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First published online August 19, 2005; 10.1104/pp.105.063198 Plant Physiology 139:287-295 (2005) © 2005 American Society of Plant Biologists A Novel Plant Major Intrinsic Protein in Physcomitrella patens Most Similar to Bacterial Glycerol Channels1Department of Plant Biochemistry, Centre for Chemistry and Chemical Engineering, Lund University, SE–221 00 Lund, Sweden (S.G., K.N., U.J.); and Unité de Biochimie Physiologique, Institut des Sciences de la Vie, Université Catholique de Louvain, B–1348 Louvain-la-Neuve, Belgium (A.-S.L., F.C.)
A gene encoding a novel fifth type of major intrinsic protein (MIP) in plants has been identified in the moss Physcomitrella patens. Phylogenetic analyses show that this protein, GlpF-like intrinsic protein (GIP1;1), is closely related to a subclass of glycerol transporters in bacteria that in addition to glycerol are highly permeable to water. A likely explanation of the occurrence of this bacterial-like MIP in P. patens is horizontal gene transfer. The expressed P. patens GIP1;1 gene contains five introns and encodes a unique C-loop extension of approximately 110 amino acid residues that has no obvious similarity with any other known protein. Based on alignments and structural comparisons with other MIPs, GIP1;1 is suggested to have retained the permeability for glycerol but not for water. Studies on heterologously expressed GIP1;1 in Xenopus laevis oocytes confirm the predicted substrate specificity. Interestingly, proteins of one of the plant-specific subgroups of MIPs, the NOD26-like intrinsic proteins, are also facilitating the transport of glycerol and have previously been suggested to have evolved from a horizontally transferred bacterial gene. Further studies on localization and searches for GIP1;1 homologs in other plants will clarify the function and significance of this new plant MIP.
The major intrinsic proteins (MIPs) form a large and ancient protein family since members of the MIP family are found in all kinds of organisms from the three domains of life: bacteria, archaea, and eukaryotes. MIPs form channels or pores through membranes to facilitate passive transport of water and other small polar molecules, such as glycerol, across membranes. According to substrate specificity, MIPs can be classified as aquaporins (AQPs; water channels) or glycerol facilitators (glycerol intrinsic proteins [GLPs]; Heymann and Engel, 1999  ). GLPs are also referred to as aquaglyceroporins because they, in addition to glycerol, are permeable to water to varying extent (Agre et al., 1998). High-resolution structures are available for AQPs from mammalians (AQP0 and AQP1) and Escherichia coli (AQPZ) along with a glycerol facilitator from E. coli (EcGlpF; Fu et al., 2000; de Groot et al., 2001; Sui et al., 2001; Savage et al., 2003; Gonen et al., 2004; Harries et al., 2004). Despite the evolutionary distance and the different substrate specificities, the structures are remarkably similar. All MIPs share the same basic topology of six transmembrane  -helices and a seventh transmembrane structure formed by two short helices entering the membrane from opposite sides and connecting at the conserved Asn-Pro-Ala (NPA) motifs. Plants have more genes encoding different MIPs than any other type of organism, possibly reflecting the importance of differential regulation of transport of water and small polar solutes in different tissues. In the model plant Arabidopsis (Arabidopsis thaliana), we have identified 35 genes coding for full-length MIPs (Johanson et al., 2001). Based on phylogenetic comparisons, plant MIPs are classified into four subfamilies, plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), Nod26-like intrinsic proteins (NIPs), and small-basic intrinsic proteins (SIPs). The PIPs and TIPs are in general regarded as AQPs, whereas the NIPs have been reported to transport both glycerol and water (for review, see Johanson et al., 2001). The specificity of the fourth subfamily is not yet known (Johanson and Gustavsson, 2002). All four subfamilies have also been identified in the primitive nonvascular moss Physcomitrella patens, suggesting that they are present in all terrestrial plants (Borstlap, 2002). Phylogenetic analyses have made it likely that the NIPs originally originated from a horizontally transferred bacterial AQP gene and were later adapted for glycerol transport (Zardoya et al., 2002). The lack of GLPs in plants was suggested to provide the driving force for the evolution of NIPs, although the physiological role of glycerol transport in plants is not completely clear. Here, we report and characterize a novel plant MIP with very low similarity to any other plant MIP but a surprisingly high similarity to a subclass of bacterial aquaglyceroporins. We suggest that this gene was also introduced into plants via horizontal gene transfer as proposed for the NIPs.
Sequence of cDNA Clone and Genes We have identified a novel plant MIP in P. patens upon searching for MIPs by TBLASTN in the P. patens expressed sequence tag (EST) database at http://www.moss.leeds.ac.uk. Two nonoverlapping EST sequences, BU052189 and BQ826917, both originating from the clone PPAS020308, were found. These ESTs encode MIP peptides that are very unlike previously known plant MIPs. Sequencing of PPAS020308 revealed a 1,602-bp-long cDNA insert with an open reading frame of 1,113 bp. Alignments with other MIPs support that this open reading frame contains the complete coding sequence. To ensure that the cDNA clone originated from P. patens and to study the gene structure, the corresponding genomic sequence was directly amplified from P. patens DNA with specific primers based on the cDNA sequence. Different primer combinations resulted in overlapping PCR products, which were sequenced with amplification primers and internal specific primers. The sequences of the exons and the cDNA are identical and confirm that the cDNA clone originates from P. patens. The gene from translation start to stop, including five introns, is 2,291 bp long. Intron lengths and positions relative to transmembrane regions in the protein are presented in Figure 1. The gene contains six exons and five introns, which all have canonical dinucleotides GT and AG for donor and acceptor sites. In the translated protein, introns I and V are positioned after H2 and in H5, respectively. Introns II, III, and IV are all located in the sequence encoding the elongated C-loop (see below).
It has previously been noted that intron positions are conserved within each of the four MIP subfamilies in Arabidopsis (Johanson et al., 2001 ). It is therefore worth mentioning that the position of the first intron is situated in the exact site as the first plant PIP intron. Part of an orthologous gene encoding the novel MIP type, 421 bp including intron V, was also successfully amplified and sequenced in the moss Funaria hygrometrica. Sequence comparison of the orthologous genes revealed six indels and approximately twice the substitution frequency in the introns relative to the coding sequences, 0.19 and 0.10, respectively (data not shown). The majority of the substitutions in the coding region are synonymous, reducing the effect in the protein to five conservative replacements in the 54 amino acids encoded by the sequenced region of the gene.
By comparing this P. patens MIP sequence to AQPs and glycerol channels, it is obvious that it belongs to the superfamily of MIPs (Fig. 2). The highly conserved NPA motifs that often are used as fingerprints of the MIP family are indeed present, and the predicted topology of six transmembrane helices agrees well with known MIP structures. However, this MIP is clearly different from all earlier identified plant MIPs and cannot be categorized to any of the earlier identified subfamilies of MIPs in plants (Johanson et al., 2001
The most striking feature of GIP1;1 is that the C-loop has an extension of around 100 amino acid residues compared to most other MIPs (112 residues compared to the most similar sequences). The insert does not show any obvious similarity with any known protein in public protein or translated DNA databases. In the structure of EcGlpF, the extension fits nicely at the very top of the extracellular protrusion of the protein. The inserted region consists of a large portion of charged and polar residues, and according to the topology prediction, the C-loop does not form any transmembrane helices.
Another odd feature of GIP1;1 is that it differs in some amino acid positions that are very well conserved among glycerol facilitators and even MIPs in general. For example, in EcGlpF, H66, T72, F89, and Q93 have been suggested to be involved in packing of the core near the first NPA-box (Fu et al., 2000
GIP1;1 has a relatively short N terminus, which is unusual for plant MIPs and only found in the SIP subfamily. In contrast, short N termini are common among bacterial glycerol channels. The C terminus of GIP1;1 is also relatively short but contains a putative phosphorylation site in resemblance to some other plant MIPs that have been suggested to be regulated by phosphorylation (Johansson et al., 2000 The most probable subcellular localization predicted by TargetP V1.0 is "other membrane" (score of 0.794), which excludes membranes from chloroplast (0.007), mitochondria (0.020), and secretory pathway (0.657). The PSORT result suggests plasma membrane localization (0.600). Second best is the chloroplast thylakoid membrane (0.421). Hence, the most probable localization of GIP1;1 is in the plasma membrane.
GIP1;1 belongs to the MIPs, but it clearly diverts from any other plant MIP characterized to date. Instead, it is more similar to bacterial glycerol transporters. To clarify the origin of the GIP1;1 gene, phylogenetic analyses were performed. Bayesian, neighbor joining, and parsimony methods were employed for analysis of a multiple alignment of 67 taxa, including mammal, plant, fungi, bacterial, and archaean MIP sequences. These particular taxa were sampled to represent all the plant MIP subfamilies, mammalian AQPs and GLPs and a wide range of bacterial glycerol transporters. The archaean sequence MthAQPM was used as an outgroup. The ingroups form two major clades that are well supported both by neighbor joining and Bayesian methods (Fig. 3). The plant MIPs, except GIP1;1, together with the bacterial and human water channels, form one major clade from which the NIPs split off first and then the bacterial AQPs. The second major clade consists mainly of bacterial and human glycerol transporters that branch into two subclades: type I glycerol transporters together with the human aquaglyceroporins and type II glycerol transporters together with GIP1;1. The bootstrap value and the posterior probability of the node joining type II glycerol transporters and GIP1;1 are 71% and 100%, respectively. This makes the clustering of GIP1;1 with the type II bacterial GLPs quite well supported and confirms our initial classification of GIP1;1. Interestingly, the human aquaglyceroporins cluster together with type I bacterial GLPs. However, with a posterior probability and a bootstrap value of 52%, this grouping is poorly supported.
In a parsimony analysis of the 67 taxa, the resulting trees were very unstable, and the topology of the trees changed upon removal of some taxa (data not shown). However, in an exhaustive parsimony search of 10 taxa of the type II bacterial GLPs and GIP1;1, using EcGlpF as outgroup, GIP1;1 appears at the top in all of the resulting five trees (data not shown). GIP1;1 is associated with Clostridium tetani or Chloroflexus aurantiacus GLPs in four trees and with C. aurantiacus and Oceanobacillus iheyensis GLPs in one tree. In the Bayesian tree, the former association is supported by a posterior probability of 96%. Although there are several differences between the trees from different methods, the major groupings are similar and most importantly support that GIP1;1 belongs to the clade of type II bacterial glycerol transporters rather than to other plant MIPs.
The divergency times of GIP1;1 and type II bacterial GLPs and of NIPs and bacterial AQPZs were estimated to 1,038 and 1,135 million years, respectively, using the nonparametric rate-smoothing method (Sanderson, 2003
In order to examine the permeability of GIP1;1, the protein was heterologously expressed in X. laevis oocytes. Oocytes were injected with water or in vitro transcribed RNA coding for GIP1;1 or a positive control. Labeling of the oocyte proteins with [35S]-Met confirmed that GIP1;1 was expressed in the oocytes (Fig. 4A). The water permeability of oocytes that expressed GIP1;1 was not significantly higher than that of the negative control (Fig. 4B). In contrast, the glycerol uptake was 4-fold higher in oocytes injected with RNA encoding GIP1;1 than for the water-injected negative controls (Fig. 4C). Although GIP1;1 is not as permeable to glycerol as the positive control EcGlpF, GIP1;1 is clearly a glycerol-specific channel.
GIP1;1 Belongs to the Bacterial Glycerol Transporters of Type II
Based on sequence comparisons, glycerol transporters of bacteria are divided into two groups that have been proposed to have different substrate specificity (Hohmann et al., 2000
In addition to the extended C-loop, there are several other deviations from previously identified MIPs. In EcGlpF, H66, T72, F89, and Q93 have been suggested to participate in packing of the core next to the first NPA-box. These amino acid residues are very well conserved throughout all MIPs with few exceptions. The corresponding residues in GIP1;1, F66, A72, C89, and E93, suggest that correlated replacements have resulted in an alternative packing core in GIP1;1. These changes might also modify the properties of the pore in this region, but the exact consequences on the substrate specificity are hard to predict.
According to available high-resolution structures, the amino acid residues in the constriction region form the narrowest part of the channel and directly interact with the substrate. The constriction regions of some of the discussed MIPs are summarized in Table I. In AQP1, amino acid residues F58, H182, C191, and R197 form the constriction region (numbering refers to bovine AQP1). A nitrogen in the His side chain makes a hydrogen bond to one of the water molecules in the channel. The same water is also bonded to the side chain of the Arg. The Phe orients the water molecule and thereby enables the hydrogen bonding from water to H182 and R197 (de Groot and Grubmuller, 2001
In EcGlpF, the corresponding amino acid residues are W48, G191, F200, and R206 (Fu et al., 2000 ; de Groot and Grubmuller, 2001; Stroud et al., 2003). Here, the Arg side chain creates hydrogen bonds with two of the substrate hydroxyl groups. The backbone of the glycerol molecule is wedged tightly against a hydrophobic corner created by the Trp and the Phe. To make space for the bulky Phe, the His is replaced by a Gly. Although the EcGlpF pore is larger than the pore of AQP1, the passage of water is relatively small. One reason for this is the hydrophobicity of the constriction region, which reduces the possibility of water molecules to pass (Sui et al., 2001).
In GlaLlac from L. lactis, which is an aquaglyceroporin with ability to facilitate both glycerol and water transport, the constriction region is Y49, V223, P232, and R238 (Froger et al., 2001
In GIP1;1, the four constriction region residues are F49, V304, P313, and R319. The only difference with GlaLlac is that the Tyr is replaced by Phe, resulting in a more hydrophobic constriction region. Thus, it is reasonable to expect GIP1;1 to have a relatively large pore and to be permeable for glycerol in a similar manner as has been shown for GlaLlac. It is more speculative to predict the water permeability of GIP1;1. The hydroxyl group of Tyr in GlaLlac might be important for water transport by providing a site for hydrogen bond formation. This would suggest that GIP1;1 is less permeable to water compared to GlaLlac. It has been proposed that aquaglyceroporins facilitating both water and glycerol have two polar residues in the constriction region, whereas glycerol channels excluding water only have one single polar residue in the constriction region (Thomas et al., 2002 Although the constriction region of GIP1;1 is quite similar to the corresponding region of GlaLlac, the MIP sequences closest related to GIP1;1 (see below) usually have Trp, Gly, Tyr, and Arg at the constriction region. This suggests that GIP1;1 has evolved from an aquaglyceroporin into a glycerol-specific channel.
Consistent with the predictions, functional analyses clearly show that GIP1;1 is a glycerol-specific channel with no or very low water permeability. However, the glycerol permeability is somewhat lower than expected. The C-loop connects the two direct repeats that form all MIPs and is one of the more variable regions in MIPs, indicating that alterations can be accommodated here without interfering with the general MIP structure. Among GLPs and GIP1;1 there is a conserved element in the last part of the C-loop that has been shown to be involved in binding of glycerol in the extracellular vestibule of EcGlpF (Fu et al., 2000
In our alignments, the MIPs most similar to GIP1;1 are GlpFs from C. tetani and Bacillus halodurans with 41.5% and 41.0% identity, respectively. In contrast, the identity of the best match in plants based on BLAST searches, NIP1;1 from Zea mays, is only 30.0%. The phylogenetic tree (Fig. 3) and the high identity to type II glycerol transporters preferentially found in the evolutionary distant gram-positive bacteria suggest that the ancestral GIP gene was incorporated in a plant genome via horizontal gene transfer.
In a recent investigation of the transcriptome of P. patens (Nishiyama et al., 2003
The plant-specific NIP subfamily has also been suggested to have evolved through a horizontal gene transfer event (Zardoya et al., 2002
If GIP1;1 was recruited by plants from a gram-positive bacterium by horizontal gene transfer, there were probably no introns in the original GIP gene. Although it is well established that intron recruitment occurs, the mechanisms are still unclear (Brady and Danforth, 2004
A novel plant MIP with unique features has been identified. The phylogenetic analyses and the high identity to bacterial glycerol transporters support that this gene has been acquired via horizontal gene transfer of a bacterial gene encoding a glycerol transporter. Sequence-based predictions of substrate specificity suggest that GIP1;1 is permeable for glycerol but not for water. This is confirmed by functional analyses of heterologously expressed GIP1;1 in X. laevis oocytes. The high divergence of GIP1;1 from the proposed gram-positive bacterial ancestors might suggest that GIP1;1 is member of a relatively old and until now unrecognized fifth subfamily of plant MIPs. Investigations of the presence of GIPs in other plant species will bring more light to the origin and possible loss of GIPs.
Sequencing EST clone PPAS20308 was achieved from the Leeds Institute for Plant Biotechnology and Agriculture. The plasmid was sequenced with T3-, T7-, and gene-specific primers. Genomic DNA was isolated from Physcomitrella patens and Funaria hygrometrica kindly provided by Hans Ronne (Swedish University of Agricultural Sciences, Uppsala, Sweden) and Nils Cronberg (Lund University, Sweden), respectively. The genomic sequence from P. patens was obtained by PCR amplification of overlapping segments from genomic DNA followed by sequencing of PCR products with amplification primers and with internal primers. Only a part of the GIP1;1 gene from F. hygrometrica was amplified and sequenced. Primer sequences are available on request. The accession numbers for cDNA, gene sequence from P. patens, and partial F. hygrometrica gene sequence are AY611236, AY611237, and DQ092355, respectively.
The topology of the translated protein was predicted by the HMMTOP2.0 method (Tusnady and Simon, 1998
A protein alignment of 67 taxa, including plant, human, fungi, bacterial, and archaean sequences, was performed with ClustalW included in MacVector 7.2 (Accelrys) and refined by eye. The alignment included all 35 Arabidopsis (Arabidopsis thaliana) MIPs, and the total length was 808 characters. N- and C-terminal regions and loop regions, except loops B and E, were excluded from the analyses. In total, 198 characters of the original alignment were included in the analyses (characters 345–372, 396–471, 611–637, 655–693, and 710–737). Phylogenetic analysis was performed with Mr Bayes V 3.0 Mac (Ronquist and Huelsenbeck, 2003
For in vitro transcription, the coding sequence of GIP1;1 was subcloned into the BglII site of pX Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AY611236, AY611237, and DQ092355.
We thank Dr. Stavros Bashiardes as part of the P. patens EST Program at the University of Leeds (Leeds, UK) and Washington University (St. Louis) for sharing the cDNA clone PPAS20308. Prof. Hans Ronne (Swedish University of Agricultural Sciences, Uppsala, Sweden) and Nils Cronberg (Lund University) are acknowledged for providing P. patens and F. hygrometrica, respectively. Received March 22, 2005; returned for revision June 12, 2005; accepted June 16, 2005.
1 This work was supported by the Erik Philip-Sörensen Foundation and the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning (FORMAS; grants to U.J.) and by the Belgian Fund for Scientific Research and the Interuniversity Attraction Poles Programme-Belgian Science Policy (grants to F.C.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.063198. * Corresponding author; e-mail urban.johanson{at}plantbio.lu.se; fax 46–46–2224116.
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G-ev1 by PCR. The construct was verified by sequencing. In vitro transcription, X. laevis oocyte preparation, injection, protein labeling, and the osmotic water permeability assay were performed as previously described (Fetter et al., 2004
