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. 2009 Dec 28;187(7):1117-32.
doi: 10.1083/jcb.200909183.

The Chlamydomonas Reinhardtii BBSome Is an IFT Cargo Required for Export of Specific Signaling Proteins From Flagella

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Free PMC article

The Chlamydomonas Reinhardtii BBSome Is an IFT Cargo Required for Export of Specific Signaling Proteins From Flagella

Karl-Ferdinand Lechtreck et al. J Cell Biol. .
Free PMC article

Abstract

In humans, seven evolutionarily conserved genes that cause the cilia-related disorder Bardet-Biedl syndrome (BBS) encode proteins that form a complex termed the BBSome. The function of the BBSome in the cilium is not well understood. We purified a BBSome-like complex from Chlamydomonas reinhardtii flagella and found that it contains at least BBS1, -4, -5, -7, and -8 and undergoes intraflagellar transport (IFT) in association with a subset of IFT particles. C. reinhardtii insertional mutants defective in BBS1, -4, and -7 assemble motile, full-length flagella but lack the ability to phototax. In the bbs4 mutant, the assembly and transport of IFT particles are unaffected, but the flagella abnormally accumulate several signaling proteins that may disrupt phototaxis. We conclude that the BBSome is carried by IFT but is an adapter rather than an integral component of the IFT machinery. C. reinhardtii BBS4 may be required for the export of signaling proteins from the flagellum via IFT.

Figures

Figure 1.
Figure 1.
Identification of C. reinhardtii bbs1, -4, and -7 mutants. (A) Map showing the genomic region near the BBS4 locus. Black arrows indicate PCR primer pairs; plus and minus signs indicate whether or not these primers gave PCR products. bbs4-1 lacks the entire BBS4 gene. The 6,797-bp fragment used to rescue strain bbs4-1 is indicated. JGI protein IDs of the predicted gene products are shown. (B) Map showing the genomic region near the BBS7 locus. Only one of the tested primer pairs failed to amplify in the ptx6-1 strain. Sequencing of this region revealed the insertion of a retrotransposal footprint into the BBS7 gene of ptx6-1, causing a premature stop after 52 codons. (C) Map showing the genomic region near the BBS1 locus. Approximately half of BBS1 is deleted in bbs1-1. Gene orientation is marked by white arrows. wt, wild type.
Figure 2.
Figure 2.
BBS4 is a flagellar protein required for phototaxis but not IFT. (A) Motion analysis of wild type, bbs4-1, and R26, a strain rescued with a genomic fragment encompassing the BBS4 gene. The direction of light (arrow) is indicated. The radial histograms show the percentage of cells moving in a particular direction relative to the light (24 bins, each 15°). (B) Flagellar length of g1, bbs4-1, bbs7-1, and bbs1-1, as determined by DIC. (C) The velocity of anterograde (a.) and retrograde (r.) IFT particles in wild-type (g1) and bbs4-1 flagella, as determined by DIC. (D) Velocity of KAP-GFP in BBS4 and bbs4-1 flagella, as determined by TIRFM. (C and D) SDs are indicated. (E) Detached flagella from wild type (a–c) or the bbs4-1 mutant (d–f) were stained with anti-IFT46 (a and d) and anti-IFT139 (b and e). (F) Western blot probing proteins of whole cells (a) or isolated flagella (b) with anti-BBS4 (a and b) and anti-IFT139 (a) or anti-IC2, which is specific for an axonemal protein (b), as loading controls. Anti-BBS4 stained a band of ∼41 kD (arrowhead) in whole-cell and flagella samples of wild type and the rescued strain R26; BBS4 was undetectable in bbs4-1. Molecular masses are indicated in kilodaltons. (G) Detached flagella from wild type and the bbs4-1 mutant were stained with anti–acetylated tubulin (ac-tubulin; a and c) and anti-BBS4 (b and d). Bars, 5 µm.
Figure 3.
Figure 3.
BBS4 has an IFT-like distribution within the cell. (A) Dish phototaxis assay of wild type, bbs4-1, and BBS4HA21 rescued by BBS4-3xHA. (B) Western blot of flagella from the strains shown in C probed with monoclonal anti-HA. (C) BBS4HA21 cells labeled by anti–α-tubulin and anti-HA. BBS4-3xHA is located near the basal bodies (arrowhead) and in dots along both flagella. (D) Western blot of BBS4HA21 to compare the amounts of various proteins in cell bodies (CB) versus isolated flagella (FLA). 1× indicates that approximately two flagella were loaded per cell body, etc. The blot was probed for BBS4-HA, IC2, and IFT57 and -81. BBS4-3xHA is about eight times more abundant in cell bodies than in flagella. (E) Western blot analyzing equivalent amounts of whole flagella, axonemes (AXO), and NP-40–soluble membrane-plus-matrix (M&M) of flagella from BBS4HA21 and wild-type cells. Antibodies were as indicated; those to IC2 and various IFT particle proteins were used as controls. (F) Western blot comparing equivalent amounts of wild-type flagella, axonemes, AP, and DP resulting from Triton X-114 phase partitioning. The blot was probed with the antibodies indicated. (B and D–F) The positions of standard proteins and their molecular masses in kilodaltons are indicated. Bar, 5 µm.
Figure 4.
Figure 4.
BBS4 forms a complex with other BBS proteins in the flagellum. (A) Proteins in the flagellar membrane-plus-matrix from BBS4HA21 (top) or wild type (bottom) were separated by sucrose gradient centrifugation, and fractions were analyzed by Western blotting using antibodies to the HA tag or BBS4 and IFT172, -139, and -46. White lines seperate images of the membranes used for this blot. (B) Silver-stained SDS gel of protein complexes affinity purified from the membrane-plus-matrix fraction of BBS4HA21 and wild-type (wt) flagella using anti-HA beads. Proteins identified by MS are indicated. (C) Western blot analysis of isolated flagella or whole-cell extracts from the strains indicated. The blots were probed with antibodies to BBS4 and, as loading controls, IC2 (flagella) or IFT139 (cells). ptx1-1 and ptx7-1 are control nonphototactic strains. (A–C) The positions of standard proteins and their molecular masses in kilodaltons are indicated.
Figure 5.
Figure 5.
BBSomes are less abundant than IFT particles in C. reinhardtii flagella. (A and B) Double immunolabeling of a BBS4HA21 cell with polyclonal anti-HA and anti-IFT139 (A) or anti-IFT46 (B). In B, cells were deflagellated by pH shock and allowed to regrow flagella for ∼10 (a–c), 20 (d–f), and 45 min (g–i). BBS4-HA and the IFT proteins colocalize at the flagellar base, and the signals partially overlap in the flagella. Arrowheads indicate flagellar tips. (C) Detached wild-type flagella were double labeled with anti-BBS4 (a) and anti-IFT139 (b). Closed arrowheads indicate anti-IFT139 signals that did not colocalize with anti-BBS4 signals; the open arrowhead indicates anti-BBS4 signal that did not colocalize with anti-IFT139 staining. (D) Relative abundance of the native and the AQUA peptide LLETLNEDVK of IFT81. The AQUA peptide ion (590.831) and a peptide ion of monoisotopic m/z = 587.323, corresponding to the native LLETLNEDVK peptide ion (theoretical m/z value of 587.322), are marked. For details see Fig. S4. (E) As in D but for the LYVEQTQR peptide of BBS1. Bars: (A and B) 5 µm; (C) 1 µm.
Figure 6.
Figure 6.
BBS4 is moved by IFT. (A) BBS4-GFP undergoes IFT. (a) Cell attached to the glass surface by its flagella (arrowheads). (b) TIRFM image of the two flagella of a BBS4-GFP1 cell. (c) Kymogram revealing the anterograde and retrograde movement of BBS4-GFP in the flagella. (B) Western blot of flagella isolated from wild-type control (g1), bbs4-1, IFT20-mCherry, and strain MxG1.3 expressing BBS4-GFP and IFT20-mCherry. The blots were probed as indicated; the positions of the immunoreactive proteins and standard proteins and their molecular masses in kilodaltons are marked. (C) The velocity of fluorescent protein–tagged IFT20 and BBS4 in flagella, as determined by TIRFM. In the BBS4-GFP/IFT20-mCherry strain, only particles with both tags were scored. n, number of particles analyzed. (D) Frequency of BBS4-GFP and fluorescent protein–tagged IFT20, as determined by TIRFM. n, number of flagella analyzed. (C and D) SDs are indicated. (E) Single frame from simultaneous recording of BBS4-GFP and IFT20-mCherry. Flagella are marked by arrowheads, and the cell body is marked by an arrow. (F) Kymograms showing the movement of BBS4-GFP (a) and IFT20-mCherry (b) in one flagellum. The image in c is a merger of a and b. Arrowheads and arrows indicate anterograde and retrograde cotransport of both proteins, respectively. Several tracks representing IFT particles are devoid of BBS4-GFP. Note the fission of a BBS4-GFP signal (arrowhead 1), a part of which is later picked up by a different IFT particle (arrowhead 2). (G) A BBS4-GFP particle falls off (arrowheads) a moving IFT20-mCherry particle (arrows). See Video 7. Bar, 10 µm.
Figure 7.
Figure 7.
Biochemical defects in bbs4-1 flagella. (A) Silver-stained 5–15% SDS–polyacrylamide gel of flagella (FLA), axonemes (AXO), AP, and DP from wild type (g1) and bbs4-1. Proteins present in the AP and DP of bbs4-1 (arrows 1–4) but not in the corresponding fractions from wild type were identified by MS analysis as PLDc (band 1), THB1 (band 2), STPK (band 3), and JGI protein ID 191821 (band 4; see Fig. S3). (B) Details of 2D gels (from Fig. S5 C) separating matrix proteins from wild type (a and c) and bbs4-1 flagella (b and d). Protein spots 1 (b) and 3 (d) were enriched in the matrix of bbs4-1 and revealed to be THB1 and STPK, respectively, by MS. Protein spot 2 (a), which was reduced in quantity in the bbs4-1 matrix, was identified as OEE3. (C) Detail of a silver-stained SDS gel of the DP from the strains indicated. The arrow indicates the position of PLDc. (D) Detail of a silver-stained SDS gel and Western blots of the AP of g1, bbs4-1, and the retrograde IFT mutant dhc1bts probed with antibodies to the indicated proteins. The arrow indicates the position of THB1. (A–D) The positions of standard proteins and their molecular masses in kilodaltons are indicated.
Figure 8.
Figure 8.
Phototaxis is partially restored in bbs4-1 during flagellar regeneration. Dish phototaxis assays of wild type (g1) and bbs4-1 during flagellar regeneration. Cells were deflagellated by pH shock and allowed to regenerate flagella. Samples were transferred to culture dishes and exposed to directional light (direction indicated by arrows) for ∼10 min. T43, T52, T69, and T120 refer to the time in minutes since deflagellation. Controls were treated identically except that the pH shock was omitted.

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