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Amino Acids 785, 787 of the Na(+)/H(+) Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation

The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulator...

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Autores principales: Li, Xiuju, Tu, Tommy, Quan, Sicheng, Quintero, Francisco J., Fahlman, Richard, Fliegel, Larry
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8583816/
https://www.ncbi.nlm.nih.gov/pubmed/34768780
http://dx.doi.org/10.3390/ijms222111349
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author Li, Xiuju
Tu, Tommy
Quan, Sicheng
Quintero, Francisco J.
Fahlman, Richard
Fliegel, Larry
author_facet Li, Xiuju
Tu, Tommy
Quan, Sicheng
Quintero, Francisco J.
Fahlman, Richard
Fliegel, Larry
author_sort Li, Xiuju
collection PubMed
description The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. Here, we investigated the effect of mutation of two amino acids of the regulatory tail, Ser(785) and Ser(787), that were similar in location and context to two amino acids of the Arabidopsis Na(+)/H(+) exchanger SOS1. Mutation of these two amino acids to either Ala or phosphomimetic Glu did not affect surface targeting but led to a slight reduction in the level of protein expressed. The activity of the NHE1 protein was reduced in the phosphomimetic mutations and the effect was due to a decrease in Vmax activity. The Ser to Glu mutations also caused a change in the apparent molecular weight of both the full-length protein and of the cytosolic tail of NHE1. A conformational change in this region was indicated by differential trypsin sensitivity. We also found that a peptide containing amino acids 783–790 bound to several more proximal regions of the NHE1 tail in in vitro protein interaction experiments. The results are the first characterization of these two amino acids and show that they have significant effects on enzyme kinetics and the structure of the NHE1 protein.
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spelling pubmed-85838162021-11-12 Amino Acids 785, 787 of the Na(+)/H(+) Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation Li, Xiuju Tu, Tommy Quan, Sicheng Quintero, Francisco J. Fahlman, Richard Fliegel, Larry Int J Mol Sci Article The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. Here, we investigated the effect of mutation of two amino acids of the regulatory tail, Ser(785) and Ser(787), that were similar in location and context to two amino acids of the Arabidopsis Na(+)/H(+) exchanger SOS1. Mutation of these two amino acids to either Ala or phosphomimetic Glu did not affect surface targeting but led to a slight reduction in the level of protein expressed. The activity of the NHE1 protein was reduced in the phosphomimetic mutations and the effect was due to a decrease in Vmax activity. The Ser to Glu mutations also caused a change in the apparent molecular weight of both the full-length protein and of the cytosolic tail of NHE1. A conformational change in this region was indicated by differential trypsin sensitivity. We also found that a peptide containing amino acids 783–790 bound to several more proximal regions of the NHE1 tail in in vitro protein interaction experiments. The results are the first characterization of these two amino acids and show that they have significant effects on enzyme kinetics and the structure of the NHE1 protein. MDPI 2021-10-21 /pmc/articles/PMC8583816/ /pubmed/34768780 http://dx.doi.org/10.3390/ijms222111349 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Li, Xiuju
Tu, Tommy
Quan, Sicheng
Quintero, Francisco J.
Fahlman, Richard
Fliegel, Larry
Amino Acids 785, 787 of the Na(+)/H(+) Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation
title Amino Acids 785, 787 of the Na(+)/H(+) Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation
title_full Amino Acids 785, 787 of the Na(+)/H(+) Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation
title_fullStr Amino Acids 785, 787 of the Na(+)/H(+) Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation
title_full_unstemmed Amino Acids 785, 787 of the Na(+)/H(+) Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation
title_short Amino Acids 785, 787 of the Na(+)/H(+) Exchanger Cytoplasmic Tail Modulate Protein Activity and Tail Conformation
title_sort amino acids 785, 787 of the na(+)/h(+) exchanger cytoplasmic tail modulate protein activity and tail conformation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8583816/
https://www.ncbi.nlm.nih.gov/pubmed/34768780
http://dx.doi.org/10.3390/ijms222111349
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