• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of plntphysLink to Publisher's site
Plant Physiol. Sep 2005; 139(1): 287–295.
PMCID: PMC1203378

A Novel Plant Major Intrinsic Protein in Physcomitrella patens Most Similar to Bacterial Glycerol Channels1

Abstract

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 α-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.

RESULTS

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).

Figure 1.
Gene structure of P. patens GIP1;1. GIP1;1 is presented. Based on alignments and structural data from E. coli, GlpF membrane spanning helices (H1–H6) and half spanning helices (HB and HE) are depicted as boxes. Corresponding helices in the direct ...

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.

General Structure and Localization

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). Most surprisingly, when performing BLASTP and TBLASTN searches, the most significant hits are bacterial glycerol channels rather than plant MIPs. In accordance with the proposed nomenclature for plant MIPs, in the following we will refer to this protein as GlpF-like intrinsic protein (GIP1;1; Johanson et al., 2001).

Figure 2.
Multiple protein alignment of GIP1;1 (PpGIP1;1), three bacterial GLPs type II, one bacterial GLP type I, AQPZ from E. coli, NIP 1;1 from maize (the closest plant homolog to GIP1;1 found by BLAST searches), and bovine AQP1. Identical residues are boxed ...

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). However, in GIP1;1, the corresponding residues are F66, A72, C89, and E93, which all have properties that are very different from the conserved residues. In addition, the loop preceding the first NPA-box is one amino acid residue shorter in GIP1;1 than in most MIPs.

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). However, the putative phosphorylation motif in GIP1;1, RXXRXS, is not identical to phosphorylation motifs in other plant MIPs. Only short motifs with high probability of occurrence by chance are found when GIP1;1 is scanned for motifs and patterns in the PROSITE database (data not shown).

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.

Phylogenetic Analysis of GIP1;1

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.

Figure 3.
Bayesian 50% majority-rule consensus tree. Posterior probabilities of each node are indicated. GIP1;1 (PpGIP1;1; marked by an asterisk) is closely associated with the bacterial GLPs of type II. Archaean MthAQPM was used as outgroup.

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).

Functional Analysis of GIP1;1 in Xenopus laevis Oocytes

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.

Figure 4.
Protein labeling and water and glycerol permeability measurements in X. laevis oocytes expressing GIP1;1, ZmPIP2;1, or GlpF. A, In vivo labeled proteins in total membrane fraction prepared from the oocytes. The arrowhead indicates the position of GIP1;1. ...

DISCUSSION

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; Froger et al., 2001). In the phylogenetic tree in Figure 3, we refer to these groups as types I and II GLPs. This classification reflects the phylogeny of bacteria since type I GLPs are preferentially found in gram-negative bacteria, whereas type II is commonly found in gram-positive bacteria (Hohmann et al., 2000). In all our phylogenetic analyses, GIP1;1 clusters with the type II GLPs. The bacterial glycerol transporter best characterized so far is EcGlpF, which belongs to type I glycerol transporters. EcGlpF has been shown to be relatively permeable to glycerol and other linear alcohols but less permeable to water (Heller et al., 1980; Maurel et al., 1994; Borgnia and Agre, 2001). Furthermore, the three-dimensional structure of EcGlpF is known at a resolution of 2.2 Å (Fu et al., 2000). The only member of type II glycerol transporters functionally characterized is GlaLlac from Lactococcus lactis. In contrast to EcGlpF, GlaLlac has been shown to be functional both as a water channel and as a glycerol transporter by complementation studies in E. coli mutants and by studies of water and glycerol uptake in X. laevis oocytes after heterologous expression (Froger et al., 2001; Thomas et al., 2002). At several amino acid residue positions, GIP1;1 shares residues with both type I, type II, and other glycerol transporting MIPs, such as NIPs and human aquaglyceroporins, that are not conserved among most other MIPs (Fig. 2). Some of these residues have been suggested to interact with the substrate/substrates (Fu et al., 2000). T137 and G195 (numbers are referring to residues in EcGlpF) are two examples of such residues. There are also positions that are conserved within the type I or type II classes, for example, residues corresponding to P141 and P196 in EcGlpF, both suggested to be involved in glycerol binding (Fu et al., 2000). In type II and in GIP1;1, the corresponding conserved residues are I243 and G309. In general, GIP1;1 is more similar to glycerol transporters of type II than type I.

Constriction Region in GIP1;1 and Substrate Specificity

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). The Cys interacts with the water molecule by hydrogen bond from the backbone carbonyl group (Sui et al., 2001).

Table I.
Amino acid residues forming the constriction region in relevant MIPs

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; Thomas et al., 2002). Replacement of W48 and F200 (EcGlpF) by the less hydrophobic Y49 and P232 (GlaLlac) is suggested to result in a channel with a constriction region that is more hydrophilic than the EcGlpF pore. Water can thereby pass through the channel. Since there is no bulky His in the GlaLlac constriction region, the pore should be large enough to fit also a glycerol molecule.

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). In line with this, we predicted that GIP1;1 is mainly a glycerol facilitator and less permeable to water.

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.

Functional Analyses of Substrate Specificity

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). Mutations in an aquaglyceroporin from Plasmodium falciparum show that this element can also modulate the water permeability (Beitz et al., 2004). It is thus possible that the extended C-loop, directly or indirectly, affects interactions with the substrate in the vestibule, resulting in lower glycerol permeability.

Horizontal Gene Transfer

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), it was found that some transcripts in P. patens are more similar to bacterial transcripts than to higher plant transcripts. These transcripts were suggested to originate from horizontally transferred genes or from genes that were lost during evolution of vascular plants. We note that among these transcripts the EST BQ826917, which encodes a part of GIP1;1, also appeared. The peptide was defined as a glycerol uptake facilitator most similar to a protein from O. iheyensis but was not investigated more closely. Although, GLP from O. iheyensis (OihGLP) is the best match in BLAST searches, OihGLP together with GLPs from Bacillus subtilis, B. halodurans, and Thermus aquaticus form a sister group to the clade containing GIP1;1 in the Bayesian analysis. This grouping is also supported by the finding of a very interesting and conserved extra transmembrane helix in the C termini of GLPs in the former group but not in the GIP1;1 clade (Figs. 2 and and33).

The plant-specific NIP subfamily has also been suggested to have evolved through a horizontal gene transfer event (Zardoya et al., 2002). In this case, a bacterial aqpZ gene is believed to have been incorporated in the genome of a common ancestor to land plants and later modified to allow the transport of glycerol. The pairwise identities of EcAQPZ and the closest NIP, NOD26 from soybean (Glycine max), and GIP1;1 and the closest GLP, OihGLP, identified in BLAST searches are 43.1% and 40.4%, respectively. The high divergences could indicate that both types of MIPs are relatively old. Using the nonparametric rate-smoothing method, it is estimated that the horizontal gene transfer of NIP and GIP ancestors into plants occurred 1,135 and 1,038 million years ago, respectively. The former estimate is in agreement with the earlier published date for the horizontal gene transfer of the NIP ancestor 1,188 million years ago (Zardoya et al., 2002). However, one has to be cautious inferring dates from sequence divergence because substitution rates vary widely between different proteins and also because both GIP1;1 and NIPs might have gone through phases of positive selection/adaptive evolution that have resulted in a modified substrate specificity. Nevertheless, it is interesting to note that P. patens harbors two different types of MIPs that both might have been acquired independently via horizontal gene transfer from bacteria and selected for as glycerol transporters. This suggests that it is a selective advantage for plants to be able to facilitate transport of glycerol across membranes. In a recent study, evidence was presented indicating that glycerol is an important carbon source transferred from a host plant to a fungal pathogen (Wei et al., 2004). We speculate that exported plant glycerol might also be important for symbiotic fungi and bacteria and that this is a possible reason why the glycerol transporters have been fixed in plant genomes.

Adaptations of GIP1;1

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). One possibility is that intron insertion into GIP1;1 did occur by homologous recombination with another MIP. In our alignment, the first GIP1;1 intron is situated exactly at the same site as the conserved first intron site of PIPs. This region immediately before the first NPA-box is conserved among MIPs, thus providing a possible site for homologous recombination. However, the sequence flanking the intron site in GIP1;1 deviates from the general MIP sequence, and there are no traces of the PIP-like sequence that would be expected to be incorporated along with the intron. Thus, there is no evidence indicating that the intron was inserted via homologous recombination with another plant MIP gene. The divergence between GIP1;1 and the closest bacterial homolog suggests that GIPs have had time to adapt a gene structure compatible with expression in plants. In line with this, we fail to detect any significant difference in codon usage or GC content compared to other genes in P. patens (data not shown). The fact that many functional important amino acid residues still are conserved after these adaptations and the higher conservation of coding versus noncoding regions in GIPs from P. patens and F. hygrometrica implicate that the gene is under negative selection (purifying selection) and, thus, fully functional in P. patens and F. hygrometrica.

CONCLUSION

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.

MATERIALS AND METHODS

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.

Protein Predictions

The topology of the translated protein was predicted by the HMMTOP2.0 method (Tusnady and Simon, 1998, 2001). The sequence was scanned for protein patterns in PROSITE (Bucher and Bairoch, 1994; Sigrist et al., 2002), and subcellular localization was predicted by TargetP V1.0 and with PSORT (Nakai and Kanehisa, 1991; Emanuelsson et al., 2000).

Multiple Alignment and Phylogenetic Analysis

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 1,000,000 generations, every 100th tree was collected and sampled, and the 1,500 first trees were excluded from the consensus tree. The amino acid model was fixed to Jones. Neighbor joining and parsimony analysis were preformed on the same data set using PAUP* 4.0b10 (Swofford, 2002). For the neighbor joining tree, bootstrap values of 1,000 replicates were calculated. Identities were calculated from pairwise alignments adjusted for gaps. AQP0, 1, 3, and 7 (accession numbers JN0557, P50501, CAA10517, and NP_001015726) from amphibians were included in the neighbor joining tree used in estimations of divergence times, applying the nonparametric rate-smoothing method (Sanderson, 2003). The split of human and amphibian orthologs was set to 360 million years, and all four pairs of orthologs were used as calibration points (Kumar and Hedges, 1998).

Water and Radiolabeled Glycerol Transport Assays in Xenopus laevis Oocytes

For in vitro transcription, the coding sequence of GIP1;1 was subcloned into the BglII site of pXβG-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). Glycerol uptake was performed 3 d after injection. A group of five oocytes was incubated at room temperature in 1 mL of modified Barth's solution (Fetter et al., 2004) containing 1 mm unlabeled glycerol and 1 μCi/mL [14C]glycerol (138 μCi/mmol; Amersham Pharmacia Biotech). After 0, 5, and 10 min, oocytes were washed rapidly four times in ice-cold Barth's solution, and individual oocytes were lysed for 2 to 3 h in 1 mL of 5% (w/v) SDS for scintillation counting (Beckman LS 1701).

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AY611236, AY611237, and DQ092355.

Acknowledgments

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.

Notes

1This 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.

References

  • Agre P, Bonhivers M, Borgnia MJ (1998) The aquaporins, blueprints for cellular plumbing systems. J Biol Chem 273: 14659–14662 [PubMed]
  • Beitz E, Pavlovic-Djuranovic S, Yasui M, Agre P, Schultz JE (2004) Molecular dissection of water and glycerol permeability of the aquaglyceroporin from Plasmodium falciparum by mutational analysis. Proc Natl Acad Sci USA 101: 1153–1158 [PMC free article] [PubMed]
  • Borgnia MJ, Agre P (2001) Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli. Proc Natl Acad Sci USA 98: 2888–2893 [PMC free article] [PubMed]
  • Borstlap AC (2002) Early diversification of plant aquaporins. Trends Plant Sci 7: 529–530 [PubMed]
  • Brady SG, Danforth BN (2004) Recent intron gain in elongation factor-1α of colletid bees (Hymenoptera: Colletidae). Mol Biol Evol 21: 691–696 [PubMed]
  • Bucher P, Bairoch A (1994) A generalized profile syntax for biomolecular sequence motifs and its function in automatic sequence interpretation. Proc Int Conf Intell Syst Mol Biol 2: 53–61 [PubMed]
  • de Groot BL, Engel A, Grubmuller H (2001) A refined structure of human aquaporin-1. FEBS Lett 504: 206–211 [PubMed]
  • de Groot BL, Grubmuller H (2001) Water permeation across biological membranes: mechanism and dynamics of aquaporin-1 and GlpF. Science 294: 2353–2357 [PubMed]
  • Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300: 1005–1016 [PubMed]
  • Fetter K, Van Wilder V, Moshelion M, Chaumont F (2004) Interactions between plasma membrane aquaporins modulate their water channel activity. Plant Cell 16: 215–228 [PMC free article] [PubMed]
  • Froger A, Rolland JP, Bron P, Lagree V, Le Caherec F, Deschamps S, Hubert JF, Pellerin I, Thomas D, Delamarche C (2001) Functional characterization of a microbial aquaglyceroporin. Microbiol 147: 1129–1135 [PubMed]
  • Fu D, Libson A, Miercke LJ, Weitzman C, Nollert P, Krucinski J, Stroud RM (2000) Structure of a glycerol-conducting channel and the basis for its selectivity. Science 290: 481–486 [PubMed]
  • Gonen T, Sliz P, Kistler J, Cheng Y, Walz T (2004) Aquaporin-0 membrane junctions reveal the structure of a closed water pore. Nature 429: 193–197 [PubMed]
  • Harries WE, Akhavan D, Miercke LJ, Khademi S, Stroud RM (2004) The channel architecture of aquaporin 0 at a 2.2-A resolution. Proc Natl Acad Sci USA 101: 14045–14050 [PMC free article] [PubMed]
  • Heller KB, Lin EC, Wilson TH (1980) Substrate specificity and transport properties of the glycerol facilitator of Escherichia coli. J Bacteriol 144: 274–278 [PMC free article] [PubMed]
  • Heymann JB, Engel A (1999) Aquaporins: phylogeny, structure, and physiology of water channels. News Physiol Sci 14: 187–193 [PubMed]
  • Hohmann I, Bill RM, Kayingo I, Prior BA (2000) Microbial MIP channels. Trends Microbiol 8: 33–38 [PubMed]
  • Johanson U, Gustavsson S (2002) A new subfamily of major intrinsic proteins in plants. Mol Biol Evol 19: 456–461 [PubMed]
  • Johanson U, Karlsson M, Johansson I, Gustavsson S, Sjovall S, Fraysse L, Weig AR, Kjellbom P (2001) The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants. Plant Physiol 126: 1358–1369 [PMC free article] [PubMed]
  • Johansson I, Karlsson M, Johanson U, Larsson C, Kjellbom P (2000) The role of aquaporins in cellular and whole plant water balance. Biochim Biophys Acta 1465: 324–342 [PubMed]
  • Kumar S, Hedges SB (1998) A molecular timescale for vertebrate evolution. Nature 392: 917–920 [PubMed]
  • Maurel C, Reizer J, Schroeder JI, Chrispeels MJ, Saier MH Jr (1994) Functional characterization of the Escherichia coli glycerol facilitator, GlpF, in Xenopus oocytes. J Biol Chem 269: 11869–11872 [PubMed]
  • Nakai K, Kanehisa M (1991) Expert system for predicting protein localization sites in gram-negative bacteria. Proteins 11: 95–110 [PubMed]
  • Nishiyama T, Fujita T, Shin IT, Seki M, Nishide H, Uchiyama I, Kamiya A, Carninci P, Hayashizaki Y, Shinozaki K, et al (2003) Comparative genomics of Physcomitrella patens gametophytic transcriptome and Arabidopsis thaliana: implication for land plant evolution. Proc Natl Acad Sci USA 100: 8007–8012 [PMC free article] [PubMed]
  • Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574 [PubMed]
  • Sanderson MJ (2003) r8s: inferring absolute rates of molecular evolution and divergence times in the absence of a molecular clock. Bioinformatics 19: 301–302 [PubMed]
  • Savage DF, Egea PF, Robles-Colmenares Y, Iii JD, Stroud RM (2003) Architecture and selectivity in aquaporins: 2.5 a x-ray structure of aquaporin z. PLoS Biol 1: E72. [PMC free article] [PubMed]
  • Sigrist CJ, Cerutti L, Hulo N, Gattiker A, Falquet L, Pagni M, Bairoch A, Bucher P (2002) PROSITE: a documented database using patterns and profiles as motif descriptors. Brief Bioinform 3: 265–274 [PubMed]
  • Stroud RM, Miercke LJ, O'Connell J, Khademi S, Lee JK, Remis J, Harries W, Robles Y, Akhavan D (2003) Glycerol facilitator GlpF and the associated aquaporin family of channels. Curr Opin Struct Biol 13: 424–431 [PubMed]
  • Sui H, Han BG, Lee JK, Walian P, Jap BK (2001) Structural basis of water-specific transport through the AQP1 water channel. Nature 414: 872–878 [PubMed]
  • Swofford DL (2002) PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, MA
  • Thomas D, Bron P, Ranchy G, Duchesne L, Cavalier A, Rolland JP, Raguenes-Nicol C, Hubert JF, Haase W, Delamarche C (2002) Aquaglyceroporins, one channel for two molecules. Biochim Biophys Acta 1555: 181–186 [PubMed]
  • Tusnady GE, Simon I (1998) Principles governing amino acid composition of integral membrane proteins: application to topology prediction. J Mol Biol 283: 489–506 [PubMed]
  • Tusnady GE, Simon I (2001) The HMMTOP transmembrane topology prediction server. Bioinformatics 17: 849–850 [PubMed]
  • Wei Y, Shen W, Dauk M, Wang F, Selvaraj G, Zou J (2004) Targeted gene disruption of glycerol-3-phosphate dehydrogenase in Colletotrichum gloeosporioides reveals evidence that glycerol is a significant transferred nutrient from host plant to fungal pathogen. J Biol Chem 279: 429–435 [PubMed]
  • Zardoya R, Ding X, Kitagawa Y, Chrispeels MJ (2002) Origin of plant glycerol transporters by horizontal gene transfer and functional recruitment. Proc Natl Acad Sci USA 99: 14893–14896 [PMC free article] [PubMed]

Articles from Plant Physiology are provided here courtesy of American Society of Plant Biologists
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...