Crystal Structure of an Active Form of Human MMP-1

The extracellular matrix is a dynamic environment that constantly undergoes remodelling and degradation during vital physiological processes such as angiogenesis, wound healing, and development. Unbalanced extracellular matrix breakdown is associated with many diseases such as arthritis, cancer and fibrosis. Interstitial collagen is degraded by matrix metalloproteinases with collagenolytic activity by MMP-1, MMP-8 and MMP-13, collectively known as the collagenases. Matrix metalloproteinase 1 (MMP-1) plays a pivotal role in degradation of interstitial collagen types I, II, and III. Here, we report the crystal structure of the active form of human MMP-1 at 2.67 Å resolution. This is the first MMP-1 structure that is free of inhibitor and a water molecule essential for peptide hydrolysis is observed coordinated with the active site zinc. Comparing this structure with the human proMMP-1 shows significant structural differences, mainly in the relative orientation of the hemopexin domain, between the pro form and active form of the human enzyme.
Introduction
Connective tissue remodeling is a complex process involving a plethora of cytokines, growth factors, and turnover of extracellular matrix (ECM). The main enzymes that degrade ECM molecules are matrix metalloproteinases (MMPs), which are also known as matrixins. Under normal physiological conditions, MMP activity can be regulated at various stages: during transcription, proteolytic processing of their inactive pro forms, zymogens, as well as by inhibition of enzyme activity by endogenous inhibitors such as tissue inhibitors of metalloproteinases or TIMPs.1,2 These enzymes have a similar domain structure: an N-terminal signal sequence to target for secretion, a pro-peptide domain to maintain latency, a catalytic domain containing the catalytic zinc, a linker region, and a C-terminal four-bladed propeller structure called the hemopexin domain. Some of the MMPs have additional domains, e.g. the fibronectin repeats in gelatinases. These domains are important in substrate recognition and in inhibitor binding.3 The human MMP family to date comprises of about 23 enzymes that are classified based on their preferred substrate and cellular localisation: collagenases, gelatinases, stromelysins, elastase, membrane-type MMPs and so forth.4
Collagenases (MMP-1, MMP-8 and MMP-13) are the key enzymes that are capable of cleaving interstitial fibrillar collagen. Apart from these enzymes MMP-2 (gelatinase A) and MMP-14 (MT1-MMP) are also able to initiate the breakdown of collagen fibrils.5,6 Interstitial collagens I, II and III are triple-helical proteins that are the essential structural components of all connective tissues such as the cartilage, bone, skin, tendons and ligaments. These extracellular glycoproteins provide scaffolding of the tissue and play an important role in cellular processes such as cell migration, proliferation and differentiation. Physiological collagenolysis is integral to several biological processes such as embryogenesis, tissue repair and remodeling, angiogenesis, organ morphogenesis and wound healing.7,9 The collagenases cleave the triple-helical collagen approximately three-quarters away from the N terminus of the substrate, resulting in three-quarters and one quarter length fragments that are unstable at body temperature and undergo denaturation, rendering them susceptible to other non-specific tissue proteinases. However, under aberrant circumstances degradation of collagen results in pathological conditions such as cancer, atherosclerosis, arthritis, aneurysm and fibrosis.8,10
The proteolytic activity of the enzyme resides in the catalytic domain but it requires the hemopexin domain in order to cleave the three chains of the triple-helical collagen.11 The crystal structure of porcine MMP-1,12 determined sometime ago, revealed details of the active site structure and the specificity pocket but this structure does not shed any light on how collagenolytic MMPs can cleave the triple-helical collagen. Recently, the X-ray structure of human proMMP-1 (MMP-1 zymogen) was elucidated.13 The structure revealed the interaction between the pro-peptide and the hemopexin domain of the enzyme, which results in a “closed” conformation of the zymogen in contrast to the “open” conformation of catalytic domain of the active MMP-1.
The three-dimensional structure of the human MMP-1 (E200A), an active site mutant, reported here takes us a step closer towards a more complete understanding of the interaction of the collagenases with the triple-helical collagens. It reveals new features of the active protease and provides a platform for understanding the structural changes that accompany zymogen activation.
Results
Overall structure
The structure of human MMP-1 (E200A) was determined at 2.67 Å resolution with two monomers (chains A and B) in the asymmetric unit of the trigonal space group, P3221 (see Table 1 for crystallographic statistics). The overall domain structure of human MMP-1 is similar to that of the previously solved full-length enzymes (Figure 1). The structure comprises of the N-terminal catalytic domain, the linker region and the C-terminal hemopexin domain. The catalytic domain of one monomer contacts the hemopexin domain of the other monomer. Interestingly, the contact site used by the two monomers in the asymmetric unit to form the dimer is not the same as the dimerisation site observed in the proMMP-1 structure13 or that for the MMP-9 hemopexin domain.14 This indicates that the dimerisation mechanism is perhaps not a general rule of thumb for the hemopexin domains and is most probably a crystallisation artefact. Monomers A and B deviate from each other with an overall r.m.s. deviation of 0.87 Å (for 367 Cα atoms). The dimer is stabilised by eight hydrogen bonds and 54 van der Waals contacts (Table 2). These contacts are facilitated by a total of 33 residues (18 residues from monomer A and 15 residues from monomer B). Only nine amino acid residues are common to both the monomers: Pro104, Asp105, Leu106, Arg183, Trp184, Thr185, Val300, Phe301 and Gln335. Apart from the asymmetry seen in the residues that participate from each monomer at the dimer interface, we also observe a non-symmetric interaction pattern. However, it is believed that this dimer is not physiologically relevant, as we found that human MMP-1 is a monomer in solution (R.V. & H. N., unpublished results).
Catalytic domain
The structure of MMP-1 catalytic domain is very similar to those of other MMPs. The metalloproteinase domain is about 160 amino acid residues in length with the catalytic zinc ion residing in the C-terminal segment of this domain. The catalytic fragment of the protease consists of three α-helices and a highly twisted five-stranded β-sheet. The active site zinc is bound in the sequence HELGHXXGXXH by the three His residues: His199, His203, His209 and a water molecule at the active site cleft (Figure 2). This is the first MMP-1 structure where a water molecule essential for peptide hydrolysis is observed at the active site because all the previous structures of the metalloproteinase domain of MMP-1 are in the inhibited state of the enzyme.
Also observed in this domain is the salt bridge between the ammonium group of the N-terminal, Phe81 and the carboxylate group of the side-chain of Asp232. The generation of the correct N terminus (Phe81) in the activation process of proMMP-1 is crucial for the enzyme to have full activity against collagen.15 If the N terminus is either longer or shorter the activity against collagen drops to 30–40%.15 Only with the correct N terminus the formation of the salt bridge is possible, and this stabilises the structure of the N terminus as was originally shown for MMP-8.16,17
Alignment of the pro-enzyme (1SU3) structure with that of the mature enzyme (2CLT here) shows Phe81 to have moved some 17 Å from its original position in the zymogen form of collagenase-1 (Figure 3(a)). The structure of proMMP-3 (PDB code: 1SLM)18 was aligned as well to help calculate the displacement of Phe81. The first seven N-terminal residues were not observed in the proMMP-1 structure because of lack of proper visible electron density.13 This deviation gradually decreases and by residue Pro88 the two structures are in register and remain remarkably similar throughout the catalytic domain. There are only slight differences, if any, in the side-chain orientation of the N-terminal residues in the catalytic domain cleft (Figure 3(b)).
The structural zinc exhibits tetrahedral coordination facilitated by residues His149, Asp151, His164 and His177. The catalytic domain of MMP-1 also contains three calcium-binding sites. Either four or five liganding residues coordinate all the calcium ions, except one in the catalytic domain of monomer A (Table 3). One calcium ion packs the S-loop (between strands s3 and s4) against the liganding residues from strand s5. The second calcium ion of the catalytic domain is sandwiched between strand s3 and the loop containing strands s4 and s5. The third calcium site is determined by the presence of the critical residue, Asp105 that comes from the loop following strand s1. The other residues (Glu180 and Glu182) that provide coordination to this calcium ion come from the loop following strand s5. One interesting feature observed in this crystal structure by virtue of dimerisation is that residue Gln335 from monomer B contributes the fourth coordinating ligand for this calcium ion. This feature, however, is not replicated in the corresponding calcium-binding site in monomer B. Details of the metal site geometries can be found in Table 3.
Linker region
In MMPs the catalytic domain is followed by a stretch of 15–65 amino acid residues referred to as the linker or the hinge region. This region is rich in proline residues and replacement of those with alanine drastically reduced the collagenolytic activity of MMP-8 (neutrophil collagenase),19 indicating that the presence of the correct linker structure is important for collagenolysis. This region in MMP-1 with 16 residues is well defined in the present structure unlike that in the full-length crystal structure of porcine collagenase. The conformation of the linker region is quite similar in both proMMP-113 and the present MMP-1 structures (Figure 4(a)). The residues of the linker region make extensive contacts (six hydrogen bonds and 73 van der Waals contacts; Table 4) with both the catalytic and the hemopexin domain of the enzyme. These interactions stabilise the domain arrangement in MMP-1, which is required for the concerted action of the two domains. Comparing the interactions of the linker region with the catalytic and the hemopexin domain in both the pro-enzyme and the active enzyme did not reveal many differences. Most of the interactions seen in proMMP-1 (seven hydrogen bonds and 71 van der Waals contacts) are still observed in the present structure, indicating that the activation of the enzyme does not result in structural changes and the overall structure of the areas around the linker region are mostly conserved. Interestingly though, the hydrogen-bonding interactions (Table 4) within this region are quite different between the two forms of the molecule. There are a total of seven hydrogen bonds within the linker region of active MMP-1 (Figure 4(b)) compared to the five observed in proMMP-1 (Figure 4(c)), with only two bonds common to both structures This difference in the number of hydrogen bonds is perhaps not of much significance when seen in the light of the extensive contacts this region makes with the other two domains.
Hemopexin-like domain
The hemopexin domain starts with Cys259 and forms a complete circle by joining to Cys447 in a disulphide bond that connects blade bI with blade bIV, giving this domain the characteristic four-bladed β-propeller structure. Each blade starts near the periphery with either the motif DAA or DAX, in which the Asp residues (Asp266, Asp359 and Asp408) coordinate the central calcium ion through their carbonyl oxygen atom. Glu310 provides the fourth coordination thus completing the acidic patch at the entrance of the central, solvent-accessible channel (Table 3). The side-chains of these residues form salt bridges to the neighbouring β-strands holding the entrance of the central channel together. Three water molecules are found trapped in the centre of this channel. These, however, are not involved in the coordination geometry of the calcium ion at the tunnel centre. Two of the water molecules are at positions corresponding to the sodium and chloride ion in the proMMP-1 structure.12 The water molecule corresponding to the sodium ion is at hydrogen-bonding distances to the carbonyl oxygen atom of Ile268, Ala312, Ala361 and Val410. The same cannot be said for the one corresponding to the chloride ion, which does not make any hydrogen bonds with the main-chain amides of the residues mentioned above. It is very likely that the presence of these ions is a consequence of the crystallisation conditions rather than a stability requirement for the hemopexin-like domain.
Comparison with the porcine MMP-1 and the human proMMP-1 structures
Full-length human MMP-1, human proMMP-113 and porcine MMP-112 were superimposed on the basis of the catalytic domain of the three structures. Alignments were also done for just the hemopexin domain of the molecules. As expected both types of alignments gave the same outcome whereby the active form of the enzyme (human and porcine) were closer in their structure as opposed to the proMMP-1 structure. The average r.m.s. deviation over 367 Cα atoms of the porcine and human MMP-1 is ∼1.4 Å, whereas the r.m.s. deviation when aligning the full-length active MMP-1 with the proMMP-1 structure is 1.6 Å. If, however, we take only the hemopexin-like domain into consideration, then the conformational differences that lie within this domain are brought to light (Figure 5(a)). The superimposed porcine and human MMP-1 hemopexin-like domains vary by 1.2 Å (maximum displacement of 5.2 Å). This value increases to 1.7 Å when superimposing the hemopexin-like domains of the active human MMP-1 and the human proMMP-1. A considerable change in conformation is reflected in the residues that interact with the pro-domain. Most of these differences are by virtue of the Phe289-Tyr290-Pro291 region of the hemopexin-like domain. Table 5 shows these residues, their interactions and the shift in Cα position observed when the pro and active forms of human MMP-1 were superimposed. The largest movement of 16 Å is observed for the residue, Phe289 (Figure 5(b)). Such a perturbation in structural conformation is not evident in the catalytic domain of the enzyme (Table 5). It seems like Arg281 acts as a pivot around which the hemopexin-like domain undergoes displacement upon activation as suggested by Jozic et al.13
One region in the catalytic domain that generates interest is the region between Tyr218 and Tyr221. The shift in the Cα position between the active and the proMMP-1 is not as dramatic as that observed for the Phe289-Tyr290-Pro291 region but they are involved in several interactions with the pro domain around the cysteine-switch region. As a result when the pro-enzyme undergoes activation, this region of the catalytic domain opens wider and further exposes the crucial cis-configured Glu190-Tyr191 peptide in MMP-1.
Discussion
Reconciliation of structural findings: pro-collagenase versus active collagenase
MMPs are multi-domain enzymes that consist of a pro domain, catalytic domain, a linker region and a hemopexin-like domain. Cleavage of the pro domain leads to a substantial rearrangement of the N-terminal residues 81–88 (Figure 3(a)). The activation process swings Phe81 towards the proteinase domain and terminates with a salt link between the amine of Phe81 and the carboxylate side-chain of Asp232. This interaction imparts several-fold greater enzymatic activity to MMP-1 than those with either an extended or a shorter N terminus, which lack the salt linkage.17
Previously an inhibitor-free structure of the catalytic domain of MMP-1 was reported.20 This structure was, however, unique in the sense that the N-terminal Leu-Thr-Glu-Gly (83–86) residues of one molecule occupied the active site of the other molecule thus forming an unnatural inhibited complex, which should not really be considered as an inhibitor-free structure. The present structure on the other hand stands unique in being elucidated in the absence of an inhibitor. This is the first unliganded structure of the full-length MMP-1 where a water molecule is seen at the active site providing the fourth ligand for the catalytic zinc in the tetrahedral coordination sphere (Figure 2). It must be mentioned here that the present structure is an active site mutant where the catalytic glutamate residue has been mutated to an alanine. It is likely that the scissile peptide bond and the catalytic glutamate (Glu200) flank the active site water molecule on either side. The favourable hydrogen bond between Glu200 and the water molecule would make it more nucleophilic. The strongly polarised carbonyl group of the scissile bond (by virtue of its interaction with the catalytic zinc) would face a properly oriented water molecule. An unstable transition state results in the transfer of one water proton to the leaving nitrogen (the amino group of the scissile bond) via the Glu200 carboxylate shuttle. This is followed by the cleavage of the peptide bond and concomitant shuttle of another water proton to the amino group. This hypothesis will, however, need to be substantiated with a transition state structure.
The linker region is considered to play an important role in collagenolysis,19,21 but its exact role is not clear. The structures of both proMMP-1 and active MMP-1 have revealed the residues of this region to be in close contact with the catalytic and the hemopexin-like domain. Comparative analysis of the extensively similar contacts made by the residues of the hinge region reveal that despite being highly exposed with no secondary structure, the conformation of the linker peptide is perhaps a signature of its sequence and does not depend on the crystal packing (Figure 4(a)). Mutagenesis studies of this region result in decreased collagenolytic activity of MMP-121 and MMP-8.19 This may be due to structural changes in this region affecting the interactions with the catalytic and hemopexin domains that are required for collagenolysis.22
The cleavage of the pro domain to form the active enzyme is accompanied by major conformational rearrangement in the residues that interact with the pro domain. Residues from both the catalytic domain and the hemopexin-like domain make interactions with the pro domain residues. The effects on the overall backbone structure of the catalytic domain is minimal; the effects on the hemopexin-like domain is, however, quite dramatic (Figure 5(a)). The Phe289-Tyr290-Pro291 region of the hemopexin-like domain undergoes the most significant conformational change (Figure 5(b)). In essence, the hemopexin-like domain undergoes a major displacement towards the catalytic domain, thus widening the cleft between the proteinase domain and the hemopexin-like domain on the active site face of the enzyme. This altered configuration makes the active site residues and the RWTNNFREY (residues 183–191) interface, a segment critical for collagenolysis,23 more accessible for native collagen.
Implications for collagenolysis
Despite growing awareness of the importance of collagen-recognising determinants it is still unclear as to which part of the collagenase molecule makes the first contact with the triple-helical collagen. Several hypotheses have been suggested to try and explain the steps involved in collagenolysis (the most recent by Jozic et al.13). Docking studies were performed using a single-stranded, collagen α1(I)-like 15mer peptide with the Gly-Ile cleavage site incorporated in an attempt to better understand the functioning of the active enzyme. The peptide was modelled on the known structure of one of the chains of the triple-helical collagen (PDB code: 1BKV).24 Several solutions were generated. We used localised interactions as our guide to help us pick one possible solution: (a) contact of the carbonyl group of the Gly-Ile bond of the peptide with the active site determinants, especially the catalytic zinc; and (b) contact with the critical cis-configured Glu190-Tyr191 peptide. This solution revealed the peptide to be aligned to the continuous bulge-edge strand (Gly160-Phe166) and the wall-forming segment (Pro219-Phe223) in an extended manner. This solution, however, upon energy minimisation moved outside of the coordination sphere of the catalytic zinc but it was still close enough to be polarised by the catalytic zinc. Since the peptide was only 15 amino acid residues long, we could not see any interactions with the C-terminal hemopexin-like domain. It has been proposed that the triple-helical collagen would run via the Glu190-Tyr191 cis-peptide exosite making extensive interactions with the residues of the hemopexin-like domain.22
The structure of the active form of human MMP-1 has provided us insights on the conformational changes that occur upon activation of the pro-enzyme and these are valuable clues towards understanding the mechanism of collagenolysis. A better understanding of the mechanism of collagen cleavage will assist towards design of inhibitors that would specifically interfere with collagenolysis without affecting (beneficial) the cleavage of other substrates.
Experimental Procedures
Cloning, protein expression and protein purification
The catalytically inactive mutant proMMP-1 (E200A) was cloned, overproduced, refolded, and purified as described.22 A catalytic site mutant of this enzyme was chosen in order to prevent autocatalytic cleavage during crystallisation. The mutant zymogen was activated as described22 at a final concentration of 554 μM in the presence of 1:80 molar ratio of MMP-3 lacking the hemopexin-like domain (MMP-3ΔC) and 1 mM 4-aminophenylmercuric acetate for 90 min at 37 °C. The activation mixture was directly applied to a Sephadex S-200 gel filtration column (diameter 26 mm, length 900 mm) in 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10 mM CaCl2, 0.02% (w/v) sodium azide, to separate the “active” form of MMP-1 (E200A) from MMP-3ΔC, 4-aminophenylmercuric acetate, and remnants of the pro-peptide. Fractions containing MMP-1 (E200A) were pooled and concentrated using a Vivacell 250 ml with a 5 kDa cutoff membrane, followed by a Vivaspin 20 with a 5 kDa membrane (Vivascience). The protein was stored at room temperature as incubation at 4 °C resulted in precipitation.
Crystallisation, data collection and processing
MMP-1 (E200A) was crystallised using the hanging drop vapour-diffusion method. The protein (2 μl at a concentration of 21 mg/ml) was mixed with 2 μl of the reservoir solution containing 0.1 M Tris (pH 7.5), 1.5 M ammonium formate and 10% (w/v) polyethylene glycol (PEG) 8000. Crystals appeared and grew to their maximum size within two weeks at 16 °C. A cryoprotectant solution prepared by supplementing the reservoir with 25% (v/v) glycerol enabled the crystals to be flash-frozen in liquid nitrogen.
Flash-cooled MMP-1 (E200A) crystals were used to collect diffraction data to a resolution of 2.67 Å on PX 14.1 at the Synchrotron Radiation Source, Daresbury (UK). The data were processed and scaled using HKL2000.25 The crystals belong to the space group P3221, with cell dimensions of a = b = 138.48 Å and c = 110.05 Å. There are two monomers in the asymmetric unit with solvent content of about 60%. Data reduction using the program TRUNCATE26 estimated an overall B-factor of 63.1 Å2/Da from the Wilson plot. Details of the data processing statistics are presented in Table 1.
Structure determination
The solution for the MMP-1 (E200A) structure was found using the program PHASER.26 Human proMMP-1 (PDB code: 1SU3)13 was used as the search model with the pro-peptide domain removed from the structure. Log-likelihood gain (LLG) of 161 for the first solution increased to an LLG value of 1091 when the second monomer in the asymmetric unit was located indicating the solutions were indeed the right ones.
Refinement
Crystallographic refinement was carried out using the program CNS27 at 2.67 Å resolution against 85.1% of the measured data. A test set of random reflections of 2.2% was excluded from the full data set for cross-validation purposes by calculating the free R-factor (Rfree) to monitor refinement trend.28 Initial round of refinement with the two monomers found by PHASER26 resulted in an Rcryst of 45.1% and an Rfree of 50.6%. Once the six N-terminal residues in both the monomers were built in and all the ions (two zinc ions and four calcium ions per monomer) were added and the model subjected to simulated annealing, the Rcryst and the Rfree dropped to 31% and 38%, respectively. Iterative cycles of refinement (energy minimisation, simulated annealing and individual temperature factor (B-factor) refinement) using CNS27 and model building with reference to 2Fo-Fc and Fo-Fc maps using the program Coot29 progressively improved the phases. In the final stages of refinement, water molecules with peaks greater than 3σ in the Fo-Fc maps and those within hydrogen bonding distances from appropriate atoms were incorporated into the structure.
The final refined structure at 2.67 Å resolution has an Rcryst of 22.3% and an Rfree of ∼26%. Both monomers, chains A and B have the full complement of the amino acids: from Phe81 to Cys447. Residues 117, 356, 369, 385, 386 and 424 of chain A and residues 257, 272, 287, 424 and 446 of chain B have been modelled as alanine residues because of the lack of visible electron density beyond Cβ atom. Analysis of the Ramachandran plot using the program PROCHECK26 indicated that ∼81% of the residues are in the most favourable region of the ϕ-φ plot and about 17% lie in the additional allowed region. The structure also consists of 198 water molecules. Details of the refinement statistics are given in Table 1.
Protein Data Bank accession codes
The atomic coordinates and the structure factors of the human MMP-1 (E200A) have been deposited with the RCSB Protein Data Bank (accession codes 2CLT and R2CLTSF, respectively).

collagen
matrix metalloproteinases
inhibitor-free
mmp, matrix metalloproteinase
ecm, extracellular matrix
fibroblast collagenase
x-ray crystallography


Comparative molecular biological analysis of membrane transport genes in organisms

Comparative analyses of membrane transport genes revealed many differences in the features of transport homeostasis in eight diverse organisms, ranging from bacteria to animals and plants. In bacteria, membrane-transport systems depend mainly on single genes encoding proteins involved in an ATP-dependent pump and secondary transport proteins that use H+ as a co-transport molecule. Animals are especially divergent in their channel genes, and plants have larger numbers of P-type ATPase and secondary active transporters than do other organisms. The secondary transporter genes have diverged evolutionarily in both animals and plants for different co-transporter molecules. Animals use Na+ ions for the formation of concentration gradients across plasma membranes, dependent on secondary active transporters and on membrane voltages that in turn are dependent on ion transport regulation systems. Plants use H+ ions pooled in vacuoles and the apoplast to transport various substances; these proton gradients are also dependent on secondary active transporters. We also compared the numbers of membrane transporter genes in Arabidopsis and rice. Although many transporter genes are similar in these plants, Arabidopsis has a more diverse array of genes for multi-efflux transport and for response to stress signals, and rice has more secondary transporter genes for carbohydrate and nutrient transport.
Introduction
Cells maintain their biological activities by importing and exporting various substances. Provision of energy and nutrients and efflux of salts, biochemicals, and ions are necessary to maintaining biological activity in prokaryotic and eukaryotic cells. Environmental situations within cells differ among organisms: unicellular organisms cannot control the ion concentrations outside the cell, but multicellular eukaryotes (especially animals) can precisely regulate the ion concentrations of their cellular environments within micromolar ranges. Therefore, we can expect organisms to differ in gene number, structure, and function according to their biological abilities and environmental situations. Recent sequence analyses of entire genomes have made it possible to confirm the existence of homologous genes by computer data analysis. It is also possible to reveal the overall patterns of gene networks. In plants, complete genomic sequences are available for Arabidopsis and rice, but gene annotation programs are not yet sufficiently accurate to determine the function of all genes Instead, full-length cDNA data are useful for precise analysis of genes. Because transport activities are required at distinct levels in most tissues, we expected that the transcripts of most transmembrane transporters would be represented in full-length cDNA libraries from plants at various developmental stages, various plant tissues, and plants exposed to various treatments.
We searched for orthologs of known membrane transport genes by using the 35,180 full-length rice cDNA sequences (Rice Full-Length cDNA Consortium 2003; Satoh et al. 2007) and genomic sequence data from Arabidopsis (Arabidopsis Genome Initiative 2000) and japonica rice (Goff et al. 2002; International Rice Genome Sequencing Project 2005) and global functional gene annotation in Arabidopsis and rice (Munich Information Center for Protein Sequences (MIPS) data service (http://mips.gsf.de/proj/plant/jsf/) (Schoof et al. 2004; Karlowski et al. 2003)) (RAP-DB = Rice Annotation Project Data Base: http://rapdb.lab.nig.ac.jp/ (Rice Annotation Project 2007, 2008)); the TIGR Rice Genome Annotation: http://www.tigr.org/tdb/e2k1/osa1/index.shtml (Ouyang et al. 2007). Transmembrane proteins have a hydrophobic structure, a pore-forming sequence, and molecule-binding sites. Because of these specific structural features, the identification of membrane transport orthologs is clear from computer calculations. Previous reports have characterized individual transporter protein families but have not extended to whole transport systems in general (Eng et al. 1998; Pao et al. 1999; Mäser et al. 2001; Sánchez-Fernández et al. 2001). In a more general analysis of various organisms, the features of prokaryotes were contrasted with those of eukaryotes (Ren and Paulsen 2005). However, differences among eukaryotes—especially animals and plants—were not a focus of that report. We also searched for orthologs of membrane transport gene in various organisms database (The Human Gene Nomenclature Database Search Engine (http://www.genenames.org/cgi-bin/hgnc_search.pl) (Wain et al. 2004); Genomic comparison of membrane transport systems (TransportDB: http://www.membranetransport.org/index.html) (Ren et al. 2004)), the functional genomics of plant transporters (PlantsT: http://plantst.genomics.purdue.edu/ (Tchieu et al. 2003) and ARAMEMNON (http://aramemnon.botanik.uni-koeln.de/) (Schwacke et al. 2003)). Here, we compare total membrane transport systems from diverse organisms and conclude that membrane transport genes exemplify evolutionary diversity of homeostatic systems. Evolutionary changes in gene families indicate the dynamics of alterations in biological systems and gene networks. Therefore, analysis of large categories of gene families may reveal many basic concepts of biological systems.
General comparisons of membrane transport genes
Membrane transport protein carries various materials for homeostasis. There are many clear features of domains (ex. Transmembrane, pore-forming, ATP binding, molecular capture), and functional features in the transport proteins. According to the structure and its functional systems, the membrane transport proteins have divided to three categories-pump, channel and secondary transporter. The pump system is the slowest (1–103 molecules/s) but environmental-independent system which consume energy (mainly ATP) for transport. The channel is the most rapid (107–108 molecules/s) and non-energy consuming systems but its need concentration gradients previously (transport directions are only according to the gradients). The secondary transport system adapts co-transport molecules movement energy to carry molecules. Therefore, it needs co-transport molecules and the transport directions have depended on environmental conditions and the speeds (102–104 molecules/s) are the middle of pump and channel. We summarized all three categories (pump, channel and secondary transporter) of genes and compared the total numbers of membrane transport genes in Escherichia coli, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Neurospora crassa, and Saccharomyces cerevisiae. The genome sizes among these organisms were diverse (4.6–3150 megabases), and the numbers of transmembrane genes ranged from 300 to 350 in E. coli, fungi, and yeast to about 1000–1200 in Arabidopsis and rice (Table 2). This indicates that a minimum number of about 300 gene species may be required to retain cell homeostasis. The greater numbers of transmembrane transport genes in Arabidopsis and rice may indicate additional redundancy as well as the modification of genes for new roles (e.g. addition of new substances, adaptation of systems for regulating transport, divergence of stage- and tissue-specific material transport), specialization for the various tissues and cells of multicellular organisms, and increased complexity of cells, which in eukaryotes have many additional organelles.
The greater relative increase (plants versus bacterium, fungus, and yeast) in the numbers of membrane transport genes was less than has been reported for other gene categories, such as transcription factor genes and metabolic enzyme genes, in higher eukaryotes (Wray et al. 2003). This may indicate that adaptations in membrane transport are critical for the survival of organisms during evolution. The total numbers of membrane transport genes in higher plants (Arabidopsis, about 1000; rice, 1200; Table 2) are 1.2–2.0 times those in animals (fly, 600; nematode, 650; human, 750; Table 2). These differences in numbers of transporter genes may be related to differences in the need for efflux and influx systems in restricted habitation environments. Because of their immobility and the simplicity of their uptake systems, plant cells have more opportunity to absorb inappropriate substances and greater amounts of substrates and to synthesize larger amounts of secondary products than do animals.
In accordance with their structures and mechanisms of action, transport proteins are classified into three classes: pump, channel, and secondary transporter (transporter). The composition ratios of these classes of protein were also compared (Table 2, Fig. 1). The numbers of pump genes in animals (72–82) were almost the same as in bacteria (70). The numbers of secondary transporter (animals, 350; bacteria, 230) and channel (animals, 160–320; bacteria, 15) genes were increased in animals. In particular, vertebrates (humans) had more (322) channel gene species than plants (130–180). We considered that this gene diversity in the development of channel systems was caused by the acquisition of a nervous system. The electrical transmission systems in nervous systems supplying organs (e.g. muscles, kidneys) need precisely controlled ion concentrations and the ability to make immediate changes in gradients. The development of active transport systems in animals allowed the regulation of rapid movements of the body and organs. Therefore, animals presumably acquired genes for the fastest transport-system channels. Plants also had more channel gene species than bacteria, although fewer than animals. Because plants do not transmit signals for quick movement of their organs, they do not need to regulate membrane voltages as precisely as animals. Additionally, signal-transmitting systems with ligand molecules (e.g. neurotransmitters) are not specific, unlike in animals. Therefore, the numbers of voltage-gated ion channels (VICs) and ligand-dependent channels were smaller in plants than in higher animals (Table 1). On the other hand, higher plants had increased numbers of genes for pumps (170–250) and secondary active transporters (660–760). Plant cells have chloroplasts, which synthesize carbohydrates for many biological activities, including protein synthesis and functioning of the ATP-dependent pumps. Plants presumably use ATP-consuming systems more easily than animals, and the pumps transport the molecules that act as the driving forces of the secondary active transporters. Additionally, plant-specific organelles and vacuoles provide pools of ions and catabolite molecules. Co-transport molecules for secondary transport also are safely and stably stored in the vacuoles. Therefore, plants are presumably able to constantly supply co-transport molecules for secondary active transporters, independently of environmental conditions. The existence of vacuoles gives plant cells more self-sufficiency than animal cells and explains the evolution of membrane transport genes for individual cell homeostasis in plants. Therefore, pump and secondary transporter systems in plants are more divergent than in animals.
Fig. 1Numbers of membrane transporter proteins of each class. Membrane transporter proteins were categorized into three classes (ATP-dependent [pump], channel, and secondary transporter) and compared among Escherichia coli K12-MG1655, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens NCBI, Neurospora crassa 74-OR23-IVA, and Saccharomyces cerevisiae S228CTable 1Comparative analysis of membrane transporter gene in many organismsGene familyE. coli K12A. thalianaO. sativaC. elegansD. melanogasterH. sapiens N. crassa 74S. cerevisiae MaterialPresent inEnergy-dependent (pump)ABC 671101534851473124VariousAllArsAB 00101111AnionAllF-ATPase (catalytic) 15621222H+, Na+AllH+-PPase 03900000H+P, a (vacuolar)IISP 03501329ProteinAllMPT 061411814818ProteinEukaryote (mitochondria)P-ATPase 446572219321916H+, Na+, Ca2+ etc.,AllChannelACC00000700Cation (Ca2+)A (neuron)Annexin 0810071310Ca2+EukaryoteBcl-2 000011200AnionA (mammal )Bestrophin 000214400Anion (Cl−)A, P, F, B (G−)CD2000000900Ca2+A (B-lymphocyte )ClC 3714631031Cl−AllConnexin 000001800VariousA (vertebrate)CSC 02100000VariousP (chloroplast)CytB 01970101319H+AllE-ClC 04400400Cl−A (mammal), P (distant homolog)ENaC0002025800Na+A (epithelial cell, brain)GIC 019179272000Metal (Ca2+, K+), GlutaminAllHsp70 017230141440Cation AllICC00000100Cl−A (mammal)ICln 01210100Anion (Cl−)A, P (distant homolog)Innexin 000228000VariousA (invertebrate)IRK-C000132200K+A, BLIC 00069234500VariousAMid100000001Cation (Ca2+)YMIP23838771114H2O, CO2, NH3AllMIT20000023Metal (Mg2+,Co2+, Ni2+)Y, a, BMscL 10000000VariousBMscS 68700000VariousP, BNSCC2 01111101CationA, Y, FO-ClC00001600Cl−APCC 00005600Cation (Na+, K+ , Ca2+) APLM00000700AnionA (mammal)RIR-CaC 00053600Ca2+ATic11001200000VariousPTRP-CC 000572301Ca2+A, Y, UT00000200UreaA (vertebrate), B VIC 1351863319022K+, Na+ or Ca2+AllPhosphotransferase System (PTS)GPTS 60000000CarbohydrateBSSPTS 230000000CarbohydrateBSecondary TransporterAAA 02300000ATPPlant (chloroplast)AAAP 0437711151347Amino acid, AuxinA, P, F, YAAE10000000Aspartate, Alanine BAbgT10000000p-Aminobenzoyl-glutamate BACR3 00000011Arsenite, Antimonite Y, BAE 073421021Na+, HCO3− , H+, Cl− , H3BO3 (P, Y)A, P, YAEC 18800000AuxinP, Y, BAGCS 10000000Alanine, GlycineB, aAmt161362443NH3, CO2AllAPC2212141111141524Amino acid, PolyamineAllArsB 20005100Arsenite, Antimonite All (with distant homolog)BASS 15602501Organic acidAllBCCT 30000000Betaine/Carnitine/CholineB, aBenE 10000000BenzoateBCaCA 21223811884Ca2+AllCCC 01265911Na+, K+, Cl−AllCDF 2811871085Zn2+ or Cd2+ etc.,AllCHR00000010SO42− , CrO42−F, BCNT30022310NucleosideA, Y, BCPA1287115332Na+, H+AllCPA2 3321600121Na+, K+AllDAACS30062700Dicarboxylate, Amino acidA, BDASS 54543503VariousA, P, Y, BDcu 20000000C4-Dicarboxylate B (G−)DcuC 20000000C4-Dicarboxylate B (G−)DMT 161215715141869Various AllENT 08453411NucleosideA, P, YESS 10000000GlutamateBFBT 09800000Folate, BiopterinP, B, aFNT 40000011Formate, NitrateY, B, aGntP70000000CarbohydrateB (E. coli, Bacillus) GPH 69811520SugarA, P, F, a, BGUP 00100012GlycerolA, P, Y, F, BHAAAP 81000000Amino acidB, P (distant homolog)KDGT 100000002-Keto-3-DeoxygluconateBKUP 1132100010K+P, F, BLCT00002211CystineA, P, FLctP20000000Lactate, Glycolate B, aLIV-E10000000Amino acid (L,I,V)B, aLIVCS 10000000Amino acid (L,I,V)BLysE 10000000LysineBMC 052663445443434Various EukaryoteMET 00000300Nucleoside, etc.AMFS 70901451371448214185Various AllMOP8564800213Various (Drugs etc.,)AllMTC 02262311Various (Anionic substrate )EukaryoteNCS1 210000310Nucleobase, Thiamine P, F, Y, B, aNCS211121151410Nucleobase,etc.AllNhaA 10000000Na+,H+ProkaryoteNhaB10000000Na+, H+AllNhaD 02100000Na+(Li+), H+P, BNiCoT 00000010Ni2+, Co2+AllNramp171021223Metals (Fe2+, Zn2+ etc.,)AllNSS 00012211800Neurotransmitters etc.,AOAT 022381100VariousA, P, F, YOPT0153400043OligopeptideP, B, aOST 00000200Organic compound EukaryoteOxa114501101ProteinAll (with distant homolog)PiT21351211HPO42−, SO42−AllPnaS 10010200Inorganic phosphate A (mammal)POT 4506133421OligopeptideAllRFC 00033400Folate, TiamineARhtB 50000000Amino acidB, aRND 821244721Various AllSSS 4113191121VariousAllSulP 11115791144SO42−AllTat 10100000ProteinB (G−)TDT 14900021Tellurite, C4-Dicarboxylate All (with distant homolog)ThrE 10000002Threonine, Serine All (with distant homolog)TRAP-T 10000000VariousB, aTrk 21100022K+P, Y, BZIP 0131865253Zn2+, Fe2+AllUnclassifiedCtr1 00000001Dipicolinic Acid Y, BCtr2 05743222Cu2+AllFeoB 10000000Fe2+B, aFeT 00000001Fe2+, (Co2+, Cd2+)YFP 00000100Fe2+A (mammal)LPI 00000500ProteinAOFeT 10000012Fe2+,Fe3+Y, BPnuC 10000000Nicotinamide mononucleotideBPPI 00006630ProteinA, F, BPUP 115400000Peptide, Fatty acidP, BNote: Present in: A = animal, P = plant, F = fungi, Y = yeast, B = bacteria, a = archea, G− = gramm minus bacteria
Energy (ATP, pyrophosphate)-dependent (pump) system
We compared the ATP-dependent transport genes in this diverse set of organisms (Table 1, Fig. 2, and Supplemental data-2). The main roles of the ATP-dependent (pump) proteins are (1) to transport molecules in specific directions independently of the environmental situation; and (2) to transport ions to form a concentration gradient between the areas outside and inside the membrane (active transport). Because bacteria cannot control the concentrations of ions or metabolites outside the cell, their pumps work mainly to transport molecules. In E. coli, most of the ATP-dependent genes (93%) encode ATP-binding cassette (ABC) proteins, and some of them are reported to encode channel proteins (Fig. 2, Table 1) (Holland et al. 2005). The ABC proteins transport various substances (e.g. ions, peptides, nucleosides, amino acids, carbohydrates, proteins) ATP-dependently in all organisms (Kolukisaoglu et al. 2002; Garcia et al. 2004). In eukaryotes, many functional units are present within one polypeptide, whereas many bacterial ABC subunits are encoded by individual genes. This results in an inverted relationship between prokaryotes (E. coli) (72) and primitive eukaryotes (yeast) (43). Many bacterial ABC proteins are located in the plasma membrane and serve as the main forces in energy-consuming transport for both import and export of substances. In higher eukaryotes, ABC proteins tend to export substances rather than function in import reactions.Fig. 2Comparison of numbers of pump genes among various organisms. Pump gene numbers were compared among Escherichia coli K12-MG1655, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens NCBI, Neurospora crassa 74-OR23-IVA, and Saccharomyces cerevisiae S228C. ABC: ATP-binding Cassette; ArsAB: Arsenite-antimonite Efflux; F-ATPase: H+ or Na+-translocating F-type, V-type and A-type ATPase; H+-Ppase: H+-translocating Pyrophosphatase; IISP: General Secretory Pathway (Sec); MPT: Mitochondrial Protein Translocase; P-ATPase: P-type ATPase
Structural analyses revealed that ABC proteins could be classified as half size (homo- or heterodimer functional) or full size (monomer functional) in accordance with their construction. The number of genes encoding ABC proteins in animals is the same as, or slightly less than, in bacteria, most of them representing whole subunits of ABC proteins. In contrast, the number of ABC genes in plants is about twice that in animals and bacteria, primarily due to the encoding of half-size proteins (Table 1, Fig. 2, and Supplemental data-2) (Sanchez-Fernandez et al. 2001; Sugiyama et al. 2006). This increase in numbers of genes encoding half-size (e.g., WBC, ABC2) and full-size (PDR; pleiotropic drug resistance) proteins supposedly permits stage- and tissue-specific regulation of various substances (including plant-specific substances). Therefore, increases in the capacity of import and export transport systems by an increase in the number of half-size ABC proteins have allowed plants to fulfill their unique roles.
In the import and efflux of ions and the creation of ion gradients for secondary transport, P-type ATPases transport many species of ion (e.g. H+, Na+, K+, Ca2+) in both directions in the cell (Table 1, Fig. 2, and Supplementary data-2) (Axelsen and Palmgren 2001; Baxter et al. 2003). Control of ion concentrations outside bacterial cells is not possible. Only a few of the bacterial genes involved in systems for the uptake or efflux of ions (uptake: K+, Mg2+; efflux: Ca2+, Ag+, Zn2+, Co2+, Pb2+, Ni2+, Cd2+; uptake or efflux, depending on the system: Cu2+) have been characterized, and each of the enzymes encoded comprises a distinct subfamily (Banci et al. 2006).
Eukaryotes have diverged in terms of their transport substances and have also adapted genes for making the ion gradients for secondary transport. One of the most important ATPases in animals—Na+/K+ATPase—does not exist in plants. Na+/K+ATPase makes a Na+ ion gradient across the plasma membrane and forms the basis of membrane voltage and secondary active transport in animals. On the other hand, plants use
H+ gradients for secondary transport, and more than 10 isoforms and vacuole-type H+ATPases are involved in stage- and tissue-specific control. This difference in adapted ion gradients reflects the ion concentrations in the cells and the demand for nutrient ions. A constant supply of Na+ ions is required to retain homeostasis of animal cells and rapid signal transmission systems (e.g. muscles, nerves). In contrast, plants use the more abundant H+ ion to make cation gradients and have special systems for transporting H+ ions into vacuolar pools. For transporting H+ into vacuoles, higher plants have a vacuole-type ATPase and an H+-translocating pyrophosphatase (H+-PPase) (Table 1, Fig. 2, and Supplementary data-2) that are comparable to bacterial ATPase and PPase, respectively (Sivula et al. 1999; Sze et al. 2002). Therefore, plants presumably adapted bacterial H+-ATPase systems to help in H+ ion concentration steps, whereas animals developed completely new systems to make Na+ ion gradients.
Ion channel systems
Ion channels are the “gates” in the membranes that open or close in response to signals such as mechanical or electrical stimulation and ligand binding. Therefore, ion channels are closely involved in determining whether or not ionic gradients are available. Multicellular organisms can control the ion concentrations in the tissues on both sides of the cell membrane, whereas monocellular organisms usually find it hard to control the ion gradient outside the cell. Thus, the use of ion channels becomes restrictive and unidirectional in these more primitive organisms. Unlike in eukaryotes, the channel system in prokaryotes is not well adapted to transport (Table 1, Fig. 3, and Supplemental data-3). Of the total number of genes encoding membrane transport proteins in prokaryotes, fewer than 5% are channel genes, and they mainly regulate osmotic homeostasis in the cell. Ions (e.g. Cl−, K+, metal), water, and osmolytes are imported or exported by the channels in their restricted role. In contrast, animals can make various ion gradients precisely and, in particular, can develop channel systems very well.Fig. 3Comparison of numbers of channel genes among various organisms. Channel gene numbers were compared among many organisms (E. coli K12-MG1655, A. thaliana, O. sativa, C. elegans, D. melanogaster, H. sapiens NCBI, N. crassa 74-OR23-IVA, and S. cerevisiae S228C). ACC: ATP-gated Cation Channel; Bcl-2: Bcl-2; Bestrophin: Anion Channel-forming Bestrophin; CD20: CD20 Ca2+ Channel; ClC: Chloride Channel; Connexin: Gap Junction-forming Connexin; CytB: gp91phox Phagocyte NADPH Oxidase-associated Cytochrome b558 (CytB) H+-channel; E-ClC: Epithelial Chloride Channel; EnaC: Epithelial Na+ Channel; GIC: Glutamate-gated Ion Channel; Hsp70: Cation Channel-forming Heat Shock Protein-70; ICC: Intracellular Chloride Channel; Icln: Nucleotide-sensitive Anion-selective Channel; Innexin: Gap Junction-forming Innexin; IRK-C: Inward Rectifier K+ Channel; LIC: Ligand-gated Ion Channel of Neurotransmitter Receptors; Mid1: Yeast Stretch-Activated, Cation-Selective Ca2+ Channel Mid1; MIP: Major Intrinsic Protein; MIT: CorA Metal Ion Transporter; MscL: Large Conductance Mechanosensitive Ion Channel; MscS: Small Conductance Mechanosensitive Ion Channel; NSCC2: Non-selective Cation Channel-2; O-ClC: Organellar Chloride Channel; PCC: Polycystin Cation Channel; PLM: Phospholemman; RIR-CaC; Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca2+ Channel; Tic110: Chloroplast Envelope Anion Channel-forming Tic110; TRP-CC: Transient Receptor Potential Ca2+ Channel; UT: Urea Transporter; VIC: Voltage-gated Ion Channel
Compared with other systems, the channel system is the fastest at transporting molecules without consuming energy (i.e. when it is not necessary to transport against electrical gradients). Therefore, animals (especially vertebrates) have adapted these systems to nerve and muscle signal transmission. Nervous-system-specific channels such as neurotransmitter-responsible channels (connexin, ENaC, innexin, LIC, RIR-CaC, TRP-CC) are found only in animals. The numbers of channels that are membrane-voltage-dependent for Ca2+, K+, and Na+ ion transport are dramatically increased in both vertebrates and invertebrates (Table 1, Fig. 3; Sheng et al. 2000; Du et al. 2002; Clapham 2003; Hua et al. 2003; Miyazawa et al. 2003). Because animals form Na+ and K+ gradients with the Na+/K+ ATPase pump system, there is also an increase in the numbers of species of Na+ and K+ ion channels in animals. Thus, the total number of channel genes in humans (320) is about twice that in plants (130–180) (Table 2, Fig. 3). This difference is supposedly related to the ecological and physiological specificity of plants. Sometimes constant acquisition of unevenly distributed resources is difficult for plants. Additionally, the ion concentrations in the vascular systems and intercellular spaces of plants, unlike those in animals, are difficult to control precisely. Therefore, some of the ion channels and membrane-voltage-dependent channels are not as divergent as in animals. Although plants lack neurotransmission systems and have fewer membrane-voltage-dependent systems that use Na+ and K+ ion channels, the channel genes for ion homeostasis and signal transduction (e.g. ClC, GIC) are as well developed as in animals, and the numbers of some channel genes (CytB and MscS) are in fact specifically increased in plants (Table 1, Fig. 3).Table 2Comparison of the genome size, total and membrane transport gene numbers in many organismsE. coli K12A. thalianaO. sativaC. elegansD. melanogasterH. sapiens N. crassa 74S. cerevisiae Genome Size (Mb)4.61254309712031504013Total gene number4,290 26,000 37,000 20,621 13,489 30,000 10,082 5,804 Total Transporter Proteins3549841200654590754344300Transporters per Mb genome76.747.822.846.754.30.248.6325.38Transporters per whole gene0.0820.0380.0330.0330.0440.0250.0340.052ATP-dependent pumps72 (20%)173 (18%)249 (21%)72 (11%)72 (12%)82 (11%)53 (15%)43 (14%)Ion Channels15 (4%)131 (13%)178 (15%)229 (35%)158 (27%)322 (43%)12 (3%)24 (8%)Phosphotransferase Systems (PTS)30 (8%)0000000Secondary Transporters233 (66%)658 (67%)762 (63%)349 (53%)351 (59%)336 (44%)273 (79%)227 (75%)Unclassified3 (1%)22 (2%)11 (1%)4 (1%)9 (1%)14 (2%)6 (2%)6 (2%)
Comparison of protein structures indicates that the fundamental structures of ion channels (numbers of transmembrane domains, pore-forming helices) are common in many organisms, but local similarities in individual regions among organisms are low (especially in the N and C terminus fragments). Some of the genes (e.g. for shaker-type K+ channel, SKOR-type K+ channel) have low levels of similarity among the whole structures of plants and animals (Supplemental data-3).
In contrast, the major intrinsic protein (MIP) gene family, which encodes water-transport proteins, is specifically well developed in plants (Zardoya 2005). The numbers of these genes in tissues and at different stages are 3–5 times those in animals (38 vs. 7–11) (Table 1, Fig. 3) (Quigley et al. 2002; Sakurai et al. 2005). MIPs are abundant in the plasma membrane (15–20% of total membrane protein) and vacuoles (30–50% of total membrane protein) of plants; therefore, a high level of water transport is carried out at plant cell membranes. The importance of the acquisition of water has presumably resulted in the diversification and development of water channels in plants. Plants also use water pressure for regulation of movement (e.g. stomatal opening, leaf and petal angle changes) and for transmitting signals in various homeostatic functions. Therefore, plants have presumably developed signal transduction systems that rely on water molecules instead of the neurotransmitters and membrane voltage changes used in animals.
Structural analyses indicate divergence in the level of conservation of MIP subfamilies (PIP, TIP, SIP, NIP, AQP, and GLP) (Supplemental data-3E). The plasma-membrane-intrinsic proteins (PIPs) are well conserved among organisms, but animal- (AQP) and plant-specific (SIP, NIP, TIP) genes have diverged, and closely related channel proteins have low levels of similarity within each organism (Supplementary data-3E). This also suggests a specific diversification of the water transport genes in plants. The structures of the channels in plants are simple compared with those in animals (Supplementary data-3). A decrease in the membrane spanning times and in the number of subunits of working systems can be detected in many channels (e.g. VIC [VDCC], CIC) (Supplementary data-3). Therefore, animals have developed channel systems especially for the exquisite control of ions, membrane voltages, and the signal transduction pathways of specific ligands (e.g. neurotransmitters).
Phosphotransferase system (bacteria)
Bacteria have specific membrane transport systems (phosphotransferase systems) for carbohydrates (sugar) and phosphates (Tables 1, 2) (Barabote and Saier 2005). These systems use phosphate phosphoenol pyruvate (PEP) as the energy source for phosphorylation and for transport of carbohydrates with the aid of enzyme complexes. Carbohydrates (e.g. glucose, fructose, mannitol, sorbitol) are transported against a concentration gradient, with concomitant phosphorylation. PEP is transferred via the soluble (and non-sugar-specific) enzymes EI and HPr to the enzyme complex EII. EII is made up of components A, B, and C, which, depending on the sugar specificity and bacterium involved, may be the domains of composite proteins; component/domain C is a permease and is anchored to the cytoplasmic membrane. Because the amount of phosphorylation of the enzymes influences other regulatory mechanisms in the cells (e.g. catabolite repression, chemotaxis), the whole cell needs a multiple-component, complex enzymatic control system. Therefore, the larger and more
complicated cells
of animals and plants have not adapted this system and instead use ATP-dependent and secondary transporter systems to transport carbohydrates.
Secondary transporter (transporter) system
The secondary transporter system works via the concentration gradient of co-transporter molecules. It is efficient to use a few abundant molecules as common co-transporter molecules for various transport substrates. Therefore, in accordance with the species of ion for which there is a gradient between the inside and outside of the cell, major co-transporter molecules were selected and secondary transporter systems developed for them. There are more than 100 species of gene families in the secondary transporter systems of all organisms, and various substances are transported. Compared with those in the pump and channel systems, the genes involved in the secondary transporter system are most divergent in plants and bacteria; in animals there are about 350 of these genes (44–59% of the total) (Tables 1, 2, Fig. 4).Fig. 4Comparison of numbers of secondary transport genes among organisms. Secondary transporter gene numbers were compared among Escherichia coli K12-MG1655, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens NCBI, Neurospora crassa 74-OR23-IVA, and Saccharomyces cerevisiae S228C. AAAP: Amino Acid/Auxin Permease; AE: Anion Exchanger; AEC: Auxin Efflux Carrier; Amt: Ammonium or Ammonia Transporter; APC: Amino Acid-Polyamine-Organocation; BASS: Bile Acid:Na+ Symporter; BCCT: Betaine/Carnitine/Choline Transporter; BenE: Benzoate:H+ Symporter; CaCA: Ca2+:Cation Antiporter; CCC: Cation-Chloride Cotransporter; CDF: Cation Diffusion Facilitator; CNT: Concentrative Nucleoside Transporter; CPA1: Monovalent Cation:Proton Antiporter-1; CPA2: Monovalent Cation:Proton Antiporter-2; DAACS: Dicarboxylate/Amino Acid:Cation (Na+ or H+) Symporter; DASS: Divalent Anion:Na+ Symporter; DcuC: C4-dicarboxylate Uptake C; DMT: Drug/Metabolite Transporter; ENT: Equilibrative Nucleoside Transporter; FBT: Folate-Biopterin Transporter; GntP: Gluconate:H+ Symporter; GPH: Glycoside-Pentoside-Hexuronide (GPH):Cation Symporter; HAAAP: Hydroxy/Aromatic Amino Acid Permease; KUP: K+ Uptake Permease; LCT: Lysosomal Cystine Transporter; MC: Mitochondrial Carrier; MET: 4 TMS Multidrug Endosomal Transporter; MFS: Major Facilitator Superfamily; MOP: Multidrug/Oligosaccharidyl-lipid/Polysaccharide Flippase Superfamily; NCS2: Nucleobase:Cation Symporter-2; Nramp: Metal Ion (Mn2+–iron) Transporter; NSS: Neurotransmitter:Sodium Symporter; OAT: Organo Anion Transporter; OPT: Oligopeptide Transporter; PnaS: Phosphate:Na+ Symporter; POT: Proton-dependent Oligopeptide Transporter; RFC: Reduced Folate Carrier; RhtB: Resistance to Homoserine/Threonine; RND: Resistance-Nodulation-Cell Division; SSS: Solute:Sodium Symporter; SulP: Sulfate Permease; TDT: Tellurite-resistance/Dicarboxylate Transporter; Trk: K+ Transporter; ZIP: Zinc (Zn2+)–Iron (Fe2+) Permease
Organism-specific gene families in bacteria include those encoding proteins (e.g. APC, HAAAP) that transport many species of amino acid or carbohydrate (GntP, Dcu), or other substances (BCCT, BenE) (Neidle et al. 1991; Golby et al. 1998; Saier et al. 1999; Jack et al. 2000; Samsonov et al. 2002; Prakash et al. 2003). Gene families in animals include genes for specific transport proteins (NSS, MET) that carry neurotransmitters and hydrophobic compounds (Beckman and Quick 1998; Hogue et al. 1999; Yamashita et al. 2005). Those in plants include genes with dramatically increased expression, encoding proteins (AAAP, CPA2, DMT, MFS, OPT, POT) that transport such substances as amino acids, cations, and carbohydrates (Fig. 4) (Fischer et al. 1998, 2002; Pao et al. 1999; Saier et al. 1999; Jack et al. 2001; Koh et al. 2002; Wipf et al. 2002). The greater number of secondary active transporters in plants is supposedly due to the existence of vacuoles. Secondary active transporters need an ion gradient across the membranes, and vacuoles can pool many substances at high concentrations and supply co-transporter molecules instantly at any time. Therefore, the secondary transporter systems easily control transport and have diverged more in plants than in animals.
The major facilitator superfamily (MFS) is the largest secondary transporter family in whole organisms; its members transport various substances (e.g. carbohydrates, phosphates, amino acids, cations) with Na+ or H+ ions (Pao et al. 1999; Burckhardt and Wolff 2000; Lemoine 2000). There are many modifications of these superfamily genes among organisms. Bacteria have amino acid and drug efflux families, animals have neurotransmitter transport families, and plants have diverse carbohydrate-transport genes (Tables 1, 2, Fig. 4, and Supplementary data-4). The proteins have structures typical of secondary active transporters: 10–14 transmembrane domains, co-transporter molecules, and material-binding domains. Comparative analyses indicate that their fundamental structure—membrane-spanning, pore-forming, and substrate-binding sites—is conserved, but similarities between individual domains are generally low among organisms (Tables 1, 2, Fig. 4 and Supplementary data-4). The transporter proteins are encoded by single transcription units and work as monomers. Therefore, this transport system is easy to adapt to new purposes than are the higher pump and ion channel systems. Although the secondary transporter system can adapt to many kinds of concentration gradients, generally few ion species are used. Most of the secondary active transporters depend on two ions: Na+ and H+. In the whole organism, more than 80% of the genes depend on these ions as co-transporter molecules (Table 3, Fig. 5). However, in many cases each ion can be substituted: animals use Na+ ions as co-transporter molecules and bacteria and plants use H+ ions. This has caused animals to make a Na+ ion gradient with Na+/K+ ATPase for membrane voltage and plants and bacteria to make H+ ion gradients with P-type ATPase and H+-PPase, respectively. The transporter genes of animals and plants have specifically adapted to Na+ and H+ co-transporter systems (Table 3, Fig. 5). Therefore, both animals and plants have developed transporter genes adapted to ion gradients.Table 3Comparison of secondary transporter genesE. coli A. thalianaO. sativaC. elegansD. melanogasterH. sapiens N. crassa S. cerevisiae Present inMaterialCotransporterDirectionAAA 02300000P (chloroplast)ATPH+ in (same)AAAP0437711151347A, P, F, YAmino acid, AuxinH+ in (same)AAE 10000000Bl-Aspartate, l-Alanine each otherboth (anti)AbgT10000000Bp-Aminobenzoyl-glutamate H+ in (same)ACR300000011B, YArsenite, Antimonite ?outAE 073421021A, P, YNa+, HCO3−, H+, Cr, H3BO3H+, Na+, Cl−both (anti)AEC 18800024P, Y, BAuxinH+ out (anti)AGCS10000000a, BAlanine, GycineNa+, H+in (same)Amt 161362443AllNH3, CO2?bothAPC 2212141111141524AllAmino acid, PolyamineH+, Solutein (same)ArAE311900000P, Y, B, aArmate acid (Malate etc.,)?outArsB 20005100All (with distant homolog)Arsenite, Antimonite ?outBASS 15602501AllOrganic acidNa+in (same)BCCT 30000000B, aBetaine/Carnitine/CholineH+ in (same)BenE 10000000BBenzoateH+ in (same)CaCA 21223811884AllCa2+H+, Na+both (anti) CCC 01265911AllNa+, K+, Cl−Na+, K+, Cl−both (same)CDF2811871085AllDivalent cation (Zn2+, Cd2+) H+, K+out (antit)CHR 00000010F, BSO42− , CrO42−H+both (anti)CNT30022310A, Y, BNucleosideH+, Na+in (same)CPA1 287115332AllNa+,H+H+, Na+, Cl−both (anti)CPA2 3321600121AllNa+, K+H+ (or itself)out (anti)DAACS 30062700A, BDicarboxylate, Amino acidH+, Na+in (same)DASS54543503A, P, Y, B (G−)Aminoacid, Sulfate, Phosphate etc.,H+, Na+in (same), both (anti)Dcu20000000B (G−)C4-Dicarboxylate Dicarboxylateboth (anti)DcuC20000000B (G−)C4-DicarboxylateH+, Dicarboxylatein (same = H+) , both (anti) DMT 161215715141869AllC3 Carbohydrate, Sugar, Nucleotide etc.,H+, Nucleotideout (anti)ENT08453411A, P, YNucleoside?bothESS 10000000BGlutamateNa+in (same)FBT 09800000P, a, BFolate, BiopterinH+ in (same)FNT 40000011Y, a, BFormate, NitrateH+ in (same)GntP 70000000B (E. coli, Bacillus)CarbohydrateH+ in (same)GPH69811520A, P, F, a, BSugarH+, Na+ , Li+in (same)GUP 00100012A, P, F, Y, BGlycerolH+ in (same)HAAAP 81000000B, P (distant homolog)Amino acidH+ in (same)KDGT 10000000B2-Keto-3-DeoxygluconateH+ in (same)KUP 1132100010P, F, BK+noneinLCT06302211A, P, FCystineH+ in (same)LctP 20000000a, BLactate, Glycolate H+ in (same)LIV-E10000000a, BAmino acid (L, I, V)H+ in (same)LIVCS10000000BAmino acid (L, I, V)H+, Na+in (same)LysE 10000000BLysineH+, OH−out (OH− = same; H+ = anti)MC 052663445443434EukaryoteC3 Carbohydrate, Sugar, Nucleotide etc.,Variousboth (anti)MET00000300ANucleoside, Hydrophobic compound H+ both (anti)MFS 71921451341368114185AllC3 Carbohydrate, Sugar etc.,H+, Na+, Variousboth (H+, Na+= same; others = both)MOP (MATE) 8564800213AllVarious (Drugs, Polysaccharides etc.,)Na+, (H+)out (anti)MTC 02262311EukaryoteVarious (Anionic substrate )H+ in (same)NCS1210000310P, F, Y, a, BNucleobase, Thiamine H+ in (same)NCS2 10121151410AllNucleobase, (Ascorbate = (mouse only)) H+, (Na+= (Ascotbate))in (same)NhaA 10000000ProkaryoteNa+, H+Na+, H+both (anti)NhaB 10000000AllNa+, H+Na+, H+both (anti)NhaD 02100000P, BNa+, (Li+), H+Na+, (Li+), H+both (anti)NiCoT 02000010AllNi2+, Co2+Ni2+, Co2+both (anti)Nramp 171021223AllFe2+, Zn2+, Mn2+, Co2+, Ca2+, Cu2+, Ni2+, Pb2+etc.,H+ in (same)NSS 00012211800ANeurotransmitters, Amino acids, Osmolytes, Cl− etc.,Na+in (same)OAT 022681100A, P, F, YVariousAnion both (anti)OPT 0153400043P, B, aOligopeptideH+ in (same)OST00000200EukaryoteOrganic compound (mostly anions)each otherboth (anti)Oxa1 14501101All (with distant homolog)Proteineach otherboth (anti)PiT 21351211AllHPO42−, SO42−H+, Na+in (same)PNaS 10010200A, B (distant homolog)Inorganic phosphate Na+in (same)POT 4506133421AllOligopeptideH+ both (same)RFC00033400AFolate, TiamineH+, OH−, anionboth (anti)RhtB50000000a, BAmino acidH+ out (anti)RND821244721AllHeavy metals, Drugs, Lipids etc.,H+ out (anti)SSS4113191121AllSugar, Amino acid, Organo cation, AnionNa+, (H+ can replace, but reduces affinity)in (same)SulP 11115791144AllSO42−H+ in (same)Tat10100000B (G−), P (distant homolog)ProteinnoneoutTDT 14900021All (with distant homolog)Tellurite, C4-Dicarboxylate H+ in (same)ThrE10000002All (with distant homolog)Threonine, Serine H+ both (anti).TRAP-T10000000a, BVariousH+ in (same)Trk 21100022P, Y, BK+H+ in (same)ZIP 0131865253AllZn2+, Fe2+noneinNote: Present in: A = animal, P = plant, F = fungi, Y = yeast, B = bacteria, a = archea, G− = gramm minus bacteria; Direction: “in” , “out”, and “both” indicate transport direction of materials through cell membrane, and “same” means material and cotranspoter move to the same dirction, “anti” means the different directionsFig. 5Comparison of co-transport molecules of secondary active transporters among various organisms. Numbers of co-transport molecules of secondary active transporters were compared among Escherichia coli K12-MG1655, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens NCBI, Neurospora crassa 74-OR23-IVA, and Saccharomyces cerevisiae S228C
In animals, many molecules are transported with different co-transporter molecules. Examples are (1) molecules (e.g. neurotransmitters, organic anions, phosphates) transported with Na+ ion (NSS, BASS, CPA2, Pnas) (Beckman and Quick 1998; Saier et al. 1999; Kramer et al. 2001; Segawa et al. 2002; Radchenko et al. 2006); (2) molecules (e.g. amino acids, nucleosides) transported with H+ ions (e.g. AAAP, APC, DMT, LCT, MET) (Steiner et al. 1994; Jack et al. 2001; Zhai et al. 2001; Gasol et al. 2004); (3) molecules (e.g. anions, cations, sugars, nucleosides) transported with either Na+ or H+ ions (e.g. AE, CaCA, DAACS, GPH, CNT) (Reinders and Ward 2001; Ritzel et al. 2001; Zhu et al. 2003; Cai and Lytton 2004; Ryan et al. 2004); and (4) others (e.g. CCC, OAT, RFC) transported with other molecules (Russell 2000; Flintoff et al. 2003; Hagenbuch and Meier 2003).
In contrast, in plants, almost all substances (K+: KUP; N (): Amt; phosphate: PiT, Pht [MFS], and TPT [DMT]; : SulP; sucrose: GPH; sugar alcohol: SAT [MFS]; monosaccharide: MST [MFS]; amino acid: APC and AAAP; nucleosides: NCS1), including many cations, anions, metals, and drugs, are co-transported with H+ ions. Only BASS (organic acid transporter) and NhaD (Na+: H+ antiporter = Na+: efflux system of vacuole) use Na+ ions as co-transporter molecules for efflux of ions from the cytoplasm, so plants do not require extensive sodium uptake (Table 3, Fig. 5) (Smith et al. 1995; Naderi and Saier 1996; Fu and Luan 1998; Nozaki et al. 1998; Pao et al. 1999; Daram et al. 1999; de Koning and Diallinas 2000; Jack et al. 2001; Khademi et al. 2004). Accumulation of H+ ions makes solutions acidic and prevents many enzymatic and biochemical reactions, so plants pool H+ ions in vacuoles inside the cytoplasm, thereby establishing a stable ion gradient for transport energy. Plants make H+ gradients not only inside cells, but also outside cells by the H+ pump, and they absorb many substances by secondary transporter systems. This H+-adapting system presumably has led to diversification of H+ co-transport in secondary transporter systems in plants.
Comparative analysis of membrane transporter systems in Arabidopsis and rice
We also compared the numbers of membrane transporter genes in Arabidopsis and rice. The rice genome size (430 Mb) is more than three times that of Arabidopsis (125 Mb), but the total number of membrane transporter proteins (1200) is only 1.20 times that in Arabidopsis (1000) (Table 2). The proportions of pump, channel, and secondary transporter proteins are almost the same, and the numbers of genes in many individual gene families are 1.1–1.3 times those in rice. However, Arabidopsis has more multi-efflux proteins and proteins involved in secondary active transport (CPA2, DMT, MOP, NCS2) and in channel-type signal transduction systems (CytB, GIC) (Fig. 6). Arabidopsis is a wild plant that lives in diverse soil conditions and under environmental stress, whereas rice is a cultivated crop plant grown under more stable environmental conditions. Therefore, Arabidopsis has diverged more than the rice cultivar Nipponbare to form transporter systems involved in multi-efflux and stress response signaling. On the other hand, rice has more pump and secondary transporter genes (ABC, P-type ATPase, MFS, POT) for carbohydrate and nutrient transport systems (Fig. 6). This divergence of carbohydrate transporters might have been influenced by artificial selection, during which individuals with larger seed size and numbers were chosen to be cultivated. Additionally, amino acid (AAAP), ammonia (Amt), sulfate (SulP), metal ion (ZIP), Ca2+ (CaCA), and K+ transport protein gene families (VIC, KUP) are specifically diverged in rice. This may have been caused by the need for nutrient supplements for rapid growth in subtropical plants. These differences may also result from dissimilarities between monocots and dicots. Although the cell structures are the same in both plants, there are many differences in the basic structure of tissues and organs. Differences in root and vascular bundle structure may be related to differences in uptake efficiency of nutrients. The levels of uptake and transport amounts of ions such as K+, Fe2+, and other nutrients differ between monocots and dicots; this might be related to differences in the numbers of membrane transport genes.
Fig. 6Comparison of membrane transport genes in Arabidopsis and rice. Numbers of the membrane transporter gene families are compared in Arabidopsis and rice. ABC: ATP-binding Cassette; P-ATPase: P-type ATPase; CytB: gp91phox Phagocyte NADPH Oxidase-associated Cytochrome b558 (CytB) H+-channel; GIC: Glutamate-gated Ion Channel; MIP: Major Intrinsic Protein; VIC: Voltage-gated Ion Channel; AAAP: Amino Acid/Auxin Permease; Amt: Ammonium or Ammonia Transporter; CaCA: Ca2+:Cation Antiporter; CPA2: Monovalent Cation:Proton Antiporter-2; DMT: Drug/Metabolite Transporter; KUP: K+ Uptake Permease; MFS: Major Facilitator Superfamily; MOP: Multidrug/Oligosaccharidyl-lipid/Polysaccharide Flippase Superfamily; NCS2: Nucleobase:Cation Symporter-2; OPT: Oligopeptide Transporter; POT: Proton-dependent Oligopeptide Transporter; SulP: Sulfate Permease; ZIP: Zinc (Zn2+)–Iron (Fe2+) Permease; PUP: Peptide Uptake or Activated Fatty Acid Export Permease
Comparison of the membrane transport genes in Arabidopsis and rice pointed to the directions of evolution of these plants in response to the selection pressures of their differing environmental situations. The level of conservation of gene families in Arabidopsis and rice varies, depending on the category. Rice membrane transporters were 72% orthologous with those of Arabidopsis; this is higher than the 50% ortholog found in transcription factors (Xiong et al. 2005). The levels of expression of membrane transport genes are limited precisely within a small range, and these genes are critical for survival. This may be the reason why the membrane transport genes were less divergent than the transcription factors. Therefore, the specifically divergent gene families in Arabidopsis and rice are related to the indispensable and unique systems in each plant. Divergence of gene patterns might indicate differences between wild weeds and crop cultivars that have been selected for growth in specific and possibly new environments.
Comparison of the overall gene compositions of bacteria (E. coli), animals (H. sapiens), and plants
In the eight diverse organisms that we compared, the number of membrane transporter genes (300–1200) varied less than that of genes in other categories. The minimum number of genes indispensable to retaining cell membrane transport homeostasis thus seems to be 300–350, as found in bacteria, yeasts, and fungi. Many of the additional newly diverged genes of higher animals and plants are channel transporter genes and secondary active transporters that facilitate adaptation to fluctuating concentration gradients present in their environments. Moreover, many newly acquired transporters are highly specifically stage- and tissue-regulated and transport special substrates such as neurotransmitters (ACC) in neural cells or carbohydrates (POT) in leaf tissue.
Comparison of the overall gene compositions of bacteria (E. coli), animals (H. sapiens), and plants (A. thaliana and O. sativa) reveals the strategies for osmotic pressure adjustment and the features of the substance-transport systems in each organism (Fig. 7). Since a monad cannot control the ion environment of its external world and has no need to communicate with other cells, the role of its transporter proteins is restricted to the control of material transport into and out of the cell. Therefore, transport depends mainly on an energy-consuming system (pump) and an internal ion-gradient-dependent system (secondary transporter). Because the bacterium has a cell wall, osmotic pressure is opposed by cell wall pressure and there is no need to form a molecular concentration gradient to prevent excessive accumulation within the cell. Additionally, bacteria are small (1–5 μm, <1/10 of the size of an animal cell) and do not have many of the organelles and membrane structures possessed by higher organisms. Therefore, transport of substances is simple and it is easy to control their concentrations in the cell. The genes encoding the ATP-dependent pump (ABC), phosphotransferase (PTS), small conductance mechanosensitive ion channel (MscS), and secondary active transporters (e.g. APC, DMT, MFS, NCS2, RND) with H+ or Na+ as co-transporter molecules have diverged in bacteria (Fig. 7).
Fig. 7Summaries of comparison of membrane transport genes in bacteria, animals, and plants. Bacteria-specific genes = blue; animal-specific genes = red; plant-specific genes = green; bacteria- and animal-specific genes = purple; bacteria- and plant-specific genes = brown; genes with divergent numbers in organisms = yellow. ABC: ATP-binding Cassette; ArsAB: Arsenite-Antimonite Efflux; F-ATPase: H+or Na+-translocating F-type, V-type and A-type ATPase; H+-Ppase: H+-translocating Pyrophosphatase; IISP: General Secretory Pathway (Sec); MPT: Mitochondrial Protein Translocase; P-ATPase: P-type ATPase; ACC: ATP-gated Cation Channel; Annexin: Annexin; Bcl-2: Bcl-2; Bestrophin: Anion Channel-forming Bestrophin; CD20: CD20 Ca2+ Channel; ClC: Chloride Channel; Connexin: Gap Junction-forming Connexin; CSC: Chloroplast Outer Envelope Solute Channel; CytB: gp91phox Phagocyte NADPH Oxidase-associated Cytochrome b558 (CytB) H+-channel; E-ClC: Epithelial Chloride Channel; EnaC: Epithelial Na+ Channel; GIC: Glutamate-gated Ion Channel; Hsp70: Cation Channel-forming Heat Shock Protein-70; ICC: Intracellular Chloride Channel; Ic ln: Nucleotide-sensitive Anion-selective Channel; Innexin: Gap Junction-forming Innexin; IRK-C: Inward Rectifier K+ Channel; LIC: Ligand-gated Ion Channel of Neurotransmitter Receptors; Mid1: Yeast Stretch-Activated, Cation-Selective Ca2+ Channel Mid1; MIP: Major Intrinsic Protein; MIT: CorA Metal Ion Transporter; MscL: Large Conductance Mechanosensitive Ion Channel; MscS: Small Conductance Mechanosensitive Ion Channel; NSCC2: Non-selective Cation Channel-2; O-ClC: Organellar Chloride Channel; PCC: Polycystin Cation Channel; PLM: Phospholemman; RIR-CaC; Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca2+ Channel; Tic110: Chloroplast Envelope Anion Channel-forming Tic110; TRP-CC: Transient Receptor Potential Ca2+ Channel; UT: Urea Transporter; VIC: Voltage-gated Ion Channel; AAA: ATP:ADP Antiporter; AAAP: Amino Acid/Auxin Permease; AAE: Aspartate:Alanine Exchanger; AbgT: p-Aminobenzoyl-glutamate Transporter; ACR3: Arsenical Resistance-3; AE: Anion Exchanger; AEC: Auxin Efflux Carrier; AGCS: Alanine or Glycine:Cation Symporter; Amt: Ammonium or Ammonia Transporter; APC: Amino Acid-Polyamine-Organocation; ArsB: Arsenite-Antimonite (ArsB) Efflux; BASS: Bile Acid:Na+ Symporter; BCCT: Betaine/Carnitine/Choline Transporter; BenE: Benzoate:H+ Symporter; CaCA: Ca2+:Cation Antiporter; CCC: Cation-Chloride Cotransporter; CDF: Cation Diffusion Facilitator; CHR: Chromate Ion Transporter; CNT: Concentrative Nucleoside Transporter; CPA1: Monovalent Cation:Proton Antiporter-1; CPA2: Monovalent Cation:Proton Antiporter-2; DAACS: Dicarboxylate/Amino Acid:Cation (Na+ or H+) Symporter; DASS: Divalent Anion:Na+ Symporter; Dcu: C4-Dicarboxylate Uptake; DcuC: C4-dicarboxylate Uptake C; DMT: Drug/Metabolite Transporter; ENT: Equilibrative Nucleoside Transporter; ESS: Glutamate:Na+ Symporter; FBT: Folate-Biopterin Transporter; FNT: Formate-Nitrite Transporter; GntP: Gluconate:H+ Symporter; GPH: Glycoside-Pentoside-Hexuronide (GPH):Cation Symporter; GUP: Glycerol Uptake; HAAAP: Hydroxy/Aromatic Amino Acid Permease; KDGT: 2-Keto-3-Deoxygluconate Transporter; KUP: K+ Uptake Permease; LCT: Lysosomal Cystine Transporter; LctP: Lactate Permease; LIV-E: Branched Chain Amino Acid Exporter; LIVCS: Branched Chain Amino Acid:Cation Symporter; LysE: L-Lysine Exporter; MC: Mitochondrial Carrier; MET: 4 TMS Multidrug Endosomal Transporter; MFS: Major Facilitator Superfamily; MOP: Multidrug/Oligosaccharidyl-lipid/Polysaccharide Flippase Superfamily; MTC: Mitochondrial Tricarboxylate Carrier; NCS1: Nucleobase:Cation Symporter-1; NCS2: Nucleobase:Cation Symporter-2; NhaA: Na+:H+ Antiporter A; NhaB: Na+:H+ Antiporter B; NhaD: Na+:H+ Antiporter D; NiCoT: Ni2+–Co2+ Transporter; Nramp: Metal Ion (Mn2+–iron) Transporter; NSS: Neurotransmitter:Sodium Symporter; OAT: Organo Anion Transporter; OPT: Oligopeptide Transporter; OST: Organic Solute Transporter; Oxa1: Cytochrome Oxidase Biogenesis; PiT: Inorganic Phosphate Transporter; PnaS: Phosphate:Na+ Symporter; POT: Proton-dependent Oligopeptide Transporter; RFC: Reduced Folate Carrier; RhtB: Resistance to Homoserine/Threonine; RND: Resistance-Nodulation-Cell Division; SSS: Solute:Sodium Symporter; SulP: Sulfate Permease; Tat: Twin Arginine Targeting; TDT: Tellurite-resistance/Dicarboxylate Transporter; ThrE: Threonine/Serine Exporter; TRAP-T: Tripartite ATP-independent Periplasmic Transporter; Trk: K+ Transporter; ZIP: Zinc (Zn2+)–Iron (Fe2+) Permease; GPTS: general carbohydrate phosphotransferase; SSPTS: sugar specific phosphotransferase; Ctr1: Dipicolinic Acid Transporter; Ctr2: Copper Transporter; FeoB: Ferrous Iron Uptake; FeT: Low Affinity Fe2+ Transporter; FP: Ferroportin; LPI: Lysosomal Protein Import; OfeT: Iron/Lead Transporter; PnuC: nicotinamide mononucleotide(NMN) uptake permease; PPI: Peroxisomal Protein Importer; PUP: Peptide Uptake or Activated Fatty Acid Export Permease
Increases in the size and complexity of cells with the emergence of organelles and an internal membrane structure arose in the process of evolution from prokaryote to eukaryote. The eukaryote’s inclusion of the prokaryote’s transport genes in the mitochondrion or chloroplast allowed the transport of homeostatic substrate to organelles, as well as other materials and the products of specific biosynthesis (tricarboxylic acid cycle, electron-transport chain, and photosynthesis). Some of the genes are indistinguishable between eukaryotic transcript and organelle transporter, but there are 50–100 typical organelle transporters (e.g. MC, MCX, Tat) in eukaryotes, and they make a large group (7–12% of all transporters). Furthermore, with the development of multicellular eukaryotes there came a need for organisms to develop a system of transmission between cells. Animals and plants developed specific systems to solve the problem. Animals lost the cell wall structure and gained cellular mobility and flexibility, which also promoted integration of the whole body and specialization of tissues and cells. The loss of the cell wall also led to the need to control osmotic pressure against the plasma membrane. Animal cells regulate osmotic pressure by raising the Na+ concentration outside the plasma membrane by active transport with Na+/K+ ATPase. This Na+ gradient is used not only for homeostasis of the osmotic pressure of the membrane, but also as an electrical potential to control the voltage-dependent channel system and for co-transport of symport or antiport molecules of secondary transportation. Animals then developed the channel system (e.g. Bcl-2, connexin, E-ClC, EnaC, ICC, Ic ln, LIC, PCC, PLM, RIR-CaC, VIC) for rapid transmission in nerve and muscle tissues, together with an Na+ gradient dependent on the secondary transport system (e.g. NSS, PnaS, SSS). This Na+ ion gradient adaptation system is a feature of the newly added transporters and has evolved with animal specificities, i.e. the need for rapid movement and adaptation to environmental stimulation.
In contrast to animals, plants have retained the cell wall instead of acquiring mobility. Because their cell structures and sizes became complicated and large, plants also evolved a means of controlling osmotic pressure; this means differed from the cell-wall-dependent system of bacteria. Because the plant cell controls osmotic pressure by the turgor pressure of the cell wall, it does not require a sodium or other ion gradient and lacks an Na+/K+ ATPase for control of osmotic pressure against the plasma membrane. On the other hand, an increase in permeability to water is necessary for smooth transmission of water pressure in the large and complicated structures of the cell. Therefore, water channels are abundant in both the plasma membrane and the vacuoles (20–50% of membrane proteins), and the diversity of water channels (MIP) is also four to five times higher than that in bacteria and animals. Compared with circulation systems in animals, whole-plant circulation systems are simple and less controlled. Plant cells also need to be more self-complementary in their homeostasis. Additionally, plants have less choice in their environmental conditions (e.g. soil, weather, stress) and must have more transport systems for cell homeostasis than animals. This is why plant cells include vacuoles to pool many substances and bioactive compounds.
Because plants obtain energy through photosynthesis, there are more ATP-dependent (pump) transporters (ABC, P-type ATPase) in their cells than in animal cells. Unlike the case in animals, voltage-dependent systems and Na+-ion-dependent secondary transport systems have not developed in plants; systems that rely on H+ rather than Na+ have developed as part of plant secondary transporter systems. Because the increase in H+ brings acidification, plants maintain the neutrality of the cytoplasm by accumulating H+ in vacuoles, which are membrane-bound organelles separated from the other cytoplasmic contents. By having an internal ion pool, plant cells can control the ion gradient easily and safely. Therefore, secondary transport genes (e.g. CPA2, DMT, MFS, MOP, POT) that use H+ as a co-transporter molecule have diverged far in plants (Fig. 7). The inclusion of pooling and energy-synthesizing organelles in the cell may be why plant cells (10–100 μm) are larger than animal cells (10–30 μm).
Conclusions
Comparative analysis of membrane transporters among these eight diverse organisms indicates the type of cell homeostasis, as evidenced by the pattern of gene conservation and diversification. Evolutionary changes in gene families, in general, indicate the dynamics of alterations in biological systems and gene networks. Therefore, analysis of large categories of gene families may reveal many basic concepts of biological systems. In practice, analyses of the membrane transporter mechanism are useful in revealing changes in the absorption of molecules by, or their efflux from, cells and tissues. This information also is useful for examining changes in soil adaptability, nutritional demand, and stress tolerance in plants. It may also help to improve the harvest of crop cultivars or extend areas habitable by plant species. Gene networks are intricately related, and analysis of the whole genetic structure is needed to gain a full understanding of biological phenomena and systems of gene regulation. We are continuing to analyze whole categories of genes in an effort to develop an overview of total gene networks.
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membrane transporter
oryza sativa
comparative molecular analysis