HA-tagging of putative flagellar proteins in Chlamydomonas reinhardtii identifies a novel protein of intraflagellar transport complex B

Abstract

Proteomic analysis of flagella from the green alga Chlamydomonas reinhardtii has identified over 600 putative flagellar proteins. The genes encoding nine of these not previously characterized plus the previously described PACRG protein were cloned, inserted into a vector adding a triple-HA tag to the C-terminus of the gene product, and transformed into C. reinhardtii. Expression was confirmed by western blotting. Indirect immunofluorescence located all 10 fusion proteins in the flagellum; PACRG was localized to a subset of outer doublet microtubules. For some proteins, additional signal was observed in the cell body. Among the latter was FAP232-HA, which showed a spotted distribution along the flagella and an accumulation at the basal bodies. This pattern is characteristic for intraflagellar transport (IFT) proteins. FAP232-HA co-localized with the IFT protein IFT46 and co-sedimented with IFT particles in sucrose gradients. Furthermore, it co-immunoprecipitated with IFT complex B protein IFT46, but not with IFT complex A protein IFT139. We conclude that FAP232 is a novel component of IFT complex B and rename it IFT25. Homologues of IFT25 are encoded in the genomes of a subset of organisms that assemble cilia or flagella; C. reinhardtii IFT25 is 37% identical to the corresponding human protein. Genes encoding IFT25 homologues are absent from the genomes of organisms that lack cilia and flagella and, interestingly, also from those of Drosophila melanogaster and Caenorhabditis elegans, suggesting that IFT25 has a specialized role in IFT that is not required for the assembly of cilia or flagella in the worm and fly. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.

Fig. 1

HA-tagging of flagellar proteins. (A) Vector and cloning strategy used for HA tagging of flagellar proteins. HSP70B, HSP70B promoter region; 3xHA, sequence encoding triple-HA tag; 3' RbcS, Rubisco small subunit terminator sequence; +1, first base of predicted 5'-untranslated region of the target gene. (B) Western blots of whole-cell extracts from the indicated transformants and a wild-type control probed with antibodies to HA (top and right panels), α-tubulin, or centrin. Numbers at left of each panel indicate molecular weights of protein standards. (C) Immunofluorescence microscopy of transformants and wild-type control probed with anti-HA. Bar = 5 µm.

Fig. 2

Co-localization studies of FAP73-HA, FAP134-HA, and PACRG-HA. (A) Methanol-fixed cell expressing FAP73-HA stained with anti-HA (a) and anti-IFT46 (b). The merged image is shown in c. (B) Isolated flagella with protruding central pair (arrowheads in b) from a strain lacking endogenous FAP134 and expressing FAP134-HA, stained with anti-HA (a) and anti-tubulin (b). The merged image is shown in c. (C) Same as in B but from strain 4.3 expressing PACRG-HA. (D) Split axonemes of strain 4.3 expressing PACRG-HA, stained with anti-HA (a, d, g) and anti-tubulin (b, e, h). The merged images (c, f, i) show that staining representing PACRG-HA is strongly reduced or absent from some outer doublet microtubules. Strain 4.3 used for the images in C and D carries a PACRG RNAi construct in addition to the PACRG-HA vector. See Supplementary Fig. 1 for a more detailed characterization of this strain. Bars = 5 µm.

Fig. 3

FAP173-HA does not co-localize with IFT46. (A) and (B) Detached flagella from a transformant expressing FAP173-HA were double labeled with antibodies to HA (a, red in c) and IFT46 (b, green in c). The right panels show a merger of the left and center panels. Numerous foci appeared to contain FAP173-HA only (open arrowheads) or IFT46 only (arrowsheads). (C) and (D) Cells expressing FAP173-HA were deflagellated and allowed to regenerate flagella for 20 min, then fixed and labeled with anti-HA (red) and anti-IFT46 (green) antibodies. IFT46 is concentrated at the flagellar tips (arrowheads) whereas FAP173-HA is concentrated in the middle region of the flagella (arrows). Bars = 5 µm.

Fig. 4

FAP232 co-localizes with IFT46. (A) A transformant expressing FAP232-HA was double labeled with anti-HA (red) and anti-IFT46 (green). The bottom panel shows a detail of another flagellum from the same strain. There is almost complete coincidence between the FAP232-HA and IFT46 foci. Bar = 1 µm. (B) Localization of FAP232-HA during flagellar regeneration. Cells were double labeled with anti-HA and anti-tubulin. The right panel is a merged image of several regenerating cells double-labeled for FAP232-HA (red) and tubulin (green). Arrowheads indicate accumulation of FAP232-HA at the tips of the growing flagella. The time after deflagellation is indicated in minutes (T0, T30, T60, T90). (C) Induction of FAP232 message in wild-type cells after deflagellation by pH shock. Message levels were assayed by quantitative PCR.

Fig. 5

FAP232 is a component of IFT complex B. (A) Proteins in the membrane + matrix fraction obtained from flagella of cells expressing FAP232-HA were separated by sucrose density gradient centrifugation and analyzed by western blotting. The positions of marker proteins are indicated. FAP232-HA co-sediments precisely with complex B protein IFT46 but not complex A protein IFT139. (B) Western blot analysis of immunoprecipitates obtained from fractions 12 and 13 of the sucrose gradient shown in A. Protein G beads loaded with anti-HA antibody, protein A beads loaded with anti-IFT46 antibody, and protein G beads alone were used for the immunoprecipitations. The anti-HA antibody co-precipitated FAP232-HA and IFT46 but not IFT139. Similarly, the anti-IFT46 antibody co-precipitated IFT46 and FAP232-HA but not IFT139. The position of the marker proteins is indicated. (C) Western blots of immunoprecipitates obtained from membrane + matrix fractions from flagella of wild-type (control) and a strain expressing FAP232-HA, using anti-HA-loaded or unloaded (−) protein G beads. The anti-HA antibody co-precipitated FAP232-HA and complex B proteins IFT46 and IFT20, but not complex A protein IFT139.

Fig. 6

Alignment of C. reinhardtii FAP232/IFT25 with homologues from D. rerio, M. musculus, and H. sapiens. The alignment was generated by ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html).