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. 2006 Mar 15;119(Pt 6):1165-74.
doi: 10.1242/jcs.02811. Epub 2006 Feb 28.

Radial Spoke Proteins of Chlamydomonas Flagella

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Free PMC article

Radial Spoke Proteins of Chlamydomonas Flagella

Pinfen Yang et al. J Cell Sci. .
Free PMC article

Abstract

The radial spoke is a ubiquitous component of '9+2' cilia and flagella, and plays an essential role in the control of dynein arm activity by relaying signals from the central pair of microtubules to the arms. The Chlamydomonas reinhardtii radial spoke contains at least 23 proteins, only 8 of which have been characterized at the molecular level. Here, we use mass spectrometry to identify 10 additional radial spoke proteins. Many of the newly identified proteins in the spoke stalk are predicted to contain domains associated with signal transduction, including Ca2+-, AKAP- and nucleotide-binding domains. This suggests that the spoke stalk is both a scaffold for signaling molecules and itself a transducer of signals. Moreover, in addition to the recently described HSP40 family member, a second spoke stalk protein is predicted to be a molecular chaperone, implying that there is a sophisticated mechanism for the assembly of this large complex. Among the 18 spoke proteins identified to date, at least 12 have apparent homologs in humans, indicating that the radial spoke has been conserved throughout evolution. The human genes encoding these proteins are candidates for causing primary ciliary dyskinesia, a severe inherited disease involving missing or defective axonemal structures, including the radial spokes.

Figures

Fig. 1
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.
Fig. 2
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.
Fig. 3
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.
Fig. 4
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.
Fig. 5
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.
Fig. 6
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).

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