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Effects of Nt-truncation and coexpression of isolated Nt domains on the membrane trafficking of electroneutral Na+/HCO3- cotransporters.

Wang DK, Liu Y, Myers EJ, Guo YM, Xie ZD, Jiang DZ, Li JM, Yang J, Liu M, Parker MD, Chen LM - Sci Rep (2015)

Bottom Line: Co-immunoprecipitation and bimolecular fluorescence complementation studies showed that the full-length and Nt-truncated NBCn2 interact with each other to form heterodimers in neuro-2A cells.Finally, we showed that the isolated Nt domain interacts with and enhances the surface abundance of the Nt-truncated NBCn2.The present study expands our knowledge of the NBCn1 and NBCn2 transcriptome, and provides insights into how the Nt domain could affect transporter function by regulating its membrane trafficking.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Molecular Physiology, Key Laboratory of Molecular Biophysics of Ministry of Education, Huazhong University of Science and Technology School of Life Science and Technology, Wuhan, Hubei 430074, China.

ABSTRACT
The SLC4 genes are all capable of producing multiple variants by alternative splicing or using alternative promoters. The physiological consequences of such diversity are of great interest to investigators. Here, we identified two novel variants of the electroneutral Na(+)/HCO3- cotransporter NBCn1, one full-length starting with "MIPL" and the other Nt-truncated starting with "MDEL". Moreover, we identified a new promoter of Slc4a10 encoding NBCn2 and a novel type of Nt-truncated NBCn2 starting with "MHAN". When heterologously expressed, the new NBCn1 variants were well localized to the plasma membrane and exhibited characteristic NBCn1 activity. However, MHAN-NBCn2 was poorly localized on the plasma membrane. By deletion mutations, we identified the Nt regions important for the surface localization of NBCn2. Interestingly, coexpressing the full-length NBCn2 greatly enhances the surface abundance of the Nt-truncated NBCn2. Co-immunoprecipitation and bimolecular fluorescence complementation studies showed that the full-length and Nt-truncated NBCn2 interact with each other to form heterodimers in neuro-2A cells. Finally, we showed that the isolated Nt domain interacts with and enhances the surface abundance of the Nt-truncated NBCn2. The present study expands our knowledge of the NBCn1 and NBCn2 transcriptome, and provides insights into how the Nt domain could affect transporter function by regulating its membrane trafficking.

No MeSH data available.


Related in: MedlinePlus

Effect of isolated Nt domain on surface expression of Nt-truncated NBCn2 in neuro-2A cells.(A) Expression of isolated Nt domain of rat NBCn2-C/G. Whole cell lysate was used for the analysis of the expression of the isolated Nt domains. (B) Biotinylation analysis of the surface expression of Nt-truncated NBCn2. (C) Biotinylation analysis of the surface expression of Na+-K+-ATPase. (D–E) Cellular localization of mutant ΔN121in neuro-2A cells in the presence of the isolated Nt domain of NBCn2-C or NBCn2-G. (F) Cellular localization of mutant ΔN121 in the absence of the isolated Nt domain of NBCn2 in neuro-2A cells. The isolated n2C-Nt consisted of the sequence of “MEIK…FLSQ” of rat NBCn2-C, and n2G-Nt consisted of the sequence of “MCDL…FLSQ” of rat NBCn2-G. For western blotting analysis, n2C-Nt and n2G-Nt were tagged with HA at the Nt end, whereas the Nt-truncated NBCn2-K and ΔN121 were tagged with Myc at the Ct end. In immunocytochemistry analysis, the isolated Nt and Nt-truncated NBCn2 proteins had no tag. The cells were stained with anti-NBCn2-Ct polyclonal antibody.
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f11: Effect of isolated Nt domain on surface expression of Nt-truncated NBCn2 in neuro-2A cells.(A) Expression of isolated Nt domain of rat NBCn2-C/G. Whole cell lysate was used for the analysis of the expression of the isolated Nt domains. (B) Biotinylation analysis of the surface expression of Nt-truncated NBCn2. (C) Biotinylation analysis of the surface expression of Na+-K+-ATPase. (D–E) Cellular localization of mutant ΔN121in neuro-2A cells in the presence of the isolated Nt domain of NBCn2-C or NBCn2-G. (F) Cellular localization of mutant ΔN121 in the absence of the isolated Nt domain of NBCn2 in neuro-2A cells. The isolated n2C-Nt consisted of the sequence of “MEIK…FLSQ” of rat NBCn2-C, and n2G-Nt consisted of the sequence of “MCDL…FLSQ” of rat NBCn2-G. For western blotting analysis, n2C-Nt and n2G-Nt were tagged with HA at the Nt end, whereas the Nt-truncated NBCn2-K and ΔN121 were tagged with Myc at the Ct end. In immunocytochemistry analysis, the isolated Nt and Nt-truncated NBCn2 proteins had no tag. The cells were stained with anti-NBCn2-Ct polyclonal antibody.

Mentions: Finally, we examined the effect of expressing the isolated Nt domain of NBCn2-C or -G on the surface abundance of the Nt-truncated NBCn2 proteins. Strikingly, the presence of just the Nt domain of NBCn2-C or -G was sufficient to substantially increase the surface abundance of the Nt-truncated NBCn2-K-Myc andΔN121-Myc (Fig. 11A–C). Consistently, immunocytochemistry staining showed that, in the presence of the isolated Nt domain, the Nt-truncated ΔN121 was well expressed in the plasma membrane of a specific population of cells (Fig. 11D–E), different from that without coexpression of the isolated Nt domain (Fig. 11F).


Effects of Nt-truncation and coexpression of isolated Nt domains on the membrane trafficking of electroneutral Na+/HCO3- cotransporters.

Wang DK, Liu Y, Myers EJ, Guo YM, Xie ZD, Jiang DZ, Li JM, Yang J, Liu M, Parker MD, Chen LM - Sci Rep (2015)

Effect of isolated Nt domain on surface expression of Nt-truncated NBCn2 in neuro-2A cells.(A) Expression of isolated Nt domain of rat NBCn2-C/G. Whole cell lysate was used for the analysis of the expression of the isolated Nt domains. (B) Biotinylation analysis of the surface expression of Nt-truncated NBCn2. (C) Biotinylation analysis of the surface expression of Na+-K+-ATPase. (D–E) Cellular localization of mutant ΔN121in neuro-2A cells in the presence of the isolated Nt domain of NBCn2-C or NBCn2-G. (F) Cellular localization of mutant ΔN121 in the absence of the isolated Nt domain of NBCn2 in neuro-2A cells. The isolated n2C-Nt consisted of the sequence of “MEIK…FLSQ” of rat NBCn2-C, and n2G-Nt consisted of the sequence of “MCDL…FLSQ” of rat NBCn2-G. For western blotting analysis, n2C-Nt and n2G-Nt were tagged with HA at the Nt end, whereas the Nt-truncated NBCn2-K and ΔN121 were tagged with Myc at the Ct end. In immunocytochemistry analysis, the isolated Nt and Nt-truncated NBCn2 proteins had no tag. The cells were stained with anti-NBCn2-Ct polyclonal antibody.
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Related In: Results  -  Collection

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f11: Effect of isolated Nt domain on surface expression of Nt-truncated NBCn2 in neuro-2A cells.(A) Expression of isolated Nt domain of rat NBCn2-C/G. Whole cell lysate was used for the analysis of the expression of the isolated Nt domains. (B) Biotinylation analysis of the surface expression of Nt-truncated NBCn2. (C) Biotinylation analysis of the surface expression of Na+-K+-ATPase. (D–E) Cellular localization of mutant ΔN121in neuro-2A cells in the presence of the isolated Nt domain of NBCn2-C or NBCn2-G. (F) Cellular localization of mutant ΔN121 in the absence of the isolated Nt domain of NBCn2 in neuro-2A cells. The isolated n2C-Nt consisted of the sequence of “MEIK…FLSQ” of rat NBCn2-C, and n2G-Nt consisted of the sequence of “MCDL…FLSQ” of rat NBCn2-G. For western blotting analysis, n2C-Nt and n2G-Nt were tagged with HA at the Nt end, whereas the Nt-truncated NBCn2-K and ΔN121 were tagged with Myc at the Ct end. In immunocytochemistry analysis, the isolated Nt and Nt-truncated NBCn2 proteins had no tag. The cells were stained with anti-NBCn2-Ct polyclonal antibody.
Mentions: Finally, we examined the effect of expressing the isolated Nt domain of NBCn2-C or -G on the surface abundance of the Nt-truncated NBCn2 proteins. Strikingly, the presence of just the Nt domain of NBCn2-C or -G was sufficient to substantially increase the surface abundance of the Nt-truncated NBCn2-K-Myc andΔN121-Myc (Fig. 11A–C). Consistently, immunocytochemistry staining showed that, in the presence of the isolated Nt domain, the Nt-truncated ΔN121 was well expressed in the plasma membrane of a specific population of cells (Fig. 11D–E), different from that without coexpression of the isolated Nt domain (Fig. 11F).

Bottom Line: Co-immunoprecipitation and bimolecular fluorescence complementation studies showed that the full-length and Nt-truncated NBCn2 interact with each other to form heterodimers in neuro-2A cells.Finally, we showed that the isolated Nt domain interacts with and enhances the surface abundance of the Nt-truncated NBCn2.The present study expands our knowledge of the NBCn1 and NBCn2 transcriptome, and provides insights into how the Nt domain could affect transporter function by regulating its membrane trafficking.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Molecular Physiology, Key Laboratory of Molecular Biophysics of Ministry of Education, Huazhong University of Science and Technology School of Life Science and Technology, Wuhan, Hubei 430074, China.

ABSTRACT
The SLC4 genes are all capable of producing multiple variants by alternative splicing or using alternative promoters. The physiological consequences of such diversity are of great interest to investigators. Here, we identified two novel variants of the electroneutral Na(+)/HCO3- cotransporter NBCn1, one full-length starting with "MIPL" and the other Nt-truncated starting with "MDEL". Moreover, we identified a new promoter of Slc4a10 encoding NBCn2 and a novel type of Nt-truncated NBCn2 starting with "MHAN". When heterologously expressed, the new NBCn1 variants were well localized to the plasma membrane and exhibited characteristic NBCn1 activity. However, MHAN-NBCn2 was poorly localized on the plasma membrane. By deletion mutations, we identified the Nt regions important for the surface localization of NBCn2. Interestingly, coexpressing the full-length NBCn2 greatly enhances the surface abundance of the Nt-truncated NBCn2. Co-immunoprecipitation and bimolecular fluorescence complementation studies showed that the full-length and Nt-truncated NBCn2 interact with each other to form heterodimers in neuro-2A cells. Finally, we showed that the isolated Nt domain interacts with and enhances the surface abundance of the Nt-truncated NBCn2. The present study expands our knowledge of the NBCn1 and NBCn2 transcriptome, and provides insights into how the Nt domain could affect transporter function by regulating its membrane trafficking.

No MeSH data available.


Related in: MedlinePlus