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The aquaporin splice variant NbXIP1;1α is permeable to boric acid and is Phosphorylated in the N-terminal domain

Ampah-Korsah, Henry LU ; Anderberg, Hanna I. LU ; Engfors, Angelica LU ; Kirscht, Andreas LU ; Nordén, Kristina LU ; Kjellström, Sven LU ; Kjellbom, Per LU and Johanson, Urban LU (2016) In Frontiers in Plant Science 7(JUNE2016).
Abstract

Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the... (More)

Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
AQP, Boric acid, Nicotiana benthamiana, NIP, Phosphorylation, Pichia pastoris, XIP
in
Frontiers in Plant Science
volume
7
issue
JUNE2016
publisher
Frontiers
external identifiers
  • Scopus:84975154511
ISSN
1664-462X
DOI
10.3389/fpls.2016.00862
language
English
LU publication?
yes
id
9292a28b-7792-4949-820c-9b160a034f01
date added to LUP
2016-07-26 09:00:27
date last changed
2016-09-20 03:07:00
@misc{9292a28b-7792-4949-820c-9b160a034f01,
  abstract     = {<p>Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel.</p>},
  author       = {Ampah-Korsah, Henry and Anderberg, Hanna I. and Engfors, Angelica and Kirscht, Andreas and Nordén, Kristina and Kjellström, Sven and Kjellbom, Per and Johanson, Urban},
  issn         = {1664-462X},
  keyword      = {AQP,Boric acid,Nicotiana benthamiana,NIP,Phosphorylation,Pichia pastoris,XIP},
  language     = {eng},
  month        = {06},
  number       = {JUNE2016},
  publisher    = {ARRAY(0x9b56188)},
  series       = {Frontiers in Plant Science},
  title        = {The aquaporin splice variant NbXIP1;1α is permeable to boric acid and is Phosphorylated in the N-terminal domain},
  url          = {http://dx.doi.org/10.3389/fpls.2016.00862},
  volume       = {7},
  year         = {2016},
}