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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.


Biotinylation analysis of the effect of coexpressing the full-length NBCn2 on the surface abundance of Nt-truncated NBCn2 in neuro-2A cells.(A,D) Expression of full-length NBCn2 (n2C and n2G) with HA tagged at the Nt end. (B,E) Expression of Nt-truncated NBCn2 (n2K and ΔN121) with Myc tagged at the Ct end. (C,F) Expression of endogenous Na+-K+-ATPase probed with anti-α1 to verify equal loading in each lane. Surface proteins were prepared by biotinylation and separated on 10% SDS-PAGE for western blotting.
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f10: Biotinylation analysis of the effect of coexpressing the full-length NBCn2 on the surface abundance of Nt-truncated NBCn2 in neuro-2A cells.(A,D) Expression of full-length NBCn2 (n2C and n2G) with HA tagged at the Nt end. (B,E) Expression of Nt-truncated NBCn2 (n2K and ΔN121) with Myc tagged at the Ct end. (C,F) Expression of endogenous Na+-K+-ATPase probed with anti-α1 to verify equal loading in each lane. Surface proteins were prepared by biotinylation and separated on 10% SDS-PAGE for western blotting.

Mentions: The above observations suggest that the presence of the full-length NBCn2 enhance the surface localization of the Nt-truncated NBCn2-K and ΔN121 at least in a specific population of cells. We performed biotinylation assay with neuro-2A cells to further examine this effect of full-length NBCn2. Figure 10A shows that, when coexpressed with the Nt-truncated NBCn2-K-Myc, the relative surface abundance of the full-length HA-NBCn2-C and -G was not substantially changed. However, the relative surface abundance of NBCn2-K-Myc was greatly increased in the presence of HA-NBCn2-C or -G (Fig. 10B). The staining of Na+-K+ ATPase showed the equal loading for each lane (Fig. 10C).


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)

Biotinylation analysis of the effect of coexpressing the full-length NBCn2 on the surface abundance of Nt-truncated NBCn2 in neuro-2A cells.(A,D) Expression of full-length NBCn2 (n2C and n2G) with HA tagged at the Nt end. (B,E) Expression of Nt-truncated NBCn2 (n2K and ΔN121) with Myc tagged at the Ct end. (C,F) Expression of endogenous Na+-K+-ATPase probed with anti-α1 to verify equal loading in each lane. Surface proteins were prepared by biotinylation and separated on 10% SDS-PAGE for western blotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f10: Biotinylation analysis of the effect of coexpressing the full-length NBCn2 on the surface abundance of Nt-truncated NBCn2 in neuro-2A cells.(A,D) Expression of full-length NBCn2 (n2C and n2G) with HA tagged at the Nt end. (B,E) Expression of Nt-truncated NBCn2 (n2K and ΔN121) with Myc tagged at the Ct end. (C,F) Expression of endogenous Na+-K+-ATPase probed with anti-α1 to verify equal loading in each lane. Surface proteins were prepared by biotinylation and separated on 10% SDS-PAGE for western blotting.
Mentions: The above observations suggest that the presence of the full-length NBCn2 enhance the surface localization of the Nt-truncated NBCn2-K and ΔN121 at least in a specific population of cells. We performed biotinylation assay with neuro-2A cells to further examine this effect of full-length NBCn2. Figure 10A shows that, when coexpressed with the Nt-truncated NBCn2-K-Myc, the relative surface abundance of the full-length HA-NBCn2-C and -G was not substantially changed. However, the relative surface abundance of NBCn2-K-Myc was greatly increased in the presence of HA-NBCn2-C or -G (Fig. 10B). The staining of Na+-K+ ATPase showed the equal loading for each lane (Fig. 10C).

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.