Escherichia coli alpha-ketoglutarate permease is a constitutively expressed proton symporter.

Escherichia coli kgtP which maps at 56.5 min codes for alpha-ketoglutarate permease (KgtP). This protein, expressed from the cloned gene using the T7 polymerase system and [35S]methionine labeling, fractionated with cell membranes. Right-side-out (RSO) membrane vesicles prepared from a kgtP negative mutant strain did not transport alpha-ketoglutarate, but RSO vesicles from the same strain expressing KgtP from a transforming plasmid transported alpha-ketoglutarate effectively as measured by uptake of the 14C-labeled substrate. E. coli JC7623 strain grown in M9 minimal medium with glucose, glycerol, or alpha-ketoglutarate as carbon source contained a 1.3-kilobase RNA which hybridized to nick-translated kgtP probe. In addition, strain MC1061 cultures grown under these same conditions were all capable of transporting alpha-ketoglutarate, demonstrating that KgtP is constitutively expressed. The Km and Vmax of KgtP assayed in strain MC1061 vesicles were 13-46 microM and 8 nmol/min/mg protein, respectively. Uncouplers that permeabilized the membrane to protons inhibited alpha-ketoglutarate transport into energized vesicles, and the addition of alpha-ketoglutarate to vesicle suspensions under non-energized conditions resulted in an increase in pH. These results indicate that KgtP is an alpha-ketoglutarate-proton symporter.     

Topological characterization of the essential Escherichia coli cell division protein FtsW

The membrane topology of Escherichia coli FtsW, a 46-kDa essential protein, was analyzed using a set of 28 ftsW-alkaline phosphatase (ftsW-phoA) and nine ftsW-beta-lactamase (ftsW-bla) gene fusions obtained by in vivo and in vitro methods. The alkaline phosphatase activities or resistance pattern of cells expressing the FtsW-PhoA or FtsW-Bla fusions confirmed only eight out of 10 transmembrane segments predicted by computational methods. After comparison with the recent topology of Streptococcus pneumoniae FtsW, we could identify all the fusions in absolute agreement with the predicted model: N-terminal and C-terminal ends in the cytoplasm, 10 transmembrane segments and one large loop of 67 amino acids (E240-E306) located in the periplasm.     

Site-directed mutants of Escherichia coli alpha-ketoglutarate permease (KgtP).

To investigate an active site(s) in the Escherichia coli alpha-ketoglutarate premease, 11 point mutants were made in the corresponding structural gene, kgtP, by oligonucleotide-directed mutagenesis and the polymerase chain reaction. On the basis of sequences conserved in KgtP and related members of a transporter superfamily [Henderson P. J. F., & Maiden, M. C. (1990) Philos. Trans. R. Soc. London B 326, 391], Arg76 was replaced with Ala, Asp, or Lys; Asp88 with Asn or Glu; His90 with Ala; Arg92 with Ala or Lys; and Arg198 with Ala, Asp, or Lys. Mutant proteins expressed using the T7 polymerase system were in each case shown to be membrane-associated. However, they differed in transport activity. Mutants H90A and R198K had activities similar to that of wild type, and R76K and R198A retained 10-60% of the wild-type activity. In all other mutants, alpha-ketoglutarate transport was abolished. The results suggest that Arg92, which is highly conserved among other members of the transporter superfamily, is necessary for activity and also that Asp88 is critical for function, as observed for the tetracycline transporter. These data show further that a positive charge is essential at position 76 and is also important, but not absolutely required, at position 198 for alpha-ketoglutarate transport. Unlike lacY permease which was inactivated by deleting the last helix [McKenna, E., Hardy, D., Pastore, J. C., & Kaback, H. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2969], a KgtP truncation mutant missing the last putative membrane-spanning region was relatively stable and also retained 10-50% of the wild-type level of alpha-ketoglutarate transport activity.     

Cleavage of urokinase receptor regulates its interaction with integrins in thyroid cells

The membrane topology of a resistance-nodulation-division (RND) family transporter, MexD of Pseudomonas aeruginosa, was determined. Although it had been predicted previously that most RND proteins contain 12 transmembrane helices, three independent computer programs used in the present study predicted that MexD possessed 11, 14 or 17 transmembrane segments. To investigate the topology of MexD more thoroughly, 25 MexD-PhoA (alkaline phosphatase) and 18 MexD-Bla (beta-lactamase) fusion plasmids were constructed and analyzed. The resulting topological model had just 12 transmembrane helices and two periplasmic loops of about 300 residues between helices 1 and 2 and helices 7 and 8. It is therefore proposed that the N- and C-termini are located in the cytoplasm and the predicted orientation is consistent with the 'positive-inside rule'. This topological model can be applied to other RND proteins.     

Membrane topology of the Streptococcus pneumoniae FtsW division protein

The topology of FtsW from Streptococcus pneumoniae, an essential membrane protein involved in bacterial cell division, was predicted by computational methods and probed by the alkaline phosphatase fusion and cysteine accessibility techniques. Consistent results were obtained for the seven N-terminal membrane-spanning segments. However, the results from alkaline phosphatase fusions did not confirm the hydropathy analysis of the C-terminal part of FtsW, whereas the accessibility of introduced cysteine residues was in agreement with the theoretical prediction. Based on the combined results, we propose the first topological model of FtsW, featuring 10 membrane-spanning segments, a large extracytoplasmic loop, and both N and C termini located in the cytoplasm.     

Topological analysis of the Rhodobacter capsulatus PucC protein and effects of C-terminal deletions on light-harvesting complex II.

A theoretical model for the cytoplasmic membrane topology of the Rhodobacter capsulatus PucC protein was derived and tested experimentally with pucC'::pho'A gene fusions. The alkaline phosphatase (AP) activities of selected fusions were assayed, and the resultant pattern of high and low activity was compared with that of the theoretical model. High AP activity correlated well with fusion joints located in regions predicted to be periplasmic, and most fusions in predicted cytoplasmic loops yield approximately 1/20th as much activity. Replacement of pho'A with lac'Z in nine of the fusions confirmed the topology, as beta-galactosidase activities were generally reciprocal to the corresponding AP activity. On the basis of the theoretical analysis and the information provided by the activities of fusions, a model for PucC topology in which there are 12 membrane-spanning segments and both the N and C termini are located in the cytoplasm is proposed. Translationally out-of-frame pucC::phoA fusions were expressed in an R. capsulatus delta pucC strain. None of the fusions missing only one or two of the proposed C-terminal transmembrane segments restored the wild-type phenotype, suggesting that the C terminus of PucC is important for function.     

Membrane topology of the beta-subunit of the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae.

The topology of the beta-subunit of the oxaloacetate Na+ pump (OadB) was probed with the alkaline phosphatase (PhoA) and beta-galactosidase (lacZ) fusion technique. Additional evidence for the topology was derived from amino acid alignments and comparative hydropathy profiles of OadB with related proteins. Consistent results were obtained for the three N-terminal and the six C-terminal membrane-spanning alpha-helices. However, the two additional helices that were predicted by hydropathy analyses between the N-terminal and C-terminal blocks did not conform with the fusion results. The analyses were therefore extended by probing the sideness of various engineered cysteine residues with the membrane-impermeant reagent 4-acetamido-4'-maleimidylstilbene-2, 2'-disulfonate. The results were in accord with those of the fusion analyses, suggesting that the protein folds within the membrane by a block of three N-terminal transmembrane segments and another one with six C-terminal transmembrane segments. The mainly hydrophobic connecting segment is predicted not to traverse the membrane fully, but to insert in an undefined manner from the periplasmic face. According to our model, the N-terminus is at the cytoplasmic face and the C-terminus is at the periplasmic face of the membrane.     

Membrane topology of Escherichia coli prolipoprotein signal peptidase (signal peptidase II)

The lsp gene of Escherichia coli encodes the inner membrane enzyme, signal peptidase II (SPase II). SPase II is comprised of 164 amino acid residues and contains four hydrophobic domains. A series of lsp-phoA and lsp-lacZ gene fusions have been constructed in vitro to determine the topology of SPase II. The fusion junction for each of these gene fusions was determined by DNA sequencing. The lengths of the SPase II fragment in the fusions varied from 12 to 159 amino acid residues. Strains containing SPase II-PhoA fusions to the two predicted periplasmic loops exhibited higher levels of alkaline phosphatase activity than fusions to the predicted cytoplasmic domains. In contrast, SPase II-LacZ fusions at the cytoplasmic and the periplasmic domains of SPase II showed high and low levels of beta-galactosidase activity, respectively, a result opposite to those shown by SPase II-PhoA fusions located at precisely the same amino acid of SPase II. Taken together, these results strongly support the predicted model for SPase II topology, i.e. this enzyme spans the cytoplasmic membrane four times with both the amino and the carboxyl termini facing the cytoplasm.     

Rapid screening of membrane topology of secondary transport proteins

Limited experimental data may be very useful to discriminate between membrane topology models of membrane proteins derived from different methods. A membrane topology screening method is proposed by which the cellular disposition of three positions in a membrane protein are determined, the N- and the C-termini and a position in the middle of the protein. The method involves amplification of the encoding genes or gene fragments by PCR, rapid cloning in dedicated vectors by ligation independent cloning, and determination of the cellular disposition of the three sites using conventional techniques. The N-terminus was determined by labeling with a fluorescent probe, the central position and the C-terminus by the reporter fusion technique using alkaline phosphatase (PhoA) and green fluorescence protein (GFP) as reporters. The method was evaluated using 16 transporter proteins of known function from four different structural classes. For 13 proteins a complete set of three localizations was obtained. The experimental data was used to discriminate between membrane topology models predicted by TMHMM, a widely used predictor using the amino acid sequence as input and by MemGen that uses hydropathy profile alignment and known 3D structures or existing models. It follows that in those cases where the models from the two methods were similar, the models were consistent with the experimental data. In those cases where the models differed, the MemGen model agreed with the experimental data. Three more recent predictors, MEMSAT3, OCTOPUS and TOPCONS showed a significantly higher consistency with the experimental data than observed with TMHMM.     

Ketoglutarate transport protein KgtP is secreted through the type III secretion system and contributes to virulence in Xanthomonas oryzae pv. oryzae

The phytopathogenic prokaryote Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight (BB) of rice and utilizes a type III secretion system (T3SS) to deliver T3SS effectors into rice cells. In this report, we show that the ketoglutarate transport protein (KgtP) is secreted in an HpaB-independent manner through the T3SS of X. oryzae pv. oryzae PXO99(A) and localizes to the host cell membrane for α-ketoglutaric acid export. kgtP contained an imperfect PIP box (plant-inducible promoter) in the promoter region and was positively regulated by HrpX and HrpG. A kgtP deletion mutant was impaired in bacterial virulence and growth in planta; furthermore, the mutant showed reduced growth in minimal media containing α-ketoglutaric acid or sodium succinate as the sole carbon source. The reduced virulence and the deficiency in α-ketoglutaric acid utilization by the kgtP mutant were restored to wild-type levels by the presence of kgtP in trans. The expression of OsIDH, which is responsible for the synthesis of α-ketoglutaric acid in rice, was enhanced when KgtP was present in the pathogen. To our knowledge, this is the first report demonstrating that KgtP, which is regulated by HrpG and HrpX and secreted by the T3SS in Xanthomonas oryzae pv. oryzae, transports α-ketoglutaric acid when the pathogen infects rice.     

Yersinia spp. HMWP2, a cytosolic protein with a cryptic internal signal sequence which can promote alkaline phosphatase export.

The iron starvation-induced, 2,042-amino-acid protein HMWP2 of Yersinia enterocolitica has two internal hydrophobic segments which might promote its export and association with the cytoplasmic membrane. To determine whether part of HMWP2 could be exported beyond the periplasmic face of the cytoplasmic membrane, we used TnphoA mutagenesis to construct 10 hybrid proteins in which periplasmic alkaline phosphatase (PhoA) was fused to the end of C-terminally truncated HMWP1 (at amino acid positions 1751 and 1753 two independent isolates]) had high alkaline phosphate activity (close to that of the native enzyme), both in Escherichia coli and in Y. pseudotuberculosis, indicating that the PhoA segment of the hybrid reached the periplasm. Deletion studies showed that the export signal resides in the second hydrophobic segment of HMWP2. This result would be compatible with the topology of the protein in the cytoplasmic membrane predicted from the distribution of charged amino acids at either end of the two hydrophobic segments. However, two hybrids in which the junction was even further toward the C terminus of HMMWP2 (at positions 1793 and 1999) had only weak alkaline phosphatase activity, suggesting that the predicted topology is incorrect. The location of HMWP2 was therefore determined by subcellular fractionation. The results indicate that HMPW2 is mainly cytoplasmic, consistent with its presumed role in the ATP-dependent, nonribosomal synthesis of an unknown peptide. We propose that the high alkaline phosphatase activity associated with some of the HMWP-2-PhoA hybrids results from the unmasking of the cryptic export signal activity in the second hydrophobic segment of HMPW2.     

Topology of membrane insertion in vitro and plasma membrane assembly in vivo of the yeast arginine permease

We have examined the topology of the yeast arginine permease, a plasma-membrane protein with multiple membrane-spanning domains. Using fusions of the permease with the glycosylatable secreted yeast protein, acid phosphatase, we identified membrane-spanning sequences that can translocate adjacent acid phosphatase across the membrane of the endoplasmic reticulum (ER), as measured by in vitro glycosylation. Examination for the presence or absence of glycosylation in a systematic series of such fusions gave an internally consistent model for the lumenal or cytoplasmic disposition of the acid phosphatase reporter, defining the topology of the permease. The phenotypes of a further set of arginine permease gene fusions with portions of the gene for the secreted protein, killer toxin, suggest that the pathways of export of membrane and secreted proteins need not be functionally distinct.     

Membrane topology of the xenobiotic-exporting subunit, MexB, of the MexA,B-OprM extrusion pump in Pseudomonas aeruginosa

The MexA,B-OprM efflux pump assembly of Pseudomonas aeruginosa consists of two inner membrane proteins and one outer membrane protein. The cytoplasmic membrane protein, MexB, appears to function as the xenobiotic-exporting subunit, whereas the MexA and OprM proteins are supposed to function as the membrane fusion protein and the outer membrane channel protein, respectively. Computer-aided hydropathy analyses of MexB predicted the presence of up to 17 potential transmembrane segments. To verify the prediction, we analyzed the membrane topology of MexB using the alkaline phosphatase gene fusion method. We obtained the following unique characteristics. MexB bears 12 membrane spanning segments leaving both the amino and carboxyl termini in the cytoplasmic side of the inner membrane. Both the first and fourth periplasmic loops had very long hydrophilic domains containing 311 and 314 amino acid residues, respectively. This fact suggests that these loops may interact with other pump subunits, such as the membrane fusion protein MexA and the outer membrane protein OprM. Alignment of the amino- and the carboxyl-terminal halves of MexB showed a 30% homology and transmembrane segments 1, 2, 3, 4, 5, and 6 could be overlaid with the segments 7, 8, 9, 10, 11, and 12, respectively. This result suggested that the MexB has a 2-fold repeat that strengthen the experimentally determined topology model. This paper reports the structure of the pump subunit, MexB, of the MexA,B-OprM efflux pump assembly. This is the first time to verify the topology of the resistant-nodulation-division efflux pump protein.     

Membrane topology of the Salmonella enterica serovar Typhimurium Group B O-antigen translocase Wzx

The O-antigen translocase, Wzx, is involved in translocation of bacterial polysaccharide repeat units across the cytoplasmic membrane, and is an unusually diverse, highly hydrophobic protein, with high numbers of predicted alpha-helical transmembrane segments (TMS). The Salmonella enterica serovar Typhimurium Group B O-antigen Wzx was an ideal candidate for topological study as the O-antigen gene cluster is one of only a few that have been well characterized. The topology profile prediction for this protein was determined using five programs, with different recognition parameters, which consistently predict that 12 TMS are present. A membrane topology model was constructed by analysis of lacZ and phoA gene fusions at randomly selected and targeted fusion sites within wzx. Enzyme activity of these, and full-length C-terminal fusion proteins, confirmed the 12-TMS topology for this Wzx, and also indicated that the C-terminus was located within the cytoplasm, which is consistent with the predicted topology.     

Membrane topology of the GltS Na+/glutamate permease of Escherichia coli

The GltS Na+/glutamate permease of Escherichia coli is the most extensively studied member of the ESS family of bacterial glutamate:Na+ symporters. This paper presents the membrane topology analysis of the GltS with translational alkaline phosphatase and beta-galactosidase gene fusions generated by TnphoA, nested deletions and targeted fusions. The topology model suggested by the translational fusion technique is compared with the MemGen model and discussed in detail.     

The membrane topology of the Rhizobium meliloti C4-dicarboxylate permease (DctA) as derived from protein fusions with Escherichia coli K12 alkaline phosphatase (PhoA) and β-galactosidase (LacZ)

The Rhizobium meliloti dctA gene encodes the C₄-dicarboxylate permease which mediates uptake of C₄-dicarboxylates, both in free-living and symbiotic cells. Based on the hydrophobicity of the DctA protein, 12 putative membrane spanning regions were predicted. The membrane topology was further analysed by isolating in vivo fusions of DctA to Escherichia coli alkaline phosphatase (PhoA) and E. coli β-galactosidase (LacZ). Of 10 different fusions 7 indicated a periplasmic and 3 a cytoplasmic location of the corresponding region of the DctA protein. From these data a two-dimensional model of DctA was constructed which comprised twelve transmembrane α-helices with the amino-terminus and the carboxy-terminus located in the cytoplasm. In addition, four conserved amino acid motifs present in many eukaryotic and prokaryotic transport proteins were observed.     

Urate/alpha-ketoglutarate exchange in avian basolateral membrane vesicles

Membrane transport pathways for transcellular secretion of urate across the proximal tubule were investigated in avian kidney. The presence of coupled urate/alpha-ketoglutarate exchange was investigated in basolateral membrane vesicles (BLMV) by [(14)C]urate and [alpha-(3)H]ketoglutarate flux measurements. An inward Na gradient induced accumulation of alpha-ketoglutarate of sufficient magnitude to suggest a Na-dicarboxylate cotransporter. An inward Na gradient also induced concentrative accumulation of urate in the presence of alpha-ketoglutarate but not in its absence, suggesting urate/alpha-ketoglutarate exchange. alpha-Ketoglutarate-dependent stimulation of urate uptake was not observed in brush-border membrane vesicles. An outward urate gradient induced concentrative accumulation of alpha-ketoglutarate. alpha-Ketoglutarate-coupled urate uptake was specific for alpha-ketoglutarate, Cl dependent, and insensitive to membrane potential. alpha-Ketoglutarate-coupled urate uptake was inhibited by increasing p-aminohippurate (PAH) concentrations, and alpha-ketoglutarate-coupled PAH uptake was observed. alpha-Ketoglutarate-coupled PAH uptake was inhibited by increasing urate concentrations, and an outward urate gradient induced concentrative accumulation of PAH. These results suggest a Cl-dependent, alpha-ketoglutarate-coupled anion exchange mechanism as a pathway for active urate uptake across the basolateral membrane of urate-secreting proximal tubule cells.     

Gene fusion analysis of membrane protein topology: a direct comparison of alkaline phosphatase and beta-lactamase fusions.

To compare two approaches to analyzing membrane protein topology, a number of alkaline phosphatase fusions to membrane proteins were converted to beta-lactamase fusions. While some alkaline phosphatase fusions near the N terminus of cytoplasmic loops of membrane proteins have anomalously high levels of activity, the equivalent beta-lactamase fusions do not. This disparity may reflect differences in the folding of beta-lactamase and alkaline phosphatase in the cytoplasm.     

Topology of the Escherichia coli uhpT sugar-phosphate transporter analyzed by using TnphoA fusions.

The Escherichia coli uhpT protein catalyzes the active transport of sugar-phosphates by an obligatory exchange mechanism. To examine its transmembrane topology, we isolated a collection of uhpT-phoA fusions encoding hybrid proteins of different lengths from the N terminus of UhpT fused to alkaline phosphatase by using transposon TnphoA. These fusions displayed different levels of alkaline phosphatase activity, although comparable levels of full-length UhpT-PhoA proteins were produced in maxicells of both high- and low-activity fusions. The full-length protein species were unstable and were degraded to the size of the alkaline phosphatase moiety in the case of a high-activity fusion or to small fragments in the case of a low-activity fusion. The enzyme activity present in low-activity fusions appeared to result from export of a small proportion of the fusion proteins to the periplasmic space. Although fusions were not obtained in all predicted extramembranous loops, the deduced topology of UhpT was consistent with a model of 12 membrane-spanning regions oriented with the amino and carboxyl termini in the cytoplasm.