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. 2015 Aug 26;27(4):1159–1173. doi: 10.1681/ASN.2014111074

Figure 4.

Figure 4.

PC1–1 PLAT phosphorylation by PKA regulates its surface expression and cilia localization. (A) Fluorescence microscopy of stably expressed YFP-PLAT and YFP-PLAT mutant proteins in MDCK II cells. YFP-PLAT was consistently localized to the PM, except for the S3164D mutation, which resulted in complete loss of surface localization. (B and C) Live–cell surface labeling of PC1 in cells transfected with wild-type and S3164/R3162 FLAG-PC1-HA–tagged plasmids. Surface PC1 expression was visualized in nonpermeabilized cells by FLAG labeling; total number of PC1-expressing cells by HA staining after permeabilization are shown. PC2-CFP coexpression was visualized by epifluorescence. Cell surface labeling of PC1 was almost completely abolished in the S3164D mutant compared with the wild type or S3164A. The pathogenic mutation R3162C showed detectable but significantly reduced surface expression. Values shown are the means±SEM. Scale bar, 10 µm. (D and E) Surface biotinylation of PC1 and S3164/R3162 mutants. Wild-type PC1 was biotinylated only in the presence of coexpressed PC2 and significantly reduced in the S3164D mutant. Controls included the PM transporter Na+-K+-ATPase and the ER resident protein calnexin. Lysates show equivalent expression of PC1 and PC2 in different samples. The cleaved PC1-CTF (detectable by HA) was used for quantification of surface biotinylation as previously described, because the PC1-NTF is detached under the assay conditions used.19,53 The bars indicate the ratio of biotinylated PC1 to total PC1 (HA blot) from three experiments. (F) Cilia localization of PC1 and PC2 in a normal human kidney cell line (UCL93/3) expressing the wild type or S3164/R3162 FLAG-PC1-HA. Wild-type PC1 was only detected in primary cilia when PC2-CFP was coexpressed (72% ciliated cells). There was a significant reduction in cilia expression for S3164D (6%) and S3162C (30%) but not S3164A (70%), even in the presence of PC2-CFP. An Arl13b antibody was used to label primary cilia. Scale bar, 10 µm. *P<0.05; **P<0.01; ****P<0.0001.