The value of theEscherichia coli expression system has long been established because of its effectiveness in characterizing the structure and function of exogenously expressed proteins. When eukaryotic membrane proteins are functionally expressed in E. coli, this organism can serve as an alternative to eukaryotic host cells. A few examples have been reported of functional expression of animal and plant membrane proteins in E. coli. This mini-review describes the following findings: 1) homologous K+ transporters exist in prokaryotic cells and in eukaryotic cells; 2) plant K+ transporters can functionally complement mutant K+ transporter genes inE. coli; and 3) membrane structures of plant K+ transporters can be elucidated in an E. colisystem. These experimental findings suggest the possibility of utilizing the E. coli bacterium as an expression system for other eukaryotic membrane transport proteins.
- potassium transporter
- potassium channel
membrane intrinsic proteins play an important role in perception, conversion, separation, and uptake of molecules between the inside space of a cell and its outside environment. Events occurring in or at the cell membrane thus result in biological signal transduction. To study the function of membrane proteins involved in this signal transduction, effective protein expression and assay systems are required.
Heterologous expression of eukaryotic membrane proteins is most often performed in eukaryotic cells such as Saccharomyces cerevisiae or Xenopus laevis oocytes (13, 22,62). When Escherichia coli is the host cell, the expression of eukaryotic membrane proteins seems often to be toxic to the bacterium. In addition, because of differences in membrane insertion mechanisms and the requirement of different endogenous factors, it is assumed that eukaryotic membrane proteins cannot be incorporated into the E. coli cell membrane in a functional form. Although properly folded eukaryotic soluble proteins have been widely produced in E. coli, functional eukaryotic membrane proteins are considered to be difficult to obtain from the E. coli system. Nevertheless, a few successful examples have been reported to date, as described later. Recently, it was found that plant K+ uptake transporters, such as KAT1 (75), AKT2 (75), AtKUP1–2 (40), and AtHKT1 (73), can functionally complement mutations in the K+ uptake systems of E. coli. In contrast, point mutants that produce nonconducting forms of KAT1 do not complement theE. coli mutations. An E. coli mutant that has very low K+ uptake properties was used to measure the K+ transport activities of these plant proteins. On the basis of the functional expression and reporter gene fusions, the membrane topology of plant K+ channels and K+transporters can be determined in this bacterial expression system.
E. coli is most amenable to the expression of foreign genes from not only prokaryotic but also eukaryotic origins. E. coli can serve as a feasible alternative to eukaryotic cells for the expression of proteins that have the following characteristics.1) Because bacteria have no subcellular membranes, membrane proteins that are normally expressed in the plasma membrane or endomembranes of eukaryotic cells may be inserted into the plasma membrane of the bacterial cell (Fig. 1). In fact, a successful example was reported in 1998 (72). A plastidic (choroplast) ATP/ADP transporter from Arabidopsis thaliana was functionally expressed, and the protein's properties characterized, in an E. coli mutant (47,72). 2) In E. coli, it is possible to mutate genes that interfere with the activity of an exogenous target protein. These knockout strains with very low background activities are useful for the precise analysis of target membrane proteins. In this respect, a bacterium could, therefore, provide the same advantages as a yeast cell. 3) Methods for the analysis of the topology and function of bacterial proteins in E. coli are well established. Also, it is easy to scale up the culture, if needed to purify the proteins, because the doubling time of bacteria is significantly shorter than that of eukaryotic cells.
Of course, the physiological background differs between E. coli and eukaryotic cells in such terms as membrane composition, membrane potential, and posttranslational modification mechanisms. In particular, because of a lack of a glycosylation machinery in E. coli, certain eukaryotic membrane proteins may fail to achieve their active form in bacteria. However, there have been reports of successful expression of eukaryotic membrane proteins in E. coli. Freissmuth et al. (21) reported successful expression of β1- and β2-adrenergic receptor. On the basis of that report, Lacatena et al. (42) determined the topology using β2-adrenergic receptor gene-expressing E. coli. The mouse multidrug-resistance protein (MDR1), which is a P-glycoprotein, also has been successfully expressed in E. coli(6). The unglycosylated form of the MDR1 in E. coli retains the same resistance to drugs as exhibited in animal cells. This example shows that E. coli can be used to analyze the nonglycosylated form of a target protein. In this case, the investigators identified a new mutant E. coli deficient in OmpT protease to avoid degradation of the exogenous membrane protein.
Probably numerous experiments remain unpublished in which functional expression of eukaryotic membrane proteins was attempted with negative results. In general, overexpression of membrane proteins is known to be detrimental to E. coli cell growth. Miroux and Walker (47) isolated E. coli mutants derived from strain BL21(DE), which is widely used for overexpression of foreign gene products. This strain allows overexpression of mitochondrial phosphate carrier as inclusion bodies. The host system enabled a large amount of animal membrane proteins of Caenorhabditis elegansand rat mitochondrial dicarboxylate transporters, which could be renatured and reconstituted functionally in proteoliposomes (20). In addition, as mentioned above, the same E. coli mutant allows a plastidic (choroplast) ATP/ADP transporter from A. thaliana to express functionally in its cell membrane (47, 72). According to our results, one possible cause of negative results may relate to the amount of eukaryotic protein produced in the bacterium. When we used a moderate copy number of plasmids for expression of the plant K+uptake channel KAT1 (6a), the gene products complemented the K+ uptake transporter-deficient E. coli as determined by increased growth rate of the bacteria, as described below. The findings will lead us to examine and apply the E. coli system to studies of animal K+ transporters and other types of membrane proteins.
FUNCTIONAL EXPRESSION OF PLANT K+ TRANSPORTERS IN E. COLI
E. coli has three major K+ uptake systems, Kdp, Trk, and Kup, expressed in the inner membrane (Fig. 1) (61). The background (endogenous) K+ influx activity in the triple K+ transport-deficient E. coli strain is so low that the increased K+ uptake permeability resulting from the exogenous gene products can be easily measured. The mutant strain cannot grow on media containing K+ concentrations <10 mM (17, 68). This mutant strain proved useful for complementation tests of the K+ uptake deficiency by the plant K+ channels KAT1 and AKT2 (75). Cells expressing KAT1 or AKT2 grew to their full growth potential in K+-limited medium, whereas an inactive KAT1 mutant with a point mutation within the pore region exhibited only a very low growth rate. KAT1 proteins were present in the cell membrane of E. coli as determined by immunochemical assay (75). The results suggest that KAT1 was both inserted into the cell membrane and functional, making inward transport of K+ possible. Likewise, other K+ transporters from higher plants also functionally complemented the mutant E. coli as shown in Table 1 and Fig.2.
Until recently, the yeast expression system and theXenopus oocyte expression system have been the main tools used to investigate plant K+ transporters. S. cerevisiae defective in the K+ uptake transporters TRK1 and TRK2 is a very useful strain to study K+ uptake transporters from plants. Several kinds of K+ uptake transporter genes have been isolated from plant cDNA libraries with the use of yeast complementation screening. In 1992, twoShaker-type hyperpolarization-activated (inward rectifying) K+ channels, KAT1 and AKT1 from A. thaliana, were cloned (2, 66). The high-affinity K+transporter gene HKT1 was isolated from a wheat cDNA library by using the same yeast complementation system (58). Other types of K+ uptake transporter (KUP/KT/HAK family) genes were obtained from Arabidopsis and barley by using the yeast complementation test or a cloning approach based on sequence similarities to E. coli or S. cerevisiaehomologues (23, 40, 52, 55, 56). These isolated plant K+ transporter genes have been characterized in the yeast mutant, in oocytes, or in insect cells.
STRUCTURAL SIMILARITIES BETWEEN K+ TRANSPORTERS FROM BACTERIA AND HIGHER PLANTS
At least three varieties of K+ uptake transporters have been identified in E. coli, and three kinds of K+ uptake proteins have been isolated from A. thaliana (Fig. 2). The topology and ion permeability of the plant homologous K+ transporters were determined in E. coli expression systems.
Kch family, Shaker-type K+ channels.
The structure of the membrane-pore-membrane (M1-P-M2) K+channel KcsA of Streptomyces lividans was resolved by X-ray diffraction analysis (11). The pore region contains the signature tripeptide sequence G(Y/F)G for ion-selective filtering that is common to all K+ channels. In animal neurons or cardiac cells, Shaker-type voltage-dependent (voltage gated) K+ channels of the M1-P-M2 structure play a crucial role in the generation of action potential (30). The K+ channel has additional transmembrane segments that include the voltage-sensing (S4) motif on the NH2-terminal side of the M1-P-M2 structure (36). Genes of voltage-dependent K+ channels have been isolated from various plants on the basis of sequence homologies to animalShaker K+ channels. In A. thaliana, hyperpolarization-activated K+ channels (KAT1 and AKT1) (5, 7, 12, 26, 32, 33, 35, 49, 59, 70, 74, 76), a depolarization-activated K+ channel (SKOR1 and GORK) (1, 27), and a weak rectification K+ channel (AKT2/3) (4, 39, 43, 45) have been characterized. Electrophysiological measurements taken on wild-type or mutant plants have shown that their K+ channels involve the modulation of opening and/or closing of guard cells or loading and/or unloading of molecules in the phloem and/or xylem of the root, stem, or leaf as well as K+ uptake from the soil (27, 31, 34, 45, 63, 64,65, 67). In E. coli, a homologous K+channel named Kch does exist, but its function in E. coliremains unclear (46, 77).
Although putative topologies of the family of Shaker-type ion channels have been predicted, there has been no experimental evidence of their entire membrane structure. On the basis of its functional expression in E. coli, the KAT1 topology has been systematically investigated by taking a gene fusion approach involving alkaline phosphatase (75). The method was developed by Manoil and Beckwith (44) not only to study the topology of proteins in the inner membrane of bacteria but also to examine the topology of eukaryotic membrane proteins (29). Alkaline phosphatase is active when it is exported into the periplasm, whereas it remains inactive in the cytoplasm (10). When the alkaline phosphatase was fused to a site facing the periplasm, the enzymatic activity was detected from the cells. Fusions with alkaline phosphatase thus led to the conclusion that KAT1 has the common six-transmembrane-spanning topology that has been predicted for theShaker superfamily of voltage-gated K+ channels (37, 75). It was found that the negatively charged residue in the third segment (S3) (9, 28, 51) is essential for the proper function of K+ channels by pairing with the positively charged residues in S4. This is consistent with the results in cyclic nucleotide-gated cation channels from bovine andDrosophila melanogaster. These data also were obtained usingE. coli (29). The E. coliexpression system, therefore, can provide a valuable tool for the functional analysis of membrane proteins (75).
Trk and AtHKT.
Many bacteria possess a single Trk K+ uptake system. However, two types of Trk homologues, TrkG and TrkH, exist in E. coli K12 (61). Their functional TrkG and TrkH molecules require peripheral cytosolic membrane proteins, such as TrkA with a NAD(H) binding site and SapD with an ATP binding motif. The Trk system of E. coli transports K+ with a low affinity but at a high rate estimated to be ∼100 times larger than that of the Kdp system. Other bacterial homologous proteins of Trk were found; KtrB was identified as one of the K+ transporter subunits of the KtrAB complex in Vibrio alginolyticus(50). The K+ uptake rate depended on external Na+ concentrations (71). NptJ inEnterococcus hirae was identified as a membranous component of a K+ and Na+ cotransport system (38,69). In S. cerevisiae, two homologous genes encoding a high-affinity K+ transporter, TRK1 and TRK2, were identified as components of the major K+ uptake system (24, 41, 53).
In plant cells, HKT1 genes (HKT1 from wheat and AtHKT1from Arabidopsis) share ∼23% amino acid sequence identity (48% similarity) with TRK1 and TRK2 (58, 73). Recently, two genes encoding homologous transporters, EcHKT1and EcHKT2, were isolated from Eucalyptus(18). Furthermore, the amino acid sequences of HKT1 and AtHKT1 show a similarity in the position of the hydrophobic segments with the bacterial K+ transporters KtrB and NptJ (15, 16, 50, 53, 57, 71). Detailed analysis of HKT1 with tracer flux experiments performed in S. cerevisiae and electrophysiological studies in Xenopus oocytes revealed that HKT1 functions as a Na+-coupled K+transporter (Fig. 3) (25,54). In contrast, AtHKT1 functioned as a K+-independent Na+ transporter inXenopus oocytes and did not reveal any K+ uptake activity in yeast (Fig. 3) (73). Although it was found that AtHKT1 has one N-linked glycosylation site, substitution at the N-glycosylated residue did not affect the translocation of AtHKT1 to the membrane or the expression of its function (37a). A study of differences in ion selectivity is now in progress. Although K+ uptake was not detected in the eukaryotic cell system, the AtHKT1-expressing E. coli cells did take up K+ ∼1.8 times faster than the control cells. K+ uptake by AtHKT1 may have been observed because of the lower amount required to complement the K+-deficientE. coli mutant compared with the yeast cells (Fig. 3).E. coli is one of the heterologous expression systems that is distinct from yeast, oocytes, and others.
The number of transmembrane segments of transporters related to Trk, Ktr, and HKT1 was predicted in 1999 (15, 16). This family of K+ transporters contains about eight predicted transmembrane segments and four loops, which are homologous to the selective filter-forming pore loops of K+ channels. The topology model of AtHKT1 was experimentally confirmed by the alkaline phosphatase fusion system in E. coli, glycosylation scanning in in vitro translation and translocation experiments employing a reticulocyte-lysate system supplemented with dog pancreas microsomes, and epitope tag detection in HEK-293 cells (37a). The data from expression systems based on animals are consistent with the results obtained by PhoA fusions in E. coli, which demonstrate that AtHKT1 folded properly into the E. coli cell membrane.
Kup and AtKUP (AtPOT).
Schleyer and Bakker (60) identified Kup in E. coli as the third K+ uptake transport system. Kup belongs to a low-affinity type of K+ uptake transporters and is likely to function as a K+/H+ symporter. The deduced amino acids sequence of the Kup gene shows that two-thirds of the gene, constituting the entire NH2-terminal part, contains about 12 hydrophobic domains, while the rest of the COOH-terminal region has hydrophilic features. A homologous protein of the fungi Schwanniomyces occidentalis, called SoHAK1, also has been isolated (3).
HvHAKT1–2 from barley (56) andAtKUP1–4 (23, 40), AtKT1–2 (52) from A. thaliana, andAtHAK5–8 (55) have been isolated as genes encoding proteins homologous to the KUP/KT/HAK (POT) transporters among the higher plants. The hydropathy profiles of these proteins also show 12 putative transmembrane segments in the NH2-terminal portion. The plant KUP/KT/HAK homologues can function as high-affinity K+ transporters in yeast and in plants, but kinetic analyses revealed that AtKUP1 also has the properties of a low-affinity mode. AtKUP1 and AtKUP2 complemented the K+ uptake transport mutations in E. coli mutant, although AtKUP1–2 and HvHAK1 failed to show K+ currents in the oocyte heterologous expression system.
E. coli has a P-type ATPase, Kdp, which has been classified as a high-affinity K+ transport system (68). Kdp was assigned to one of the three main K+ transporters containing Trk and Kup. The Kdp system consists of KdpA, -B, -C, and -F. The 10 transmembrane segments of KdpA have been determined by the alkaline phosphatase fusion method (6a). Durell et al. (14) predicted the four pore regions of the polypeptide of KdpA, but to date, there has not yet been a report of a Kdp-ATPase transporter in any plants.
KefB and KefC are considered K+ efflux transporters (19, 48). For the Arabidopsis genome, the KEA1 gene has been registered as encoding a homologous protein (GenBank accession no. AF003382), but its function has not yet been thoroughly characterized.
This article compares structural and functional similarity among K+ transport proteins of E. coli, yeast, plants, and animals. Although they share low homology on the amino acid level, their membrane topologies are similar. This suggests that the structure and function of the bacterial and the plant/animal ion transporters are more closely related to each other than previously thought. It may, therefore, be possible and reasonable to attempt to incorporate the eukaryotic membrane proteins into the E. coli membrane in an active form.
I thank Drs. E. P. Bakker, T Nakamura, M. Lynn Lamoreux, and J. I. Schroeder for critical reading of the manuscript. I also thank Dr. A. Mizutani for preparation of the figures.
This work was supported by Grants-in-Aid for Scientific Research and by Grant-in-Aids for the Center of Excellence (COE) research from the Ministry of Education, Science, Sports and Culture of Japan (12019227, 12206042, and 13660088).
Address for reprint requests and other correspondence: N. Uozumi, Bioscience Center, Nagoya Univ., Nagoya 464-8601, Japan (E-mail:).
- Copyright © 2001 the American Physiological Society