Dataset Information

PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.

ABSTRACT: Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.

SUBMITTER: Smith EF

PROVIDER: S-EPMC2120723 | biostudies-other | 1996 Feb

REPOSITORIES: biostudies-other

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PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.

Smith E F EF Lefebvre P A PA

The Journal of cell biology 19960201 3

Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the centr ...[more]

PMID: 8636214

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Project description:BackgroundFlagella and cilia are fine thread-like organelles protruding from cells that harbour them. The typical '9 + 2' cilia confer motility on these cells. Although the mechanistic details of motility remain elusive, the dynein-driven motility is regulated by various kinases and phosphatases. A-kinase anchoring proteins (AKAPs) are scaffolds that bind to a variety of such proteins. Usually, they are known to possess a dedicated domain that in vitro interacts with the regulatory subunits (RI and RII) present in the cAMP-dependent protein kinase (PKA) holoenzyme. These subunits conventionally harbour contiguous stretches of a.a. residues that reveal the presence of the Dimerization Docking (D/D) domain, Catalytic interface domain and cAMP-Binding domain. The Chlamydomonas reinhardtii flagella harbour two AKAPs; viz., the radial spoke AKAP97 or RSP3 and the central pair AKAP240. Both these were identified on the basis of their RII-binding property. Interestingly, AKAP97 binds in vivo to two RII-like proteins (RSP7 and RSP11) that contain only the D/D domain.ResultsWe found a Chlamydomonas Flagellar Associated Protein (FAP174) orthologous to MYCBP-1, a protein that binds to organellar AKAPs and Myc onco-protein. An in silico analysis shows that the N-terminus of FAP174 is similar to those RII domain-containing proteins that have binding affinities to AKAPs. Binding of FAP174 was tested with the AKAP97/RSP3 using in vitro pull down assays; however, this binding was rather poor with AKAP97/RSP3. Antibodies were generated against FAP174 and the cellular localization was studied using Western blotting and immunoflourescence in wild type and various flagella mutants. We show that FAP174 localises to the central pair of the axoneme. Using overlay assays we show that FAP174 binds AKAP240 previously identified in the C2 portion of the central pair apparatus.ConclusionIt appears that the flagella of Chlamydomonas reinhardtii contain proteins that bind to AKAPs and except for the D/D domain, lack the conventional a.a. stretches of PKA regulatory subunits (RSP7 and RSP11). We add FAP174 to this growing list.

Dataset Information

PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.

Publications

PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.

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