Distinct transport selectivity of two structural subclasses of the nodulin-like intrinsic protein family of plant aquaglyceroporin channels

Major intrinsic proteins (MIPs) are a diverse class of integral membrane proteins that facilitate the transport of water and some small solutes across cellular membranes. X-ray structures of MIPs indicate that a tetrad of residues (the ar/R region) form a narrow pore constriction that constitutes the selectivity filter. In comparison with mammalian and microbial species, plants have a greater number and diversity of MIPs with greater than 30 genes encoding four phylogenetic subfamilies with eight different classes of ar/R sequences. The nodulin 26-like intrinsic protein (NIP) subfamily in Arabidopsis can be subdivided into two ar/R subgroups: the NIP subgroup I, which resembles the archetype of the family, soybean nodulin 26, and the NIP subgroup II, which is represented by the Arabidopsis protein AtNIP6;1. These two NIPs differ principally by the substitution of a conserved alanine (NIP subgroup II) for a conserved tryptophan (NIP subgroup I) in the helix 2 position (H2) of the ar/R filter. A comparison of the water and solute tranport properties of the two proteins was performed by expression in Xenopus laevis oocytes. Nodulin 26 is an aquaglyceroporin with a modest osmotic water permeability (P(f)) and the ability to transport uncharged solutes such as glycerol and formamide. In constrast, AtNIP6;1 showed no measurable water permeability but transported glycerol, formamide, as well as larger solutes that were impermeable to nodulin 26. By site-directed mutagenesis, we show that the H2 position is the crucial determinant that confers these transport behaviors. A comparison of the NIPs and tonoplast-intrinsic proteins (TIP) shows that the H2 residue can predict the transport profile for water and glycerol with histidine found in TIP-like aquaporins, tryptophan found in aquaglyceroporins (NIP I), and alanine found in water-impermeable glyceroporins (AtNIP6;1).     

Homology modeling of major intrinsic proteins in rice,maize and <Emphasis Type="Italic">Arabidopsis</Emphasis>: comparative analysis of transmembrane helix association and aromatic/arginine selectivity filters

Background  

The major intrinsic proteins (MIPs) facilitate the transport of water and neutral solutes across the lipid bilayers. Plant MIPs are believed to be important in cell division and expansion and in water transport properties in response to environmental conditions. More than 30 MIP sequences have been identified in Arabidopsis thaliana, maize and rice. Plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), Nod26-like intrinsic protein (NIPs) and small and basic intrinsic proteins (SIPs) are subfamilies of plant MIPs. Despite sequence diversity, all the experimentally determined structures belonging to the MIP superfamily have the same "hour-glass" fold.     

The structure, function and regulation of the nodulin 26-like intrinsic protein family of plant aquaglyceroporins

The nodulin 26-like intrinsic protein family is a group of highly conserved multifunctional major intrinsic proteins that are unique to plants, and which transport a variety of uncharged solutes ranging from water to ammonia to glycerol. Based on structure-function studies, the NIP family can be subdivided into two subgroups (I and II) based on the identity of the amino acids in the selectivity-determining filter (ar/R region) of the transport pore. Both subgroups appear to contain multifunctional transporters with low to no water permeability and the ability to flux multiple uncharged solutes of varying sizes depending upon the composition of the residues of the ar/R filter. NIPs are subject to posttranslational phosphorylation by calcium-dependent protein kinases. In the case of the family archetype, soybean nodulin 26, phosphorylation has been shown to stimulate its transport activity and to be regulated in response to developmental as well as environmental cues, including osmotic stresses. NIPs tend to be expressed at low levels in the plant compared to other MIPs, and several exhibit cell or tissue specific expression that is subject to spatial and temporal regulation during development.     

The structure, function and regulation of the nodulin 26-like intrinsic protein family of plant aquaglyceroporins

The nodulin 26-like intrinsic protein family is a group of highly conserved multifunctional major intrinsic proteins that are unique to plants, and which transport a variety of uncharged solutes ranging from water to ammonia to glycerol. Based on structure-function studies, the NIP family can be subdivided into two subgroups (I and II) based on the identity of the amino acids in the selectivity-determining filter (ar/R region) of the transport pore. Both subgroups appear to contain multifunctional transporters with low to no water permeability and the ability to flux multiple uncharged solutes of varying sizes depending upon the composition of the residues of the ar/R filter. NIPs are subject to posttranslational phosphorylation by calcium-dependent protein kinases. In the case of the family archetype, soybean nodulin 26, phosphorylation has been shown to stimulate its transport activity and to be regulated in response to developmental as well as environmental cues, including osmotic stresses. NIPs tend to be expressed at low levels in the plant compared to other MIPs, and several exhibit cell or tissue specific expression that is subject to spatial and temporal regulation during development.     

Plasma membrane intrinsic proteins from maize cluster in two sequence subgroups with differential aquaporin activity

The transport of water through membranes is regulated in part by aquaporins or water channel proteins. These proteins are members of the larger family of major intrinsic proteins (MIPs). Plant aquaporins are categorized as either tonoplast intrinsic proteins (TIPs) or plasma membrane intrinsic proteins (PIPs). Sequence analysis shows that PIPs form several subclasses. We report on the characterization of three maize (Zea mays) PIPs belonging to the PIP1 and PIP2 subfamilies (ZmPIP1a, ZmPIP1b, and ZmPIP2a). The ZmPIP2a clone has normal aquaporin activity in Xenopus laevis oocytes. ZmPIP1a and ZmPIP1b have no activity, and a review of the literature shows that most PIP1 proteins identified in other plants have no or very low activity in oocytes. Arabidopsis PIP1 proteins are the only exception. Control experiments show that this lack of activity of maize PIP1 proteins is not caused by their failure to arrive at the plasma membrane of the oocytes. ZmPIP1b also does not appear to facilitate the transport of any of the small solutes tried (glycerol, choline, ethanol, urea, and amino acids). These results are discussed in relationship to the function and regulation of the PIP family of aquaporins.     

Arabidopsis thaliana NIP7;1: an anther-specific boric acid transporter of the aquaporin superfamily regulated by an unusual tyrosine in helix 2 of the transport pore

Plant nodulin-26 intrinsic proteins (NIPs) are members of the aquaporin superfamily that serve as multifunctional transporters of uncharged metabolites. In Arabidopsis thaliana, a specific NIP pore subclass, known as the NIP II proteins, is represented by AtNIP5;1 and AtNIP6;1, which encode channel proteins expressed in roots and leaf nodes, respectively, that participate in the transport of the critical cell wall nutrient boric acid. Modeling of the protein encoded by the AtNIP7;1 gene shows that it is a third member of the NIP II pore subclass in Arabidopsis. However, unlike AtNIP5;1 and AtNIP6;1 proteins, which form constitutive boric acid channels, AtNIP7;1 forms a channel with an extremely low intrinsic boric acid transport activity. Molecular modeling and molecular dynamics simulations of AtNIP7;1 suggest that a conserved tyrosine residue (Tyr81) located in transmembrane helix 2 adjacent to the aromatic arginine (ar/R) pore selectivity region stabilizes a closed pore conformation through interaction with the canonical Arg220 in ar/R region. Substitution of Tyr81 with a Cys residue, characteristic of established NIP boric acid channels, results in opening of the AtNIP7;1 pore that acquires a robust, transport activity for boric acid as well as other NIP II test solutes (glycerol and urea). Substitution of a Phe for Tyr81 also opens the channel, supporting the prediction from MD simulations that hydrogen bond interaction between the Tyr81 phenol group and the ar/R Arg may contribute to the stabilization of a closed pore state. Expression analyses show that AtNIP7;1 is selectively expressed in developing anther tissues of young floral buds of A. thaliana, principally in developing pollen grains of stage 9-11 anthers. Because boric acid is both an essential nutrient as well as a toxic compound at high concentrations, it is proposed that Tyr81 modulates transport and may provide an additional level of regulation for this transporter in male gametophyte development.     

Substitution of a single amino acid residue in the aromatic/arginine selectivity filter alters the transport profiles of tonoplast aquaporin homologs

Aquaporins are integral membrane proteins that facilitate the transport of water and some small solutes across cellular membranes. X-ray crystallography of aquaporins indicates that four amino acids constitute an aromatic/arginine (ar/R) pore constriction known as the selectivity filter. On the basis of these four amino acids, tonoplast aquaporins called tonoplast intrinsic proteins (TIPs) are divided into three groups in Arabidopsis. Herein, we describe the characterization of two group I TIP1s (TgTIP1;1 and TgTIP1;2) from tulip (Tulipa gesneriana). TgTIP1;1 and TgTIP1;2 have a novel isoleucine in loop E (LE2 position) of the ar/R filter; the residue at LE2 is a valine in all group I TIPs from model plants. The homologs showed mercury-sensitive water channel activity in a fast kinetics swelling assay upon heterologous expression in Pichia pastoris. Heterologous expression of both homologs promoted the growth of P. pastoris on ammonium or urea as sole sources of nitrogen and decreased growth and survival in the presence of H(2)O(2). TgTIP1;1- and TgTIP1;2-mediated H(2)O(2) conductance was demonstrated further by a fluorescence assay. Substitutions in the ar/R selectivity filter of TgTIP1;1 showed that mutants that mimicked the ar/R constriction of group I TIPs could conduct the same substrates that were transported by wild-type TgTIP1;1. In contrast, mutants that mimicked group II TIPs showed no evidence of urea or H(2)O(2) conductance. These results suggest that the amino acid residue at LE2 position is critical for the transport selectivity of the TIP homologs and group I TIPs might have a broader spectrum of substrate selectivity than group II TIPs.     

The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants

Major intrinsic proteins (MIPs) facilitate the passive transport of small polar molecules across membranes. MIPs constitute a very old family of proteins and different forms have been found in all kinds of living organisms, including bacteria, fungi, animals, and plants. In the genomic sequence of Arabidopsis, we have identified 35 different MIP-encoding genes. Based on sequence similarity, these 35 proteins are divided into four different subfamilies: plasma membrane intrinsic proteins, tonoplast intrinsic proteins, NOD26-like intrinsic proteins also called NOD26-like MIPs, and the recently discovered small basic intrinsic proteins. In Arabidopsis, there are 13 plasma membrane intrinsic proteins, 10 tonoplast intrinsic proteins, nine NOD26-like intrinsic proteins, and three small basic intrinsic proteins. The gene structure in general is conserved within each subfamily, although there is a tendency to lose introns. Based on phylogenetic comparisons of maize (Zea mays) and Arabidopsis MIPs (AtMIPs), it is argued that the general intron patterns in the subfamilies were formed before the split of monocotyledons and dicotyledons. Although the gene structure is unique for each subfamily, there is a common pattern in how transmembrane helices are encoded on the exons in three of the subfamilies. The nomenclature for plant MIPs varies widely between different species but also between subfamilies in the same species. Based on the phylogeny of all AtMIPs, a new and more consistent nomenclature is proposed. The complete set of AtMIPs, together with the new nomenclature, will facilitate the isolation, classification, and labeling of plant MIPs from other species.     

A new subfamily of major intrinsic proteins in plants

The major intrinsic proteins (MIPs) form a large protein family of ancient origin and are found in bacteria, fungi, animals, and plants. MIPs act as channels in membranes to facilitate passive transport across the membrane. Some MIPs allow small polar molecules like glycerol or urea to pass through the membrane. However, the majority of MIPs are thought to be aquaporins (AQPs), i.e., they are specific for water transport. Plant MIPs can be subdivided into the plasma membrane intrinsic protein, tonoplast intrinsic protein, and NOD26-like intrinsic protein subfamilies. By database mining and phylogenetic analyses, we have identified a new subfamily in plants, the Small basic Intrinsic Proteins (SIPs). Comparisons of sequences from the new subfamily with conserved amino acid residues in other MIPs reveal characteristic features of SIPs. Possible functional consequences of these features are discussed in relation to the recently solved structures of AQP1 and GlpF. We suggest that substitutions at conserved and structurally important positions imply a different substrate specificity for the new subfamily.     

Genome-Wide Identification and Expression Analysis of Aquaporins in Tomato

The family of aquaporins, also called water channels or major intrinsic proteins, is characterized by six transmembrane domains that together facilitate the transport of water and a variety of low molecular weight solutes. They are found in all domains of life, but show their highest diversity in plants. Numerous studies identified aquaporins as important targets for improving plant performance under drought stress. The phylogeny of aquaporins is well established based on model species like Arabidopsis thaliana, which can be used as a template to investigate aquaporins in other species. In this study we comprehensively identified aquaporin encoding genes in tomato (Solanum lycopersicum), which is an important vegetable crop and also serves as a model for fleshy fruit development. We found 47 aquaporin genes in the tomato genome and analyzed their structural features. Based on a phylogenetic analysis of the deduced amino acid sequences the aquaporin genes were assigned to five subfamilies (PIPs, TIPs, NIPs, SIPs and XIPs) and their substrate specificity was assessed on the basis of key amino acid residues. As ESTs were available for 32 genes, expression of these genes was analyzed in 13 different tissues and developmental stages of tomato. We detected tissue-specific and development-specific expression of tomato aquaporin genes, which is a first step towards revealing the contribution of aquaporins to water and solute transport in leaves and during fruit development.     

Comparative phylogenetic analysis of aquaporins provides insight into the gene family expansion and evolution in plants and their role in drought tolerant and susceptible chickpea cultivars

Aquaporins (AQPs) are water channel proteins that play a significant role in drought stress. Although the AQPs identified in multiple plant species, there is no detailed evolutionary and comparative study of AQPs regarding chickpea plant. The current study involved evolutionary analyses coupled with promoter and expression analyses of chickpea AQPs (CaAQPs). A total of 924 non-redundant AQPs were studied in 24 plant species including algae, mosses, lycophytes, monocots and dicots. Phylogenetic analysis demonstrated a clear divergence of eight AQP subfamilies (LIPs, SIPs, GIPs, NIPs, XIPs, PIPs, HIPs and TIPs). The comparative phylogenetic trees of AQP subfamilies among Arabidopsis, soybean, common bean, maize and chickpea demonstrated that the AQPs were highly species-specific. Interestingly, the dual NPA motif was conserved in all species. However, the ar/R selectivity filter signatures [W/T/S/N/G/A]-[V/S/L/I/A]-[S/G/A]-R (in NIPs), F-H-T-R (in PIPs), [H/N/Q/S]-[A/I/L/S/V]-[A/G]-[A/C/L/M/R/V] (in TIPs) and [V/I/L/M]-[V/I/A/F/M]-[A/S/F/C]-[N/F/L/I/A/S (in SIPs) were found in five species. Moreover, the Froger's positions (P1-P5) were found as [F/L/Y]-[S/T]-A-Y-[L/I/M/V/F] (in NIPs), [Q/E/M]-S-A-F-W (in PIPs), [A/L/S/T/V]-[A/C/N/S/T/V]-[P/R/S]-[Y/N/F]-[W/Q] (in TIPs) and [I/M/F]-[A/V]-[A/V]-Y-W (in SIPs). The MEME motif analyses showed that most of the motifs were specific to subfamily and subgroups. Tissue-specific expression profiling of CaAQPs revealed that CaTIPs and CaPIPs are highly expressed in most of the tissues, while CaNIPs and CaSIPs have low expression. In promoter analysis of CaAQPs, multiple stress-related cis-acting elements e.g. MYB, MYC, ABRE, etc. were found. Semi-quantitative RT-PCR analysis showed that CaPIP2;3 and CaNIP3;1 are positive regulator, while CaSIP1;1 and CaPIP2;1 have a negative role in drought tolerance. The findings and implications of this study are discussed in detail.     

Aquaporins constitute a large and highly divergent protein family in maize

Aquaporins (AQPs) are an ancient family of channel proteins that transport water and neutral solutes through a pore and are found in all eukaryotes and most prokaryotes. A comparison of the amino acid sequences and phylogenetic analysis of 31 full-length cDNAs of maize (Zea mays) AQPs shows that they comprise four different groups of highly divergent proteins. We have classified them as plasma membrane intinsic proteins (PIPs), tonoplast intrinsic proteins, Nod26-like intrinsic proteins, and small and basic intrinsic proteins. Amino acid sequence identities vary from 16% to 100%, but all sequences share structural motifs and conserved amino acids necessary to stabilize the two loops that form the aqueous pore. Most divergent are the small and basic integral proteins in which the first of the two highly conserved Asn-Pro-Ala motifs of the pore is not conserved, but is represented by alanine-proline-threonine or alanine-proline-serine. We present a model of ZmPIP1-2 based on the three-dimensional structure of mammalian AQP1. Tabulation of the number of times that the AQP sequences are found in a collection of databases that comprises about 470,000 maize cDNAs indicates that a few of the maize AQPs are very highly expressed and many are not abundantly expressed. The phylogenetic analysis supports the interpretation that the divergence of PIPs through gene duplication occurred more recently than the divergence of the members of the other three subfamilies. This study opens the way to analyze the function of the proteins in Xenopus laevis oocytes, determine the tissue specific expression of the genes, recover insertion mutants, and determine the in planta function.