Dimeric novel HSP40 is incorporated into the radial spoke complex during the assembly process in flagella

• Published in: Molecular biology of the cell 2005-02, Vol.16 (2), p.637-648
• Main Authors: Yang, Chun, Compton, Mark M, Yang, Pinfen
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Blotting, Western; Cell Fractionation; Chlamydomonas; Chlamydomonas - chemistry; Chlamydomonas - genetics; Chlamydomonas - metabolism; Chromatography, Affinity; Chromatography, Liquid; Chromosome Mapping; Cloning, Molecular; Dimerization; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Flagella - chemistry; Flagella - metabolism; Heat-Shock Proteins - chemistry; Heat-Shock Proteins - metabolism; HSP40 Heat-Shock Proteins; Molecular Sequence Data; Mutation; Phylogeny; Protein Structure, Tertiary; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Recombinant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid
• ISSN: 1059-1524; 1939-4586; 1059-1524
• DOI: 10.1091/mbc.E04-09-0787

The radial spoke is a stable structural complex in the 9 + 2 axoneme for the control of flagellar motility. However, the spokes in Chlamydomonas mutant pf24 are heterogeneous and unstable, whereas several spoke proteins are reduced differentially. To elucidate the defective mechanism, we clone RSP16, a prominent spoke protein diminished in pf24 axonemes. Unexpectedly, RSP16 is a novel HSP40 member of the DnaJ superfamily that assists chaperones in various protein-folding-related processes. Importantly, RSP16 is uniquely excluded from the 12S spoke precursor complex that is packaged in the cell body and transported toward the flagellar tip to be converted into mature 20S axonemal spokes. Rather, RSP16, transported separately, joins the precursor complex in flagella. Furthermore, RSP16 molecules in vitro and in flagella form homodimers, a characteristic required for the cochaperone activity of HSP40. We postulate that the spoke HSP40 operates as a cochaperone to assist chaperone machinery at the flagellar tip to actively convert the smaller spoke precursor and itself into the mature stable complex; failure of the interaction between the spoke HSP40 and its target polypeptide results in heterogeneous unstable radial spokes in pf24.