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BBS4 and BBS5 show functional redundancy in the BBSome to regulate the degradative sorting of ciliary sensory receptors.

Xu Q, Zhang Y, Wei Q, Huang Y, Li Y, Ling K, Hu J - Sci Rep (2015)

Bottom Line: However, the mechanisms underlying the ciliary homeostasis of sensory receptors remain elusive.Here, we demonstrate that BBS-4 and BBS-5, two distinct BBSome components, show unexpected functional redundancy in the context of cilia in C. elegans.Further analyses indicate that co-depletion of BBS-4 and BBS-5 disrupts the lysosome-targeted degradative sorting of ciliary sensory receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA.

ABSTRACT
Cilia harbor sensory receptors for various signaling cascades critical for vertebrate development. However, the mechanisms underlying the ciliary homeostasis of sensory receptors remain elusive. Here, we demonstrate that BBS-4 and BBS-5, two distinct BBSome components, show unexpected functional redundancy in the context of cilia in C. elegans. BBS-4 directly interacts with BBS-5 and the interaction can be disrupted by a conserved mutation identified in human BBS4. Surprisingly, we found that BBS-4 and BBS-5 act redundantly in the BBSome to regulate the ciliary removal, rather than the ciliary entry or retrograde IFT transport, of various sensory receptors. Further analyses indicate that co-depletion of BBS-4 and BBS-5 disrupts the lysosome-targeted degradative sorting of ciliary sensory receptors. Moreover, mammalian BBS4 and BBS5 also interact directly and coordinate the ciliary removal of polycystin 2. Hence, we reveal a novel and highly conserved role for the BBSome in fine-tuning ciliary signaling by regulating the ciliary removal of sensory receptors for lysosomal degradation.

No MeSH data available.


Related in: MedlinePlus

BBS-4 and BBS-5 directly associate, and the interaction is abolished by a conserved ciliopathy mutation identified in BBS4 patients.(a) BBS-4 directly interacts with BBS-5 via its carboxyl-terminus. GST pull down assay was performed to determine the association between GST fused BBS-5 and MBP fused BBS-4 variants. BBS-4 wild type (WT), BBS-4 with two human ciliopathy related mutations (E107Q and A388E) and BBS-4 fragments (N-Term, amino-terminus, a.a. 1-280; C-Term, carboxyl-terminus, a.a. 270-462) were included. Upper panel, blotted with anti-MBP antibody. Lower panel, loading of GST and GST-BBS5 proteins was shown by Commassie Blue staining. (b) BBS-4-BBS-5 interaction was impaired by ciliopathy mutation A388E in BBS-4. The results shown in (a) were quantified. (c) BiFC was used to visualize the in vivo BBS-4-BBS-5 association. Stable fluorescence complementation between BBS-4 and BBS-5 was impaired by A388E, but not E107Q, mutation in BBS-4. (d) A388E, but not E107Q, compromises the ciliary targeting of BBS-4. Arrows and arrowheads indicate the base and tip of cilia, respectively. (e) BBS-4A388E was unable to rescue ciliogenesis defect in bbs-4; bbs-5 mutants. # denotes under-detectable dye-filling. Data represent three or more experiments. In each experiment, n > 40 were counted in each group. Results represented as mean ± SD. N.S., not statistically significant. ***p < 0.001. Scale bars, 5 μm.
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f3: BBS-4 and BBS-5 directly associate, and the interaction is abolished by a conserved ciliopathy mutation identified in BBS4 patients.(a) BBS-4 directly interacts with BBS-5 via its carboxyl-terminus. GST pull down assay was performed to determine the association between GST fused BBS-5 and MBP fused BBS-4 variants. BBS-4 wild type (WT), BBS-4 with two human ciliopathy related mutations (E107Q and A388E) and BBS-4 fragments (N-Term, amino-terminus, a.a. 1-280; C-Term, carboxyl-terminus, a.a. 270-462) were included. Upper panel, blotted with anti-MBP antibody. Lower panel, loading of GST and GST-BBS5 proteins was shown by Commassie Blue staining. (b) BBS-4-BBS-5 interaction was impaired by ciliopathy mutation A388E in BBS-4. The results shown in (a) were quantified. (c) BiFC was used to visualize the in vivo BBS-4-BBS-5 association. Stable fluorescence complementation between BBS-4 and BBS-5 was impaired by A388E, but not E107Q, mutation in BBS-4. (d) A388E, but not E107Q, compromises the ciliary targeting of BBS-4. Arrows and arrowheads indicate the base and tip of cilia, respectively. (e) BBS-4A388E was unable to rescue ciliogenesis defect in bbs-4; bbs-5 mutants. # denotes under-detectable dye-filling. Data represent three or more experiments. In each experiment, n > 40 were counted in each group. Results represented as mean ± SD. N.S., not statistically significant. ***p < 0.001. Scale bars, 5 μm.

Mentions: It is believed that during the assembly of the BBSome, BBS2, 7, and 9 form the core, then BBS1, 5, 8, and finally BBS4 are added in a stepwise manner1336. Given that BBS-4 and BBS-5 act redundantly and both are probably peripheral components of the BBSome, we hypothesized that BBS-4 and BBS-5 may directly interact with each other. Indeed, we observed the interaction in GST pull-down assay. We further mapped down the binding region in BBS-4 to the C-terminal 193 amino acids (a.a. 270–462) (Fig. 3a). To test whether BBS-4-BBS-5 association exists in vivo, we performed bimolecular fluorescence complementation (BiFC) assay in worms. BiFC assay was developed for direct visualization of protein-protein interaction in the same macromolecular complex in their natural environment, and we have successfully applied this approach to examine the in vivo association between IFT components within worm cilia2537. As expected, strong fluorescence complementation between BBS-4 and BBS-5 was observed (Fig. 3c), indicative of in vivo BBS-4-BBS-5 association.


BBS4 and BBS5 show functional redundancy in the BBSome to regulate the degradative sorting of ciliary sensory receptors.

Xu Q, Zhang Y, Wei Q, Huang Y, Li Y, Ling K, Hu J - Sci Rep (2015)

BBS-4 and BBS-5 directly associate, and the interaction is abolished by a conserved ciliopathy mutation identified in BBS4 patients.(a) BBS-4 directly interacts with BBS-5 via its carboxyl-terminus. GST pull down assay was performed to determine the association between GST fused BBS-5 and MBP fused BBS-4 variants. BBS-4 wild type (WT), BBS-4 with two human ciliopathy related mutations (E107Q and A388E) and BBS-4 fragments (N-Term, amino-terminus, a.a. 1-280; C-Term, carboxyl-terminus, a.a. 270-462) were included. Upper panel, blotted with anti-MBP antibody. Lower panel, loading of GST and GST-BBS5 proteins was shown by Commassie Blue staining. (b) BBS-4-BBS-5 interaction was impaired by ciliopathy mutation A388E in BBS-4. The results shown in (a) were quantified. (c) BiFC was used to visualize the in vivo BBS-4-BBS-5 association. Stable fluorescence complementation between BBS-4 and BBS-5 was impaired by A388E, but not E107Q, mutation in BBS-4. (d) A388E, but not E107Q, compromises the ciliary targeting of BBS-4. Arrows and arrowheads indicate the base and tip of cilia, respectively. (e) BBS-4A388E was unable to rescue ciliogenesis defect in bbs-4; bbs-5 mutants. # denotes under-detectable dye-filling. Data represent three or more experiments. In each experiment, n > 40 were counted in each group. Results represented as mean ± SD. N.S., not statistically significant. ***p < 0.001. Scale bars, 5 μm.
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Related In: Results  -  Collection

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f3: BBS-4 and BBS-5 directly associate, and the interaction is abolished by a conserved ciliopathy mutation identified in BBS4 patients.(a) BBS-4 directly interacts with BBS-5 via its carboxyl-terminus. GST pull down assay was performed to determine the association between GST fused BBS-5 and MBP fused BBS-4 variants. BBS-4 wild type (WT), BBS-4 with two human ciliopathy related mutations (E107Q and A388E) and BBS-4 fragments (N-Term, amino-terminus, a.a. 1-280; C-Term, carboxyl-terminus, a.a. 270-462) were included. Upper panel, blotted with anti-MBP antibody. Lower panel, loading of GST and GST-BBS5 proteins was shown by Commassie Blue staining. (b) BBS-4-BBS-5 interaction was impaired by ciliopathy mutation A388E in BBS-4. The results shown in (a) were quantified. (c) BiFC was used to visualize the in vivo BBS-4-BBS-5 association. Stable fluorescence complementation between BBS-4 and BBS-5 was impaired by A388E, but not E107Q, mutation in BBS-4. (d) A388E, but not E107Q, compromises the ciliary targeting of BBS-4. Arrows and arrowheads indicate the base and tip of cilia, respectively. (e) BBS-4A388E was unable to rescue ciliogenesis defect in bbs-4; bbs-5 mutants. # denotes under-detectable dye-filling. Data represent three or more experiments. In each experiment, n > 40 were counted in each group. Results represented as mean ± SD. N.S., not statistically significant. ***p < 0.001. Scale bars, 5 μm.
Mentions: It is believed that during the assembly of the BBSome, BBS2, 7, and 9 form the core, then BBS1, 5, 8, and finally BBS4 are added in a stepwise manner1336. Given that BBS-4 and BBS-5 act redundantly and both are probably peripheral components of the BBSome, we hypothesized that BBS-4 and BBS-5 may directly interact with each other. Indeed, we observed the interaction in GST pull-down assay. We further mapped down the binding region in BBS-4 to the C-terminal 193 amino acids (a.a. 270–462) (Fig. 3a). To test whether BBS-4-BBS-5 association exists in vivo, we performed bimolecular fluorescence complementation (BiFC) assay in worms. BiFC assay was developed for direct visualization of protein-protein interaction in the same macromolecular complex in their natural environment, and we have successfully applied this approach to examine the in vivo association between IFT components within worm cilia2537. As expected, strong fluorescence complementation between BBS-4 and BBS-5 was observed (Fig. 3c), indicative of in vivo BBS-4-BBS-5 association.

Bottom Line: However, the mechanisms underlying the ciliary homeostasis of sensory receptors remain elusive.Here, we demonstrate that BBS-4 and BBS-5, two distinct BBSome components, show unexpected functional redundancy in the context of cilia in C. elegans.Further analyses indicate that co-depletion of BBS-4 and BBS-5 disrupts the lysosome-targeted degradative sorting of ciliary sensory receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA.

ABSTRACT
Cilia harbor sensory receptors for various signaling cascades critical for vertebrate development. However, the mechanisms underlying the ciliary homeostasis of sensory receptors remain elusive. Here, we demonstrate that BBS-4 and BBS-5, two distinct BBSome components, show unexpected functional redundancy in the context of cilia in C. elegans. BBS-4 directly interacts with BBS-5 and the interaction can be disrupted by a conserved mutation identified in human BBS4. Surprisingly, we found that BBS-4 and BBS-5 act redundantly in the BBSome to regulate the ciliary removal, rather than the ciliary entry or retrograde IFT transport, of various sensory receptors. Further analyses indicate that co-depletion of BBS-4 and BBS-5 disrupts the lysosome-targeted degradative sorting of ciliary sensory receptors. Moreover, mammalian BBS4 and BBS5 also interact directly and coordinate the ciliary removal of polycystin 2. Hence, we reveal a novel and highly conserved role for the BBSome in fine-tuning ciliary signaling by regulating the ciliary removal of sensory receptors for lysosomal degradation.

No MeSH data available.


Related in: MedlinePlus