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First published online 14 March 2006
doi: 10.1242/jcs.02818

Journal of Cell Science 119, 1383-1395 (2006)
Published by The Company of Biologists 2006
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Polycystin-2 traffics to cilia independently of polycystin-1 by using an N-terminal RVxP motif

Lin Geng1,*, Dayne Okuhara1,*, Zhiheng Yu1, Xin Tian1, Yiqiang Cai1, Sekiya Shibazaki1 and Stefan Somlo1,2,3,{ddagger}

1 Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA
2 Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA
3 Section of Nephrology, Yale University School of Medicine, P.O. Box 208029, New Haven, CT 06520, USA


Figure 1
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  		Fig. 1. Trafficking of full-length and truncated PC2 to cilia. (A) Expression of full-length PC2 with a COOH-terminal EGFP tag in the cilia of LLC-PK1 (top panel) and MDCK (bottom panel) cells. (B) Expression of PC2-L703X with a COOH-terminal EGFP in the cilia of LLC-PK1 (top panel) and MDCK (bottom panel) cells. Cells expressing PC2-L703X form longer cilia more readily than cells expressing only native PC2 or the full-length PC2 construct. (Left panels) EGFP epifluorescence (green) shows expression of PC2, (middle panels) acetylated {alpha}-tubulin (red) marks the cilia; (right panels) merged images. Bars, 10 µm.

 

Figure 2
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  		Fig. 2. PC2 traffics to cilia independently of PC1. (A) PC2 is expressed in cilia at the embryonic node in Pkd1-/- mice. (B) PC2 is expressed in cilia (arrow) of cells lining kidney cysts formed in Pkd1 mutant mice. (C) PC2 is expressed in the cilia (arrows) in cultured mouse kidney cells lacking PC1. (D) PC2-GFP fusion protein expressed in cells lacking PC1 traffics into cilia as evidenced by EFFP epifluorescence in cilia. Adjacent cells not expressing the transfected protein do not show EGFP epifluorescence. (A-C) Anti-PC2 (YCC2), green; (D) EGFP epifluorescence, green; (A-D) anti-acetylated {alpha}-tubulin, red; right panels, merged images. Bars, A, C, D, 10 µm; B, 5 µm. Insets show selected cilia in each field.

 

Figure 3
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  		Fig. 3. Amino acids 5-72 of PC2 are necessary for its trafficking to cilia. Deletion of amino acids 5-72 in either (A) the PC2-L703X truncated peptide [({Delta}5-72)PC2-L703X] or (D) the full-length PC2 [({Delta}5-72)PC2] with an intact COOH terminus results in PC2 absence in the cilia. Robust expression of the respective proteins at the level of the cell body is shown in the right-most panel as well as in the xz-plane reconstructions in these confocal images. (B,C) Deletion of amino acids (B) 72-130 [({Delta}72-130)PC2-L703X] or (C) 130-220 [({Delta}130-220)PC2-L703X] does not abrogate trafficking of the respective peptides into cilia. Anti-HA epitope (green) indirect immunofluorescence indicates expression of PC2 constructs and acetylated {alpha}-tubulin (red) marks the cilia; right panels, merged images. The cell lines were made in LLC-PK1. Bars, 10 µm.

 

Figure 4
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  		Fig. 4. Aberrant trafficking of N-terminal mutant forms of PC2. (A) PC2-L703X (anti-HA, green) shows partial co-localization (arrows) with the Golgi marker GM130 (red) in LLC-PK1 cells. (B) {Delta}(5-72)PC2-L703X (anti-HA, green) shows a typical ER pattern of expression but does not co-localize with GM130 (red), indicating that the protein does not traffic to the Golgi. Bars, 10 µm. (C) Immunoblot using anti-HA on lysates of LLC-PK1 cell lines stably expressing PC2-L703X or its respective N-terminal deleted forms ({Delta}72-130)PC2-L703X, ({Delta}5-72)PC2-L703X and ({Delta}130-220)PC2-L703X. Each cell line shows similar expression levels of the respective proteins, indicating comparable stability of the various deleted forms (20 µg of protein from total cell lysate in each lane). (D) Immunoblots using anti-HA of lysates incubated either with enzyme buffer alone or Endo H or PNGase F. PC2-L703X, ({Delta}72-130)PC2-L703X and ({Delta}130-220)PC2-L703X have slower migrating species (arrows) that show resistance to Endo H as indicated by the persistence of the bands after Endo H digestion. The Endo H resistance indicates that these peptides traffic past the middle Golgi. By contrast, ({Delta}5-72)PC2-L703X does not have an Endo H-resistant component, suggesting that it does not traffic past the middle Golgi. The latter finding is consistent with absence of co-localization with the Golgi marker GM130 (B). All proteins are completely sensitive PNGase F indicating the migration of the deglycosylated peptide backbone. (E) Expression of PC2, PC2-L703X and ({Delta}5-72)PC2-L703X on the cell surface, evaluated by selective apical and basolateral biotinylation in living, ciliated LLC-PK1 cells. Live cells forming confluent monolayers on semipermeable supports were biotinylated from either the apical or basolateral surface, followed by pull-down with streptavidin-conjugated agarose beads. Lanes on the immunoblots were alternately loaded with total protein from the starting material after cell lysis but before streptavidin pull down (lysate) or with eluted protein after streptavidin pull down from cells biotinylated from either the apical or basolateral surfaces, respectively. Immunoblotting with anti-HA was used to detect PC2 related peptides. Na+,K+-ATPase, which is expressed exclusively on the basolateral surface, was used to show the selectivity of the biotinylation reaction. No biotinylated PC2 was detectable in either membrane compartment (left panel), consistent with the conclusion that PC2 is only expressed in the cilial plasma membrane in amounts not detectable by biotinylation (see text for details). PC2-L703X is biotinylated on the both basolateral and apical surfaces (middle panel). It is noteworthy that only the slower migrating band corresponding to the Endo H resistant form of PC2-L703X is biotinylated, confirming the expression of the Endo H resistant form on the plasma membrane. {Delta}(5-72)PC2-L703X is not biotinylated on the cell surface in either membrane compartment (right panel). Coupled with the immunofluorescence data showing lack of expression in cilia, the failure to co-localize with Golgi markers and the absence of Endo H resistance, we conclude that {Delta}(5-72)PC2-L703X is not expressed in the plasma membrane.

 

Figure 5
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  		Fig. 5. The trafficking properties of PKD2L1. (A) Confocal images above the apical surface of the cell and in the xz plane show cilia devoid of PKD2L1 with either COOH-terminal EGFP (A) or triple-HA (B) epitope tags. EGFP epifluorescence (A) and double indirect anti-HA immunofluorescence (B) are shown in green; anti-acetylated {alpha}-tubulin, red. (A*,B*) Confocal images at the level of the nucleus showing that all cells express pPKD2L1. Bars, 10 µm. (C) PKD2L1 does not acquire detectable Endo H resistance (left panel) and is not detectably biotinylated on the cell surface in either LLC-PK1 of CHO-K1 cells (middle panel). Truncation of the predicted COOH terminus of PKD2L1 does not result in trafficking to the cell surface as indicated by the absence of a biotinylated species (right panel). `Lysate' is the total cellular protein from the starting material before streptavidin pull down; `biotin' is the eluted material after streptavidin pull down. PKD2L1 was detected by anti-HA in immunoblots. CD4 was co-transfected with PKD2L1 as a positive control for live cell surface biotinylation and streptavidin immunoprecipitation and was detected with anti-CD4 monoclonal antibody 1F6.

 

Figure 6
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  		Fig. 6. The N-terminal domain of PC2 can direct cilial localization of heterologous non-cilial proteins. (A) Addition of the first 72 amino acids of PC2 to PKD2L1 results in trafficking of the chimeric protein, (1-72-PC2)-PKD2L1, into cilia (arrows). (B) Replacing the N-terminal region of PC2-L703X with the N-terminal region of PKD2L1 results in failure of the chimeric protein, PKD2L1-{Delta}(1-328)PC2-L703X, to traffic to cilia. (C) A doubly chimeric protein in which the first 72 amino acids of PC2 are fused to the PKD2L1-{Delta}(1-328)PC2-L703X chimera localizes to cilia. Anti-HA, green; anti-acetylated {alpha}-tubulin, red; right panels, merged images. Bars, 10 µm.

 

Figure 7
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  		Fig. 7. A heterologous cilia trafficking assay using transferrin receptor. LLC-PK1 cells stably over-expressing (A) wild-type transferrin receptor (hTFR), (B) (1-56)PKD2L1-hTFR and (C) (1-72)PC2-hTFR. All heterologous proteins have a triple HA epitope at their COOH-termini and are immunolabeled by anti-HA (green). Cilial axonemes are marked by anti-acetylated {alpha}-tubulin (red). (A) The wild-type transferrin receptor (hTFR) does not traffic into cilia. (B) Replacement of the cytosolic N-terminus of hTFR with the first 56 amino acids of PKD2L1 does not result in trafficking of the chimeric protein to cilia. The right-most panels in A and B are confocal sections through the cell body at the level of the nucleus to show the robust expression of the heterologous protein in all cells in the field. (C) Replacement of the cytosolic N-terminus of hTFR with the first 72 amino acids of PC2 results in expression of the chimeric protein in cilia, indicating this domain contains cilia-targeting information. Bars, 10 µm.

 

Figure 8
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  		Fig. 8. Refinement of the cilia trafficking domain of PC2. (A) Deletion of amino acids 31-72 from PC2-L703X does not alter trafficking to cilia, whereas (B) deletion of amino acids 5-31 results in complete absence of {Delta}(5-31)PC2-L703X from cilia. Replacement of the cytosolic N-terminus of hTFR with either (C) the first 31 amino acids or (D) the first 15 amino acids of PC2 is sufficient to traffic the chimeric reporter molecule to cilia. (E) Non-permeabilized living cells have epitopes available to anti-HA on cilia and (E*) at the level of the apical plasma membrane. Anti-HA was applied on both the apical and basolateral aspects but there was no detectable immunoreactivity on the basolateral surfaces, indicating that the chimeric protein only trafficked to cilia and the apical membrane. Anti-acetylated {alpha}-tubulin gave no signal in non-permeabilized, living cells (not shown) confirming that the observed signal is indeed extracellular. Anti-HA, green; anti-acetylated {alpha}-tubulin, red. Bars, 10 µm.

 

Figure 9
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  		Fig. 9. RVxP is necessary for cilial targeting of PC2. (A) Alignment of the first 31 amino acids of PC2 from several species. *, location of mutations in the most conserved residues whose cilial trafficking properties are shown in B-E. +, other mutation sites tested that had no effect on cilial localization. Identical residues are highlighted and boxed; residues in red abrogated cilial location of PC2-L703X. (B) Amino acid substitution S4Adoes not affect trafficking of PC2-L703X to cilia. (C-E) Substitutions R6G (C) V7A (D) and P9A (E) result in loss of cilial localization of PC2-L703X. Green, anti-HA; red, anti-acetylated {alpha}-tubulin, third row, merged images; far right panels in C-E are confocal sections at the level of the nucleus showing robust expression of the respective mutant proteins in all cells. Bars, 10 µm.

 

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© The Company of Biologists Ltd 2006