Molecular and Functional Characterization of Grapevine NIPs through Heterologous Expression in aqy-Null Saccharomyces cerevisiae

Abstract

Plant Nodulin 26-like Intrinsic Proteins (NIPs) are multifunctional membrane channels of the Major Intrinsic Protein (MIP) family. Unlike other homologs, they have low intrinsic water permeability. NIPs possess diverse substrate selectivity, ranging from water to glycerol and to other small solutes, depending on the group-specific amino acid composition at aromatic/Arg (ar/R) constriction. We cloned three NIPs (NIP1;1, NIP5;1, and NIP6;1) from grapevine (cv. Touriga Nacional). Their expression in the membrane of aqy-null Saccharomyces cerevisiae enabled their functional characterization for water and glycerol transport through stopped-flow spectroscopy. VvTnNIP1;1 demonstrated high water as well as glycerol permeability, whereas VvTnNIP6;1 was impermeable to water but presented high glycerol permeability. Their transport activities were declined by cytosolic acidification, implying that internal-pH can regulate NIPs gating. Furthermore, an extension of C-terminal in VvTnNIP6;1M homolog, led to improved channel activity, suggesting that NIPs gating is putatively regulated by C-terminal. Yeast growth assays in the presence of diverse substrates suggest that the transmembrane flux of metalloids (As, B, and Se) and the heavy metal (Cd) are facilitated through grapevine NIPs. This is the first molecular and functional characterization of grapevine NIPs, providing crucial insights into understanding their role for uptake and translocation of small solutes, and extrusion of toxic compounds in grapevine.

Insights into the Selectivity Mechanisms of Grapevine NIP Aquaporins

“…To investigate the functional significance of the two conserved residues in putative ar/R filter (H2 and H5) of the pore region of NIP subgroups (I, II, and III) proteins, amino acid substitution within the NIP subgroup was performed by site-directed mutagenesis in VvTnNIP1;1, VvTnNIP2;1, VvTnNIP5;1, and VvTnNIP6;1 [ 17 ]. In the case of VvTnNIP6;1, a mutated version (VvTnNIP6;1M) with extended C-terminal, possessing water conductivity and improved glycerol permeability, was chosen, to investigate the influence of ar/R substitution on both glycerol and water transport.…”

“…Stopped-flow fluorescence spectroscopy was employed to compare the water and glycerol transport activity of yeast cells expressing various ar/R mutant constructs of functional grapevine NIPs, namely VvTnNIP1;1 and VvTnNIP6;1 from the previous study [ 17 ]. In detail, we constructed three mutated versions of VvTnNIP1;1, either at H2 (W86T) or H5 (V206I) positions, with a double mutant at both H2 and H5 positions (W86T/V206I) in the present study.…”

“…We recently demonstrated that VvTnNIP1;1 is an aquaglyceroporin with high glycerol and moderate water permeability, respectively. Differently from VvTnNIP1;1, VvTnNIP6;1 is an aquaglyceroporin with low water transport capability, although featuring less pronounced glycerol permeation than VvTnNIP1;1 [ 17 ]. The ar/R filter of aquaglyceroporin VvTnNIP1;1 is comprised of WVAR.…”

“…Our recent study revealed that native grapevine NIPs might have a putative role in transport of arsenic, boron and selenium [ 17 ]. Yeast cells expressing VvTnNIP1;1 and VvTnNIP6;1 showed sensitivity towards arsenous and boronic acid.…”

“…However, only a few studies have addressed NIPs, despite being considered as crucial channels for transport of various beneficial (like B and Si), as well as toxic (like As), metalloids in plants [ 23 , 24 , 25 , 26 ]. Thus, the present study aimed to explore the role of ar/R filter in the regulation of substrate selectivity in grapevine NIPs [ 17 , 18 ]. Site directed mutagenesis in their r/R region was performed and the resulting functional divergence caused by possible changes in the pore structural architecture was investigated by stopped-flow fluorescence [ 27 ].…”