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Figure 1. Linear model and functional properties of Xenopus channel-activating protease (xCAP-1) mutants and schematic structure of xCAP-1 protein. The amino acids of the catalytic triad (*), the n-glycosylation site (⧫), and the position of the Flag insertion (DYKDDDDK) are indicated. Gray boxes represent hydrophobic sequences; SS, signal sequence; PM, plasma membrane. Results of the experiments performed in this article are summarized for each mutant. Activity means the ability of xCAP-1 protein to increase ENaC activity by coexpression in Xenopus oocytes.
Figure 2. Effects of endoglycosidase H (EndoH) and n-glycosidase F (PNGase [EndoF]) treatment on the electrophoretic mobility of xCAP-1. Injected oocytes were pulse-labeled for 16 h with modified Barth saline (MBS) containing 100 μCi/ml [35S]methionine. Microsomal membranes were immunoprecipitated with anti-Flag antibody. Immunoprecipitates were digested with EndoH or PNGase before gel loading. Migration positions of the size markers are indicated on the left.
Figure 3. Effects of inhibitors of n-glycosylation on wild type (WT) xCAP-1 n-glycosylation. Oocytes were injected with WT xCAP-1 cRNA diluted in either tunicamycine (TUN), castanospermine (CST), 1-deoxymannojirimycin (DMJ), or swainsonine (SW). After immunoprecipitation, proteins were either treated with EndoH or not to identify glycosylated and nonglycosylated forms of xCAP-1.
Figure 4. Cell surface expression of xCAP-1 by immunofluorescence. (A) Immunofluorescence on cryosections of noninjected versus flagged xCAP-1 injected oocytes. Detection of FLAG-tagged xCAP-1 with an anti-FLAG monoclonal antibody and of microvillar F actin with rhodamin-conjugated phalloidin. (B) Immunocytochemical detection of FLAG-tagged xCAP-1 in WT, S223A, N288Q, or G305Stop xCAP-1-injected oocytes. Scale bars, 40 μm.
Figure 5. Cell-surface expression by binding to flagged xCAP-1. Iodinated anti-Flag-binding assay on flagged xCAP-1 constructs-injected oocytes. Cell-surface expression of xCAP-1 was determined by using the 125I-labeled anti-Flag M2Ab antibody and expressed as femtomol per oocyte. Each bar represents the mean of two experiments in which the radioactivity bound to 6 to 10 individual oocytes have been counted for each condition.
Figure 6. Immunoprecipitation on MBS-conditioned medium. Injected oocytes were incubated for 16 h in 100 μl of MBS medium containing [35S]methionine. MBS was then recovered, clarified by 10,000-rpm centrifugation, and submitted to immunoprecipitation by using anti-Flag antibody.
Figure 7. Functional properties of xCAP-1 on ENaC activity. Oocytes were coinjected with cRNA encoding the three subunits of ENaC and one of the xCAP-1 mutant. Absolute values of amiloride-sensitive sodium current (Iam) measured before (−) and after (+) 2-min trypsin perfusion (2 μg/ml) of the oocyte. Each bar represents the mean of two experiments (two different batches of oocytes) in which 8 to 10 oocytes have been measured for each condition.
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