Figure 5. GPS cleavage is required for the ciliary trafficking of polycystin complex.

(a) Confocal images of collecting duct cells isolated from Pkd1^V/V kidneys (CD^V/V) stained with antibodies against PC1(E4) or PC2. Arl13b or Ac-tubulin was used as a ciliary marker. The graph compares the percentage of cells with positive PC1 or PC2 in the wild-type CD (as shown in Fig. 1e or f) and CD^V/V cells, from three independent experiments and presented as the mean±s.e.m.; ***P<0.001. The number of cells analysed (n) is indicated. (b) Confocal images of induced MDCK^PC1V cells show the absence of ciliary PC1^V and endogenous PC2. The graph compares the percentage of cells with positive PC1 or PC2 ciliary staining in MDCK^PC1WT (as shown in Fig. 2f) and MDCK^PC1V cells from three independent experiments, presented as the mean±s.e.m.; ***P<0.001. The number of cells analysed (n) is indicated. The white line in the XY scan in (a,b) indicates the path of the XZ scan. Scale bar, 10 μm. (c) Confocal images of cystic collecting ducts of the Pkd1^V/V kidney stained with anti-PC2. Note that PC2 was not detectable in 17 of 17 cilia analysed. (d) N-glycosylation pattern of polycystin complex in Pkd1^V/V MEFs versus wild-type MEFs. Polycystin complex was immunoprecipitated and analysed as in Fig. 3, with PC1 and PC2 bands indicated with colour code. Note that the co-precipitated PC2 from Pkd1^V/V MEFs (lanes 4–6) was entirely EndoH sensitive, lacking EndoH-resistant PC2₁₃₀ seen in wild-type MEFs (lanes 1–3). (e) N-glycosylation pattern of polycystin complex in Pkd1^V/V kidney versus wild-type kidney. Polycystin complex was immunoprecipitated and analysed as in (d), expect that an anti-CC antibody was used to detect PC1. Note the presence of cleaved PC1_CTF in wild-type kidney (lanes 1–3) but not in Pkd1^V/V kidneys (lanes 4–6).