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The cytoplasmic tail of fibrocystin contains a ciliary targeting sequence.

Follit JA, Li L, Vucica Y, Pazour GJ - J. Cell Biol. (2010)

Bottom Line: This motif is sufficient to target green fluorescent protein (GFP) to cilia of ciliated cells and targets GFP to lipid rafts if the cells are not ciliated.Dominant-negative Rab8 interacts more strongly than wild-type or constitutively active Rab8, and coexpression of this dominant-negative mutant Rab8 blocks trafficking to the cilium.This suggests that the CTS functions by binding regulatory proteins like Rab8 to control trafficking through the endomembrane system and on to the cilium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.

ABSTRACT
Sensory functions of primary cilia rely on ciliary-localized membrane proteins, but little is known about how these receptors are targeted to the cilium. To further our understanding of this process, we dissected the ciliary targeting sequence (CTS) of fibrocystin, the human autosomal recessive polycystic kidney disease gene product. We show that the fibrocystin CTS is an 18-residue motif localized in the cytoplasmic tail. This motif is sufficient to target green fluorescent protein (GFP) to cilia of ciliated cells and targets GFP to lipid rafts if the cells are not ciliated. Rab8, but not several other Rabs implicated in ciliary assembly, binds to the CTS in a coimmunoprecipitation assay. Dominant-negative Rab8 interacts more strongly than wild-type or constitutively active Rab8, and coexpression of this dominant-negative mutant Rab8 blocks trafficking to the cilium. This suggests that the CTS functions by binding regulatory proteins like Rab8 to control trafficking through the endomembrane system and on to the cilium.

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The CTS is associated with lipid. (A) Comparison of the CTS from fibrocystin to the lipid raft targeting sequence of SNAP25 (Gonzalo et al., 1999). (B) Colocalization of GFP-CTS with lipid rafts. (a–a″) Live cells stained with Alexa Fluor 594–conjugated cholera toxin. In nonciliated cells, cholera toxin shows extensive colocalization with the GFP-CTS in the cell body (the arrows point at one example). (b–b″) Cholera toxin was cross-linked by antibody before fixation, which caused the toxin and GFP to cluster (arrows). (c–c″) In ciliated cells, GFP and cholera toxin colocalize in the cilium (arrows). (d–d″) The lipid raft targeting sequence of Snap25 does not target GFP to the cilium (arrows). Bar, 5 µm. (C) Tritiated palmitate is incorporated into the wild-type but not the cysteine-mutated CTS (arrow). (D) The CTS cysteines mediate interaction with membranes. Cells expressing either wild-type or cysteine-mutated CTS-GFP were lysed and fractionated by an OptiPrep gradient.
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fig2: The CTS is associated with lipid. (A) Comparison of the CTS from fibrocystin to the lipid raft targeting sequence of SNAP25 (Gonzalo et al., 1999). (B) Colocalization of GFP-CTS with lipid rafts. (a–a″) Live cells stained with Alexa Fluor 594–conjugated cholera toxin. In nonciliated cells, cholera toxin shows extensive colocalization with the GFP-CTS in the cell body (the arrows point at one example). (b–b″) Cholera toxin was cross-linked by antibody before fixation, which caused the toxin and GFP to cluster (arrows). (c–c″) In ciliated cells, GFP and cholera toxin colocalize in the cilium (arrows). (d–d″) The lipid raft targeting sequence of Snap25 does not target GFP to the cilium (arrows). Bar, 5 µm. (C) Tritiated palmitate is incorporated into the wild-type but not the cysteine-mutated CTS (arrow). (D) The CTS cysteines mediate interaction with membranes. Cells expressing either wild-type or cysteine-mutated CTS-GFP were lysed and fractionated by an OptiPrep gradient.

Mentions: Although we did not detect any significant homology between the fibrocystin CTS and other nonfibrocystin sequences, we noted similarity between the CTS amino acid composition and a lipid raft targeting sequence in SNAP25 (Fig. 2 A; Salaün et al., 2005). This suggested that the punctuate spots to which the CTS localized might be lipid rafts. To test this, live CTS-GFP–expressing cells were labeled with fluorescent cholera toxin B (Fig. 2 B). Cholera toxin B binds GM1 gangliosides and is a marker for membrane domains enriched in these lipids. In nonciliated cells, there is strong colocalization between the CTS-GFP spots and the cholera toxin-binding sites (Fig. 2 B, a). The colocalization is also observed if the cholera toxin is cross-linked with an antibody and then fixed (Fig. 2 B, b). Cholera toxin B labels the cilium (Fig. 2 B, c), confirming a previous report that this organelle is enriched in GM1 gangliosides (Janich and Corbeil, 2007). SNAP25 has been reported to localize to cilia (Low et al., 1998), but the lipid raft targeting sequence of SNAP25 was not sufficient to target GFP to cilia (Fig. 2 B, d), indicating that a lipid raft targeting sequence alone is not sufficient for ciliary targeting.


The cytoplasmic tail of fibrocystin contains a ciliary targeting sequence.

Follit JA, Li L, Vucica Y, Pazour GJ - J. Cell Biol. (2010)

The CTS is associated with lipid. (A) Comparison of the CTS from fibrocystin to the lipid raft targeting sequence of SNAP25 (Gonzalo et al., 1999). (B) Colocalization of GFP-CTS with lipid rafts. (a–a″) Live cells stained with Alexa Fluor 594–conjugated cholera toxin. In nonciliated cells, cholera toxin shows extensive colocalization with the GFP-CTS in the cell body (the arrows point at one example). (b–b″) Cholera toxin was cross-linked by antibody before fixation, which caused the toxin and GFP to cluster (arrows). (c–c″) In ciliated cells, GFP and cholera toxin colocalize in the cilium (arrows). (d–d″) The lipid raft targeting sequence of Snap25 does not target GFP to the cilium (arrows). Bar, 5 µm. (C) Tritiated palmitate is incorporated into the wild-type but not the cysteine-mutated CTS (arrow). (D) The CTS cysteines mediate interaction with membranes. Cells expressing either wild-type or cysteine-mutated CTS-GFP were lysed and fractionated by an OptiPrep gradient.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812845&req=5

fig2: The CTS is associated with lipid. (A) Comparison of the CTS from fibrocystin to the lipid raft targeting sequence of SNAP25 (Gonzalo et al., 1999). (B) Colocalization of GFP-CTS with lipid rafts. (a–a″) Live cells stained with Alexa Fluor 594–conjugated cholera toxin. In nonciliated cells, cholera toxin shows extensive colocalization with the GFP-CTS in the cell body (the arrows point at one example). (b–b″) Cholera toxin was cross-linked by antibody before fixation, which caused the toxin and GFP to cluster (arrows). (c–c″) In ciliated cells, GFP and cholera toxin colocalize in the cilium (arrows). (d–d″) The lipid raft targeting sequence of Snap25 does not target GFP to the cilium (arrows). Bar, 5 µm. (C) Tritiated palmitate is incorporated into the wild-type but not the cysteine-mutated CTS (arrow). (D) The CTS cysteines mediate interaction with membranes. Cells expressing either wild-type or cysteine-mutated CTS-GFP were lysed and fractionated by an OptiPrep gradient.
Mentions: Although we did not detect any significant homology between the fibrocystin CTS and other nonfibrocystin sequences, we noted similarity between the CTS amino acid composition and a lipid raft targeting sequence in SNAP25 (Fig. 2 A; Salaün et al., 2005). This suggested that the punctuate spots to which the CTS localized might be lipid rafts. To test this, live CTS-GFP–expressing cells were labeled with fluorescent cholera toxin B (Fig. 2 B). Cholera toxin B binds GM1 gangliosides and is a marker for membrane domains enriched in these lipids. In nonciliated cells, there is strong colocalization between the CTS-GFP spots and the cholera toxin-binding sites (Fig. 2 B, a). The colocalization is also observed if the cholera toxin is cross-linked with an antibody and then fixed (Fig. 2 B, b). Cholera toxin B labels the cilium (Fig. 2 B, c), confirming a previous report that this organelle is enriched in GM1 gangliosides (Janich and Corbeil, 2007). SNAP25 has been reported to localize to cilia (Low et al., 1998), but the lipid raft targeting sequence of SNAP25 was not sufficient to target GFP to cilia (Fig. 2 B, d), indicating that a lipid raft targeting sequence alone is not sufficient for ciliary targeting.

Bottom Line: This motif is sufficient to target green fluorescent protein (GFP) to cilia of ciliated cells and targets GFP to lipid rafts if the cells are not ciliated.Dominant-negative Rab8 interacts more strongly than wild-type or constitutively active Rab8, and coexpression of this dominant-negative mutant Rab8 blocks trafficking to the cilium.This suggests that the CTS functions by binding regulatory proteins like Rab8 to control trafficking through the endomembrane system and on to the cilium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.

ABSTRACT
Sensory functions of primary cilia rely on ciliary-localized membrane proteins, but little is known about how these receptors are targeted to the cilium. To further our understanding of this process, we dissected the ciliary targeting sequence (CTS) of fibrocystin, the human autosomal recessive polycystic kidney disease gene product. We show that the fibrocystin CTS is an 18-residue motif localized in the cytoplasmic tail. This motif is sufficient to target green fluorescent protein (GFP) to cilia of ciliated cells and targets GFP to lipid rafts if the cells are not ciliated. Rab8, but not several other Rabs implicated in ciliary assembly, binds to the CTS in a coimmunoprecipitation assay. Dominant-negative Rab8 interacts more strongly than wild-type or constitutively active Rab8, and coexpression of this dominant-negative mutant Rab8 blocks trafficking to the cilium. This suggests that the CTS functions by binding regulatory proteins like Rab8 to control trafficking through the endomembrane system and on to the cilium.

Show MeSH
Related in: MedlinePlus