15S spoke stalks. Similar results were obtained for RSP7 (data not shown). (C) Antibodies raised against RSPs 1 and 5 purified from spots on 2D gels recognize the recombinant proteins (R-RSP1 and R-RSP5). In addition, the recombinant proteins, which are His tagged, migrate at the expected size relative to the native proteins in WT axonemes. The proteins are absent in pf14 axonemes.

View larger version (15K): [in a new window]   Fig. 4. Predicted domain architecture in RSPs. MN, MORN domain; DPY, Dpy-30 motif; GAF, cyclic GMP, adenylyl cyclase, FhlA domain; CAM, 1-8-14 calmodulin-binding motif; AKAP, A-kinase anchoring protein motif; RIIa, RII alpha motif; EFH, EF-hand domain; PPI, peptidyl-prolyl isomerase motif; LRR, leucine-rich repeat; DnaJ and DnaJ-C, DnaJ-J and DnaJ-C molecular chaperone homology domains; NDK, nucleotide diphosphate kinase domain; IQ, IQ calmodulin-binding motif. Coiled-coil domains are indicated by an open bar. Spoke head proteins are indicated by white characters on a black background.

View larger version (27K): [in a new window]   Fig. 5. The five canonical EF-hand motifs in RSP7. An asterisk indicates a precise match to the Ca2+-binding loop consensus sequence D-x-[DNS]-{ILVFYW}-[DENSTG]-[DNQGHRK]-{GP}-[LIVMC]-[DENQSTAGC]-x(2)-[DE]-[LIVMFYW], where x = any amino acid, [] = either/or, and {} = any amino acid except those in brackets (PROSITE accession number PS00018). The Ca2+ ligands that contribute oxygen atoms are denoted as X, Y, Z, -Y, -X and -Z.

View larger version (35K): [in a new window]   Fig. 6. Model for radial spoke structure. The diagram illustrates the probable locations of the RSPs (A) and their molecular modules (B) relative to a central pair microtubule (right) and an inner dynein arm on an outer doublet microtubule (left). LC8, dynein light chain 8; MN, MORN domain Ox/Re, aldo-keto reductase domain. The locations of the RSPs are based on studies of mutants (Huang et al., 1981; Patel-King et al., 2004; Yang et al., 2005) and chemical dissection of the spoke (Piperno et al., 1981); RSPs 18 and 19 are not included because of insufficient experimental evidence to locate them within the radial spoke. The stoichiometry of subunits is speculative but is based on the homodimerization of RSPs 3, 16 and 22 (Benashski et al., 1997; Yang et al., 2005) (W.S. and P.Y., unpublished data), the presence of homodimerization domains in RSPs 2, 7 and 11, and other data discussed in Yang et al. (Yang et al., 2004).