Myc-binding protein orthologue interacts with AKAP240 in the central pair apparatus of the Chlamydomonas flagella.
Flagella 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.



We 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.



It 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.
UM-DAE Centre for Excellence in Basic Sciences, Kalina campus, Santacruz (E), Mumbai, 400098, India.
Venkatramanan G
VG
Rao
UM-DAE Centre for Excellence in Basic Sciences, Kalina campus, Santacruz (E), Mumbai, 400098, India.
Ruhi B
RB
Sarafdar
UM-DAE Centre for Excellence in Basic Sciences, Kalina campus, Santacruz (E), Mumbai, 400098, India.
Twinkle S
TS
Chowdhury
Wehr Life Sciences, Marquette University, P.O. Box 1881, Milwaukee, WI, 53201-1881, USA.
Priyanka
P
Sivadas
Wehr Life Sciences, Marquette University, P.O. Box 1881, Milwaukee, WI, 53201-1881, USA.
Pinfen
P
Yang
Department of Biophysics, University of Mumbai, Kalina campus, Santacruz (E), Mumbai, 400098, India.
Prabhakar M
PM
Dongre
UM-DAE Centre for Excellence in Basic Sciences, Kalina campus, Santacruz (E), Mumbai, 400098, India. jacinta@cbs.ac.in.
Jacinta S
JS
D'Souza
D000595:Amino Acid Sequence; D002696:Chlamydomonas / Q000378:metabolism*; D005407:Flagella / Q000378:metabolism*; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D000072417:Protein Domains; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D010940:Plant Proteins; D011994:Recombinant Proteins
A-kinase anchoring proteins (AKAPs); Central pair; Chlamydomonas reinhardtii; FAP174; Flagella; MYCBP-1