Article Figures & Data
Figures
- FIG. 1.
Enrichment and purification of flagellar proteins and analysis of phosphoproteins in the flagellar MMA fraction. Totals of 20 μg and 25 μg of proteins from a crude extract (CE) and the flagellar MMA fraction, respectively, were separated by 9% SDS-PAGE along with a molecular mass standard and immunoblotted with specific antibodies against the C1 subunit of CHLAMY1 (A), VIPP1 (B), CK1 (C), phospho-Thr (E), and phospho-Tyr (F) or stained with Coomassie brilliant blue (D).
- FIG. 2.
Distribution of in vivo phosphorylation sites of flagellar proteins belonging to different functional categories.
- FIG. 3.
Examples of variances and clustering of phosphorylation sites. Black boxes mark identified phosphopeptides. (A) Predicted domain structure of tektin along with its tektin domain (TEK). (B) Variances of phosphorylation sites in phosphopeptide 1 of tektin. The amino acid positions are mentioned at the top. Dark and light gray backgrounds mark peptides with four and three phosphorylation sites, respectively. No background is used for the peptides with two phosphorylation sites. “p” indicates in vivo phosphorylation sites. (C) Variances of phosphorylation sites in phosphopeptide 2 of tektin. (D) Predicted structure of FAP190 along with its SAM domain. Black circles indicate nonvariable in vivo phosphorylation sites, and open circles indicate variable in vivo phosphorylation sites on Ser residues at different positions (numbers in parentheses) within the phosphopeptides.
- FIG. 4.
In vivo phosphorylation sites in structural flagellar phosphoproteins. Identified phosphopeptides are marked by black boxes in 4-fold (A, B, D, E, and G), 8-fold (C), and 16-fold (F) amplitudes. Circles indicate Ser residues, squares Thr residues, and triangles Tyr residues, which are phosphorylated at positions (numbers in parentheses) within the amino acid sequence of Vs2 protein models. Black circles, squares, or triangles mark nonvariable in vivo phosphorylation sites, and white circles, squares, or triangles mark variable in vivo phosphorylation sites (see Table S1 in the supplemental material). The domain structure was analyzed by NCBI protein BLAST homology searches. *, the depicted protein represents the Vs3 model for IC138.
- FIG. 5.
Positions of in vivo phosphorylation sites of flagellar kinases and one ATPase. Identified phosphopeptides are marked by black boxes in fourfold (A and D) and eightfold (B and C) amplitudes. Circles indicate Ser residues, squares Thr residues, and triangles Tyr residues, which are phosphorylated at positions (numbers in parentheses) within the amino acid sequence of Vs2 protein models. Black circles, squares, or triangles mark nonvariable in vivo phosphorylation sites, and open circles, squares, or triangles mark variable in vivo phosphorylation sites. The domain structure was adapted from NCBI protein BLAST homology searches. CAP, cAMP receptor protein effector domain; Ser/Thr Kin, Ser/Thr protein kinase catalytic domain; E1-E2 ATPase, E1-E2 ATPase domain; P-ATP, soluble P-type ATPase domain; A-C, cation-transporting ATPase C-terminal domain.
- FIG. 6.
Components of the flagella and the positions of its phosphoproteins (in gray). A schematic diagram of a cross section of a flagellum from C. reinhardtii (A) and a more detailed view (B), represented by the black box in panel A, are shown. The locations of the components were adapted from several studies (27, 28, 32, 33, 45, 48, 54, 58, 61). FM, flagellar membrane; O, outer dynein arm; I, inner dynein arm; R, radial spoke; N, nexin bridge; C1P, C1 central pair projection; C2P, C2 central pair projection. (B) Identified phosphorylated proteins have been depicted in gray. C1, C1 central pair of microtubules; C2, C2 central pair of microtubules; PF6, PF6 protein; H, hydin; RSP17, radial spoke protein 17; DC1, outer dynein arm docking complex 1; DC2, outer dynein arm docking complex 2; T, tektin; IC138, inner dynein arm intermediate chain protein 138; IFT, intraflagellar transport particle including intraflagellar transport protein 43.
Tables
- TABLE 1.
Functional categorization of flagellar phosphoproteinsa
Category and protein Abbreviation(s) Structural proteins of the flagella Outer dynein arm docking complex subunit 1 ODA3, ODA-DC1 Outer dynein arm docking complex protein 2 ODA1, ODA-DC2 Inner dynein arm I1 intermediate chain IC138 BOP5 Tektin, associated with inner arm dynein Radial spoke protein 17 Flagellar central pair-associated protein PF6 Hydin-like proteinb Intraflagellar transport protein IFT43 MOT41 Enzymes cGMP-dependent protein kinase Mitogen-activated protein kinase 7 FAP262; Ser/Thr protein kinase domainc FAP39; plasma membrane calcium-transporting ATPasec FAPs FAP254; putative ankyrin-like protein FAP228; callose synthase-like protein; 1,3-beta-glucan synthase component FAP230; ankyrin repeats; ion transport protein domain FAP288; EF hand FAP190; sterile alpha motif FAP33d; ankyrin repeats FAP59d; RecF/RecN/SMC N-terminal domain FAP116d; microtubule-binding protein MIP-T3 domain FAP217 FAP83 FAP1b FAP152 FAP93 FAP55 FAP18 FAP147 FAP154 FAP184 FAP263 Hypothetical protein (partially overlaps with dynein heavy chain 9) ↵ a The functions of depicted proteins are given as determined by NCBI BLASTp, along with their conserved domains.
↵ b Protein was present in the cellular phosphoproteome of C. reinhardtii.
↵ c Two FAPs with a conserved kinase and ATPase domain were included in the enzyme category.
↵ d Predicted functional domains are present only in the Vs3 model.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Table S1. Functional categorization of flagellar phosphoproteins.
PDF document, 34.3K.
- Supplemental file 1 -
Table S1. Functional categorization of flagellar phosphoproteins.
