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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

The redundant role of BBS4 and BBS5 in the context of cilia is highly conserved.(a) Human BBS4 protein directly interacts with BBS5. GST pull down assay between MBP fused human BBS4 and GST fused human BBS5. Upper panel, blotted with anti-MBP antibody. Lower panel, loading of GST and GST-BBS5 proteins was shown by Ponceau S staining. (b, c) The human ciliopathy mutation A364E in BBS4 significantly reduces the association between BBS4 and BBS5. HEK293T cells were co-transfected with Myc-tagged BBS5 and Flag-tagged BBS4 wild type (WT) or A364E mutant. The cell lysate was immunoprecipitated with normal mouse I gG or anti-Flag antibody followed by immunoblotting with anti-Flag and anti-Myc tag antibodies, respectively. Data are representative of three or more experiments. The relative amount of Myc-BBS5 or Myc-BBS5A364E was quantified. Results represented as mean ± SD. ***p < 0.001. (d) BBS4 and BBS5 co-depletion impairs ciliogenesis in hTERT-RPE-1 (RPE) cells. The percentage of ciliated cells was shown as mean ± SD. Data represent three or more experiments. **p < 0.01. n>200. (e, f) BBS4 and BBS5 co-depletion cause ~2 fold increasing for the ciliary level of PC2 in ciliated RPE cells. Anti-Acetylated tubulin (Ac-tub) antibody was used to label cilia. DNA was stained with DAPI. Scale bar, 5 μm. Dot plot shows the relative fluorescence intensity of ciliary PC2. Bars in red indicate mean ± SEM. ***p < 0.001. n > 40. (g) PC2 protein level remains unchanged after knockdown of BBS4, BBS5 or BBS8 along, or knockdown of both BBS4 and BBS5. Cell lysates were analyzed for immunoblotting using indicated antibodies. Actin was used as a loading control.
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f6: The redundant role of BBS4 and BBS5 in the context of cilia is highly conserved.(a) Human BBS4 protein directly interacts with BBS5. GST pull down assay between MBP fused human BBS4 and GST fused human BBS5. Upper panel, blotted with anti-MBP antibody. Lower panel, loading of GST and GST-BBS5 proteins was shown by Ponceau S staining. (b, c) The human ciliopathy mutation A364E in BBS4 significantly reduces the association between BBS4 and BBS5. HEK293T cells were co-transfected with Myc-tagged BBS5 and Flag-tagged BBS4 wild type (WT) or A364E mutant. The cell lysate was immunoprecipitated with normal mouse I gG or anti-Flag antibody followed by immunoblotting with anti-Flag and anti-Myc tag antibodies, respectively. Data are representative of three or more experiments. The relative amount of Myc-BBS5 or Myc-BBS5A364E was quantified. Results represented as mean ± SD. ***p < 0.001. (d) BBS4 and BBS5 co-depletion impairs ciliogenesis in hTERT-RPE-1 (RPE) cells. The percentage of ciliated cells was shown as mean ± SD. Data represent three or more experiments. **p < 0.01. n>200. (e, f) BBS4 and BBS5 co-depletion cause ~2 fold increasing for the ciliary level of PC2 in ciliated RPE cells. Anti-Acetylated tubulin (Ac-tub) antibody was used to label cilia. DNA was stained with DAPI. Scale bar, 5 μm. Dot plot shows the relative fluorescence intensity of ciliary PC2. Bars in red indicate mean ± SEM. ***p < 0.001. n > 40. (g) PC2 protein level remains unchanged after knockdown of BBS4, BBS5 or BBS8 along, or knockdown of both BBS4 and BBS5. Cell lysates were analyzed for immunoblotting using indicated antibodies. Actin was used as a loading control.

Mentions: Since all BBSome components are highly conserved across ciliated species during evolution, we asked whether our unexpected discoveries for BBS-4 and BBS-5 in C. elegans are also relevant in mammalian cells. We first validated the direct interaction between human BBS4 and BBS5 via GST pull-down assay (Fig. 6a). We then confirmed that Myc-tagged BBS5 could co-immunoprecipitate with Flag-tagged BBS4 after co-transfected into HEK293T cells (Fig. 6b). Similar to what we observed for worm proteins, introducing A364E mutation in BBS4 significantly reduced the association between BBS4 and BBS5 (Fig. 6b,c).


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)

The redundant role of BBS4 and BBS5 in the context of cilia is highly conserved.(a) Human BBS4 protein directly interacts with BBS5. GST pull down assay between MBP fused human BBS4 and GST fused human BBS5. Upper panel, blotted with anti-MBP antibody. Lower panel, loading of GST and GST-BBS5 proteins was shown by Ponceau S staining. (b, c) The human ciliopathy mutation A364E in BBS4 significantly reduces the association between BBS4 and BBS5. HEK293T cells were co-transfected with Myc-tagged BBS5 and Flag-tagged BBS4 wild type (WT) or A364E mutant. The cell lysate was immunoprecipitated with normal mouse I gG or anti-Flag antibody followed by immunoblotting with anti-Flag and anti-Myc tag antibodies, respectively. Data are representative of three or more experiments. The relative amount of Myc-BBS5 or Myc-BBS5A364E was quantified. Results represented as mean ± SD. ***p < 0.001. (d) BBS4 and BBS5 co-depletion impairs ciliogenesis in hTERT-RPE-1 (RPE) cells. The percentage of ciliated cells was shown as mean ± SD. Data represent three or more experiments. **p < 0.01. n>200. (e, f) BBS4 and BBS5 co-depletion cause ~2 fold increasing for the ciliary level of PC2 in ciliated RPE cells. Anti-Acetylated tubulin (Ac-tub) antibody was used to label cilia. DNA was stained with DAPI. Scale bar, 5 μm. Dot plot shows the relative fluorescence intensity of ciliary PC2. Bars in red indicate mean ± SEM. ***p < 0.001. n > 40. (g) PC2 protein level remains unchanged after knockdown of BBS4, BBS5 or BBS8 along, or knockdown of both BBS4 and BBS5. Cell lysates were analyzed for immunoblotting using indicated antibodies. Actin was used as a loading control.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493597&req=5

f6: The redundant role of BBS4 and BBS5 in the context of cilia is highly conserved.(a) Human BBS4 protein directly interacts with BBS5. GST pull down assay between MBP fused human BBS4 and GST fused human BBS5. Upper panel, blotted with anti-MBP antibody. Lower panel, loading of GST and GST-BBS5 proteins was shown by Ponceau S staining. (b, c) The human ciliopathy mutation A364E in BBS4 significantly reduces the association between BBS4 and BBS5. HEK293T cells were co-transfected with Myc-tagged BBS5 and Flag-tagged BBS4 wild type (WT) or A364E mutant. The cell lysate was immunoprecipitated with normal mouse I gG or anti-Flag antibody followed by immunoblotting with anti-Flag and anti-Myc tag antibodies, respectively. Data are representative of three or more experiments. The relative amount of Myc-BBS5 or Myc-BBS5A364E was quantified. Results represented as mean ± SD. ***p < 0.001. (d) BBS4 and BBS5 co-depletion impairs ciliogenesis in hTERT-RPE-1 (RPE) cells. The percentage of ciliated cells was shown as mean ± SD. Data represent three or more experiments. **p < 0.01. n>200. (e, f) BBS4 and BBS5 co-depletion cause ~2 fold increasing for the ciliary level of PC2 in ciliated RPE cells. Anti-Acetylated tubulin (Ac-tub) antibody was used to label cilia. DNA was stained with DAPI. Scale bar, 5 μm. Dot plot shows the relative fluorescence intensity of ciliary PC2. Bars in red indicate mean ± SEM. ***p < 0.001. n > 40. (g) PC2 protein level remains unchanged after knockdown of BBS4, BBS5 or BBS8 along, or knockdown of both BBS4 and BBS5. Cell lysates were analyzed for immunoblotting using indicated antibodies. Actin was used as a loading control.
Mentions: Since all BBSome components are highly conserved across ciliated species during evolution, we asked whether our unexpected discoveries for BBS-4 and BBS-5 in C. elegans are also relevant in mammalian cells. We first validated the direct interaction between human BBS4 and BBS5 via GST pull-down assay (Fig. 6a). We then confirmed that Myc-tagged BBS5 could co-immunoprecipitate with Flag-tagged BBS4 after co-transfected into HEK293T cells (Fig. 6b). Similar to what we observed for worm proteins, introducing A364E mutation in BBS4 significantly reduced the association between BBS4 and BBS5 (Fig. 6b,c).

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