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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

Subcellular localization of AQP11 mutants at Cys227. (A–L) Subcellular localizations of hAQP1-GFP (A–C), GFP-hAQP11 (D–F), GFP-hAQP11-C227S (G–I), and GFP-hAQP11-C227A (J–L) were evaluated by fluorescence microscopy. CHO cells were co-transfected with each AQP expression plasmid and a plasmid encoding an ER marker protein. Green (A, D, G, and J) and red (B, E, H, and K) colors indicate AQPs and ER marker proteins, respectively. The merged images are also shown (C, F, I, and L). The images were obtained 24 h after transfection. Scale bars = 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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f0005: Subcellular localization of AQP11 mutants at Cys227. (A–L) Subcellular localizations of hAQP1-GFP (A–C), GFP-hAQP11 (D–F), GFP-hAQP11-C227S (G–I), and GFP-hAQP11-C227A (J–L) were evaluated by fluorescence microscopy. CHO cells were co-transfected with each AQP expression plasmid and a plasmid encoding an ER marker protein. Green (A, D, G, and J) and red (B, E, H, and K) colors indicate AQPs and ER marker proteins, respectively. The merged images are also shown (C, F, I, and L). The images were obtained 24 h after transfection. Scale bars = 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Mentions: AQP11 has been shown to be localized at the ER membrane [7,8]. We investigated whether the introduction of a mutation at Cys227 replacing it with Ser (C227S) or Ala (C227A) affected the subcellular localization of AQP11. In this experiment, we used C-terminal GFP-tagged human AQP1 (hAQP1-GFP) as a control protein and N-terminal GFP-tagged human AQP11 (GFP-hAQP11), because it has been reported that the N-terminus of AQP1 functions as a signal for the proper localization, and tagging of GFP to the N- and C-terminus of AQP11 resulted in virtually the same subcellular localization [8,12]. When CHO cells were transfected with hAQP1-GFP expression plasmid together with a plasmid encoding an ER marker protein, hAQP1-GFP was clearly localized at the plasma membrane as well as at the ER (Fig. 1A–C), in line with a previous observation [8]. In contrast, the localization of GFP-hAQP11 virtually overlapped that of the ER marker (Fig. 1D–F). Similarly to GFP-hAQP11, GFP-hAQP11-C227S, and -C227A were clearly localized at the ER.


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)

Subcellular localization of AQP11 mutants at Cys227. (A–L) Subcellular localizations of hAQP1-GFP (A–C), GFP-hAQP11 (D–F), GFP-hAQP11-C227S (G–I), and GFP-hAQP11-C227A (J–L) were evaluated by fluorescence microscopy. CHO cells were co-transfected with each AQP expression plasmid and a plasmid encoding an ER marker protein. Green (A, D, G, and J) and red (B, E, H, and K) colors indicate AQPs and ER marker proteins, respectively. The merged images are also shown (C, F, I, and L). The images were obtained 24 h after transfection. Scale bars = 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
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f0005: Subcellular localization of AQP11 mutants at Cys227. (A–L) Subcellular localizations of hAQP1-GFP (A–C), GFP-hAQP11 (D–F), GFP-hAQP11-C227S (G–I), and GFP-hAQP11-C227A (J–L) were evaluated by fluorescence microscopy. CHO cells were co-transfected with each AQP expression plasmid and a plasmid encoding an ER marker protein. Green (A, D, G, and J) and red (B, E, H, and K) colors indicate AQPs and ER marker proteins, respectively. The merged images are also shown (C, F, I, and L). The images were obtained 24 h after transfection. Scale bars = 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Mentions: AQP11 has been shown to be localized at the ER membrane [7,8]. We investigated whether the introduction of a mutation at Cys227 replacing it with Ser (C227S) or Ala (C227A) affected the subcellular localization of AQP11. In this experiment, we used C-terminal GFP-tagged human AQP1 (hAQP1-GFP) as a control protein and N-terminal GFP-tagged human AQP11 (GFP-hAQP11), because it has been reported that the N-terminus of AQP1 functions as a signal for the proper localization, and tagging of GFP to the N- and C-terminus of AQP11 resulted in virtually the same subcellular localization [8,12]. When CHO cells were transfected with hAQP1-GFP expression plasmid together with a plasmid encoding an ER marker protein, hAQP1-GFP was clearly localized at the plasma membrane as well as at the ER (Fig. 1A–C), in line with a previous observation [8]. In contrast, the localization of GFP-hAQP11 virtually overlapped that of the ER marker (Fig. 1D–F). Similarly to GFP-hAQP11, GFP-hAQP11-C227S, and -C227A were clearly localized at the ER.

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