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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 function redundantly in the downregulation of sensory receptors from cilia.(a) Cartoons illustrating the constructs expressing Ubi-PKD-2, Ubi-PKD-2S534D or UbiK48R-PKD-2 proteins. All constructs were tagged with GFP to visualize in live animals. (b-d) The degradative sorting of Ubi-PKD-2, Ubi-PKD-2S534D, and UbiK48R-PKD-2 are compromised in bbs-4; bbs-5 double and bbs-7 single mutants. mCherry-tagged MKS-5 was co-expressed as a transition zone marker to label the ciliary base. (e-f) The degradative sorting of Ubi-OSM-9 and UbiK48R-OSM-9 are compromised in amphid OLQ cilia in bbs-4; bbs-5 double and bbs-7 single mutants. (g-h) The degradative sorting of Ubi-ODR-10 and UbiK48R-ODR-10 are compromised in amphid AWA fan-shaped cilia in bbs-4; bbs-5 double and bbs-7 single mutants. Arrows and arrowheads indicate the cilia base and tip, respectively. Brackets indicate distal dendrites. Scale bars, 5 μm.
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f4: BBS-4 and BBS-5 function redundantly in the downregulation of sensory receptors from cilia.(a) Cartoons illustrating the constructs expressing Ubi-PKD-2, Ubi-PKD-2S534D or UbiK48R-PKD-2 proteins. All constructs were tagged with GFP to visualize in live animals. (b-d) The degradative sorting of Ubi-PKD-2, Ubi-PKD-2S534D, and UbiK48R-PKD-2 are compromised in bbs-4; bbs-5 double and bbs-7 single mutants. mCherry-tagged MKS-5 was co-expressed as a transition zone marker to label the ciliary base. (e-f) The degradative sorting of Ubi-OSM-9 and UbiK48R-OSM-9 are compromised in amphid OLQ cilia in bbs-4; bbs-5 double and bbs-7 single mutants. (g-h) The degradative sorting of Ubi-ODR-10 and UbiK48R-ODR-10 are compromised in amphid AWA fan-shaped cilia in bbs-4; bbs-5 double and bbs-7 single mutants. Arrows and arrowheads indicate the cilia base and tip, respectively. Brackets indicate distal dendrites. Scale bars, 5 μm.

Mentions: Direct conjugation of an ubiquitin to the target protein has been successfully used as a tool to study the endocytic removal of membrane proteins642. We previously reported that Ubi-PKD-2-GFP (a GFP tagged PKD-2 with an ubiquitin conjugated to the amino terminus, see Fig. 4a) was largely absent from cilia due to the enhanced degradative sorting6. As expected, Ubi-PKD-2-GFP was not detected in WT, bbs-4 or bbs-5 cilia (Fig. 4b). However, in bbs-4; bbs-5 or bbs-7 mutants, Ubi-PKD-2-GFP strongly accumulated both inside and below cilia (Fig. 4b), suggesting that the degradative sorting of ubiquitinated PKD-2 is compromised in these bbs mutants. The detectable signal for Ubi-PKD-2-GFP inside cilia indicates that the ciliary import of receptors is not disrupted in bbs-4; bbs-5 or bbs-7 mutants. We reported that Casein Kinase 2 modulates PKD-2 activity by phosphorylating its S534 site7. PKD-2S534D mutant protein mimics the constitutively phosphorylated PKD-2 at S534 site and may represent a hyperactive PKD-2 channel7. Similar to ubiquitinated PKD-2, PKD-2S534D is absent from WT cilia due to the enhanced degradative sorting67. In good agreement with the observations made with Ubi-PKD-2-GFP, the downregulation of PKD-2S534D-GFP from cilia was also disrupted in bbs-4; bbs-5 or bbs-7 mutants (Fig. 4c).


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 function redundantly in the downregulation of sensory receptors from cilia.(a) Cartoons illustrating the constructs expressing Ubi-PKD-2, Ubi-PKD-2S534D or UbiK48R-PKD-2 proteins. All constructs were tagged with GFP to visualize in live animals. (b-d) The degradative sorting of Ubi-PKD-2, Ubi-PKD-2S534D, and UbiK48R-PKD-2 are compromised in bbs-4; bbs-5 double and bbs-7 single mutants. mCherry-tagged MKS-5 was co-expressed as a transition zone marker to label the ciliary base. (e-f) The degradative sorting of Ubi-OSM-9 and UbiK48R-OSM-9 are compromised in amphid OLQ cilia in bbs-4; bbs-5 double and bbs-7 single mutants. (g-h) The degradative sorting of Ubi-ODR-10 and UbiK48R-ODR-10 are compromised in amphid AWA fan-shaped cilia in bbs-4; bbs-5 double and bbs-7 single mutants. Arrows and arrowheads indicate the cilia base and tip, respectively. Brackets indicate distal dendrites. Scale bars, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: BBS-4 and BBS-5 function redundantly in the downregulation of sensory receptors from cilia.(a) Cartoons illustrating the constructs expressing Ubi-PKD-2, Ubi-PKD-2S534D or UbiK48R-PKD-2 proteins. All constructs were tagged with GFP to visualize in live animals. (b-d) The degradative sorting of Ubi-PKD-2, Ubi-PKD-2S534D, and UbiK48R-PKD-2 are compromised in bbs-4; bbs-5 double and bbs-7 single mutants. mCherry-tagged MKS-5 was co-expressed as a transition zone marker to label the ciliary base. (e-f) The degradative sorting of Ubi-OSM-9 and UbiK48R-OSM-9 are compromised in amphid OLQ cilia in bbs-4; bbs-5 double and bbs-7 single mutants. (g-h) The degradative sorting of Ubi-ODR-10 and UbiK48R-ODR-10 are compromised in amphid AWA fan-shaped cilia in bbs-4; bbs-5 double and bbs-7 single mutants. Arrows and arrowheads indicate the cilia base and tip, respectively. Brackets indicate distal dendrites. Scale bars, 5 μm.
Mentions: Direct conjugation of an ubiquitin to the target protein has been successfully used as a tool to study the endocytic removal of membrane proteins642. We previously reported that Ubi-PKD-2-GFP (a GFP tagged PKD-2 with an ubiquitin conjugated to the amino terminus, see Fig. 4a) was largely absent from cilia due to the enhanced degradative sorting6. As expected, Ubi-PKD-2-GFP was not detected in WT, bbs-4 or bbs-5 cilia (Fig. 4b). However, in bbs-4; bbs-5 or bbs-7 mutants, Ubi-PKD-2-GFP strongly accumulated both inside and below cilia (Fig. 4b), suggesting that the degradative sorting of ubiquitinated PKD-2 is compromised in these bbs mutants. The detectable signal for Ubi-PKD-2-GFP inside cilia indicates that the ciliary import of receptors is not disrupted in bbs-4; bbs-5 or bbs-7 mutants. We reported that Casein Kinase 2 modulates PKD-2 activity by phosphorylating its S534 site7. PKD-2S534D mutant protein mimics the constitutively phosphorylated PKD-2 at S534 site and may represent a hyperactive PKD-2 channel7. Similar to ubiquitinated PKD-2, PKD-2S534D is absent from WT cilia due to the enhanced degradative sorting67. In good agreement with the observations made with Ubi-PKD-2-GFP, the downregulation of PKD-2S534D-GFP from cilia was also disrupted in bbs-4; bbs-5 or bbs-7 mutants (Fig. 4c).

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