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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.


Cellular localization of NBCn2 variants and mutants heterologously expressed in neuro-2A cells.(A) NBCn2-C (starting with MEIK). (B) NBCn2-G (starting with MCDL). (C) NBCn2-K (starting with MHAN). (D–K) NBCn2 mutants truncated to various positions in the Nt domain. As an example, ΔN43 (truncated to “GHRT”) represents a mutant with the first 43 aa residues removed based on NBCn2-C. The cDNA encoding NBCn2 variants or mutants was subcloned into pcDNA3.1. The cells were transfected with the corresponding construct, incubated for 24 hours for transient expression, and then fixed with 4% PFA for immunocytochemistry. NBCn2 proteins were probed with anti-NBCn2-Ct antibody in combination with Dylight 549 goat-anti-rabbit secondary antibody.
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f7: Cellular localization of NBCn2 variants and mutants heterologously expressed in neuro-2A cells.(A) NBCn2-C (starting with MEIK). (B) NBCn2-G (starting with MCDL). (C) NBCn2-K (starting with MHAN). (D–K) NBCn2 mutants truncated to various positions in the Nt domain. As an example, ΔN43 (truncated to “GHRT”) represents a mutant with the first 43 aa residues removed based on NBCn2-C. The cDNA encoding NBCn2 variants or mutants was subcloned into pcDNA3.1. The cells were transfected with the corresponding construct, incubated for 24 hours for transient expression, and then fixed with 4% PFA for immunocytochemistry. NBCn2 proteins were probed with anti-NBCn2-Ct antibody in combination with Dylight 549 goat-anti-rabbit secondary antibody.

Mentions: We then examined the effect of truncation in the Nt domain on the membrane trafficking of different NCBTs in neuro-2A cells. When heterologously expressed in neuro-2A cells, rat NBCn2-C and -G were both well localized in the plasma membrane (Fig. 7A,B). However, rat NBCn2-K, the natural Nt-truncated variant, was nearly all retained in the cytosol (Fig. 7C), although its surface localization was occasionally observed in very few cells (data not shown). The same was true for the natural Nt-truncated variant rat NBCn2-M when expressed in neuro-2A cells (data not shown). The results suggested that the Nt portion omitted in MHAN-NBCn2 contain information critical for efficient surface expression of the transporter. To test this hypothesis, we made a series of truncation mutations to the Nt of NBCn2-C and tested the cellular localization of these constructs in neuro-2A cells.


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)

Cellular localization of NBCn2 variants and mutants heterologously expressed in neuro-2A cells.(A) NBCn2-C (starting with MEIK). (B) NBCn2-G (starting with MCDL). (C) NBCn2-K (starting with MHAN). (D–K) NBCn2 mutants truncated to various positions in the Nt domain. As an example, ΔN43 (truncated to “GHRT”) represents a mutant with the first 43 aa residues removed based on NBCn2-C. The cDNA encoding NBCn2 variants or mutants was subcloned into pcDNA3.1. The cells were transfected with the corresponding construct, incubated for 24 hours for transient expression, and then fixed with 4% PFA for immunocytochemistry. NBCn2 proteins were probed with anti-NBCn2-Ct antibody in combination with Dylight 549 goat-anti-rabbit secondary antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507446&req=5

f7: Cellular localization of NBCn2 variants and mutants heterologously expressed in neuro-2A cells.(A) NBCn2-C (starting with MEIK). (B) NBCn2-G (starting with MCDL). (C) NBCn2-K (starting with MHAN). (D–K) NBCn2 mutants truncated to various positions in the Nt domain. As an example, ΔN43 (truncated to “GHRT”) represents a mutant with the first 43 aa residues removed based on NBCn2-C. The cDNA encoding NBCn2 variants or mutants was subcloned into pcDNA3.1. The cells were transfected with the corresponding construct, incubated for 24 hours for transient expression, and then fixed with 4% PFA for immunocytochemistry. NBCn2 proteins were probed with anti-NBCn2-Ct antibody in combination with Dylight 549 goat-anti-rabbit secondary antibody.
Mentions: We then examined the effect of truncation in the Nt domain on the membrane trafficking of different NCBTs in neuro-2A cells. When heterologously expressed in neuro-2A cells, rat NBCn2-C and -G were both well localized in the plasma membrane (Fig. 7A,B). However, rat NBCn2-K, the natural Nt-truncated variant, was nearly all retained in the cytosol (Fig. 7C), although its surface localization was occasionally observed in very few cells (data not shown). The same was true for the natural Nt-truncated variant rat NBCn2-M when expressed in neuro-2A cells (data not shown). The results suggested that the Nt portion omitted in MHAN-NBCn2 contain information critical for efficient surface expression of the transporter. To test this hypothesis, we made a series of truncation mutations to the Nt of NBCn2-C and tested the cellular localization of these constructs in neuro-2A cells.

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.