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The role of Cysteine 227 in subcellular localization, water permeability, and multimerization of aquaporin-11.

Takahashi S, Muta K, Sonoda H, Kato A, Abdeen A, Ikeda M - FEBS Open Bio (2014)

Bottom Line: Interestingly, cells expressing the mutants had significantly higher osmotic water permeability.In contrast, the mutation lowered the cell surface expression and multimerization levels.Our observations suggest that Cys(227) is crucial for the proper molecular function of AQP11.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pharmacology, University of Miyazaki, Miyazaki 889-2192, Japan.

ABSTRACT
Aquaporin-11 (AQP11) is the latest member of the mammalian water channel protein family to be described. Recent in vivo studies have shown that mutation at Cys(227) causes renal failure. However the importance of Cys(227) for the molecular function of AQP11 is largely unknown. In this study, we examined the subcellular localization, water permeability, and multimerization of AQP11 with a mutation at Cys(227). Interestingly, cells expressing the mutants had significantly higher osmotic water permeability. In contrast, the mutation lowered the cell surface expression and multimerization levels. Our observations suggest that Cys(227) is crucial for the proper molecular function of AQP11.

No MeSH data available.


Related in: MedlinePlus

Cell surface expression level of AQP11 mutants at Cys227. (A) Cell surface expression levels of Myc-AQP11, Myc-hAQP11-C227S, and Myc-hAQP11-C227A were assessed by a biotinylation experiment. CHO cells were transfected with Myc-AQP11 or its mutant protein expression plasmid, and 24 h after transfection the cell surface proteins were labeled with membrane-impermeable biotin. The biotin-labeled proteins were precipitated by NeutrAvidin beads. The biotin-labeled cell surface proteins, as well as the total cell lysates, were analyzed by Western blotting using anti-Myc antibody. The Western blot image is separately shown, because the original image included data for the other sample in the middle lane between the Myc-hAQP11 and Myc-hAQP11-C227S, or Myc-hAQP11-C227A samples. The separate image originated from the same blot, while retaining the original quality. (B) The absolute cell surface expression level is summarized. Each value is expressed as a percentage of the mean value for Myc-hAQP11. Data are shown as means ± SE. The numbers of experiments are given in parentheses. ∗P < 0.05 compared with the Myc-hAQP11 group (Mann–Whitney U test). (C) The ratio of biotinylated to total Myc-tagged proteins was taken as an index of the relative surface expression level. Values are presented as mean ± SE. No significant difference was found between Myc-hAQP11 and Myc-hAQP11-C227S, or Myc-hAQP11-C227A (Student’s t test).
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f0010: Cell surface expression level of AQP11 mutants at Cys227. (A) Cell surface expression levels of Myc-AQP11, Myc-hAQP11-C227S, and Myc-hAQP11-C227A were assessed by a biotinylation experiment. CHO cells were transfected with Myc-AQP11 or its mutant protein expression plasmid, and 24 h after transfection the cell surface proteins were labeled with membrane-impermeable biotin. The biotin-labeled proteins were precipitated by NeutrAvidin beads. The biotin-labeled cell surface proteins, as well as the total cell lysates, were analyzed by Western blotting using anti-Myc antibody. The Western blot image is separately shown, because the original image included data for the other sample in the middle lane between the Myc-hAQP11 and Myc-hAQP11-C227S, or Myc-hAQP11-C227A samples. The separate image originated from the same blot, while retaining the original quality. (B) The absolute cell surface expression level is summarized. Each value is expressed as a percentage of the mean value for Myc-hAQP11. Data are shown as means ± SE. The numbers of experiments are given in parentheses. ∗P < 0.05 compared with the Myc-hAQP11 group (Mann–Whitney U test). (C) The ratio of biotinylated to total Myc-tagged proteins was taken as an index of the relative surface expression level. Values are presented as mean ± SE. No significant difference was found between Myc-hAQP11 and Myc-hAQP11-C227S, or Myc-hAQP11-C227A (Student’s t test).

Mentions: It has been shown that although the level of AQP11 expression is less than that at the ER, a significant amount of the protein is expressed at the plasma membrane [8]. Therefore, we checked the cell surface expression levels of Myc-hAQP11, Myc-hAQP11-C227S, and Myc-hAQP11-C227A using a cell surface biotinylation assay. The levels of both total and biotinylated protein for Myc-hAQP11-C227S and Myc-hAQP11-C227A were significantly lower than those of the wild type (Fig. 2A and B). On the other hand, the ratio of the level of biotinylated protein to that of total protein was not significantly altered between the wild type, Myc-hAQP11-C227S, and Myc-hAQP11-C227A (Fig. 2C).


The role of Cysteine 227 in subcellular localization, water permeability, and multimerization of aquaporin-11.

Takahashi S, Muta K, Sonoda H, Kato A, Abdeen A, Ikeda M - FEBS Open Bio (2014)

Cell surface expression level of AQP11 mutants at Cys227. (A) Cell surface expression levels of Myc-AQP11, Myc-hAQP11-C227S, and Myc-hAQP11-C227A were assessed by a biotinylation experiment. CHO cells were transfected with Myc-AQP11 or its mutant protein expression plasmid, and 24 h after transfection the cell surface proteins were labeled with membrane-impermeable biotin. The biotin-labeled proteins were precipitated by NeutrAvidin beads. The biotin-labeled cell surface proteins, as well as the total cell lysates, were analyzed by Western blotting using anti-Myc antibody. The Western blot image is separately shown, because the original image included data for the other sample in the middle lane between the Myc-hAQP11 and Myc-hAQP11-C227S, or Myc-hAQP11-C227A samples. The separate image originated from the same blot, while retaining the original quality. (B) The absolute cell surface expression level is summarized. Each value is expressed as a percentage of the mean value for Myc-hAQP11. Data are shown as means ± SE. The numbers of experiments are given in parentheses. ∗P < 0.05 compared with the Myc-hAQP11 group (Mann–Whitney U test). (C) The ratio of biotinylated to total Myc-tagged proteins was taken as an index of the relative surface expression level. Values are presented as mean ± SE. No significant difference was found between Myc-hAQP11 and Myc-hAQP11-C227S, or Myc-hAQP11-C227A (Student’s t test).
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Related In: Results  -  Collection

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f0010: Cell surface expression level of AQP11 mutants at Cys227. (A) Cell surface expression levels of Myc-AQP11, Myc-hAQP11-C227S, and Myc-hAQP11-C227A were assessed by a biotinylation experiment. CHO cells were transfected with Myc-AQP11 or its mutant protein expression plasmid, and 24 h after transfection the cell surface proteins were labeled with membrane-impermeable biotin. The biotin-labeled proteins were precipitated by NeutrAvidin beads. The biotin-labeled cell surface proteins, as well as the total cell lysates, were analyzed by Western blotting using anti-Myc antibody. The Western blot image is separately shown, because the original image included data for the other sample in the middle lane between the Myc-hAQP11 and Myc-hAQP11-C227S, or Myc-hAQP11-C227A samples. The separate image originated from the same blot, while retaining the original quality. (B) The absolute cell surface expression level is summarized. Each value is expressed as a percentage of the mean value for Myc-hAQP11. Data are shown as means ± SE. The numbers of experiments are given in parentheses. ∗P < 0.05 compared with the Myc-hAQP11 group (Mann–Whitney U test). (C) The ratio of biotinylated to total Myc-tagged proteins was taken as an index of the relative surface expression level. Values are presented as mean ± SE. No significant difference was found between Myc-hAQP11 and Myc-hAQP11-C227S, or Myc-hAQP11-C227A (Student’s t test).
Mentions: It has been shown that although the level of AQP11 expression is less than that at the ER, a significant amount of the protein is expressed at the plasma membrane [8]. Therefore, we checked the cell surface expression levels of Myc-hAQP11, Myc-hAQP11-C227S, and Myc-hAQP11-C227A using a cell surface biotinylation assay. The levels of both total and biotinylated protein for Myc-hAQP11-C227S and Myc-hAQP11-C227A were significantly lower than those of the wild type (Fig. 2A and B). On the other hand, the ratio of the level of biotinylated protein to that of total protein was not significantly altered between the wild type, Myc-hAQP11-C227S, and Myc-hAQP11-C227A (Fig. 2C).

Bottom Line: Interestingly, cells expressing the mutants had significantly higher osmotic water permeability.In contrast, the mutation lowered the cell surface expression and multimerization levels.Our observations suggest that Cys(227) is crucial for the proper molecular function of AQP11.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pharmacology, University of Miyazaki, Miyazaki 889-2192, Japan.

ABSTRACT
Aquaporin-11 (AQP11) is the latest member of the mammalian water channel protein family to be described. Recent in vivo studies have shown that mutation at Cys(227) causes renal failure. However the importance of Cys(227) for the molecular function of AQP11 is largely unknown. In this study, we examined the subcellular localization, water permeability, and multimerization of AQP11 with a mutation at Cys(227). Interestingly, cells expressing the mutants had significantly higher osmotic water permeability. In contrast, the mutation lowered the cell surface expression and multimerization levels. Our observations suggest that Cys(227) is crucial for the proper molecular function of AQP11.

No MeSH data available.


Related in: MedlinePlus