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J. Mol. Microbiol. Biotechnol. (2001) 3(3): 381-384. JMMB Symposium PTS Regulation of Galactoside Transport 381 Regulation of Galactoside Transport by the PTS Masayuki Kuroda1,2, Thomas H. Wilson3, and Tomofusa Tsuchiya1,* Model for the Regulation of non-PTS Sugar Transport System by the PTS 1 Since the PTS had been discovered (Kundig et al., 1964), various mutants defective in sugar utilization have been isolated and extensively characterized in enteric bacteria such as E. coli and Salmonella typhimurium. Some of mutations were mapped in genes for enzyme I and HPr, common components of the PTS. Although it had been suggested that the PTS is responsible for the uptake of many carbohydrates, it has been later found that there are several sugars (lactose, melibiose, maltose, and glycerol) which are not transported by the PTS. These sugars are now generally called non-PTS sugars. Interestingly, it has been demonstrated that mutations in enzyme I and HPr resulted in loss of utilization of not only PTS-sugars but also non-PTS sugars. This finding directed researchers to study the interaction between the utilization system of nonPTS sugars and the PTS. Regulation of catabolic enzyme synthesis appeared to be through the inhibition of inducer sugar uptake and cAMP synthesis. Based on numerous observations, a mechanism for PTS-mediated regulation has been proposed (Saier and Feucht, 1975; Saier and Stiles, 1975; Saier, 1977; Figure 1.). In this model, IIAGlc is a central regulatory protein whose phosphorylation state is crucial for the regulatory functions. According to this model, non-PTS sugar transport systems such as lactose transport system and melibiose transport system are negatively controlled by non-phosphorylated form of IIAGlc (inducer exclusion), whereas adenylate cyclase is activated by phosphorylated form of IIAGlc. Gene Research Center, Okayama University, Tsushima, Okayama 700-8530, Japan 2 Current address: Department of Microbiology, Kochi Medical School, Kohasu, Okoh, Nankoku, Kochi 783-8505, Japan 3 Department of Cell Biology, Harvard Medical School, Boston, MA, 021152, USA Abstract Inducer exclusion, regulation of activity of transporter, is mediated by phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). To elucidate the molecular mechanism of the inducer exclusion, numerous biochemical and genetic studies have been performed. It is now well known that non-phosphorylated IIAGlc inhibits the transport via direct binding to the transporter. Analysis of inducer exclusion resistant mutants of lactose transporter and melibiose transporter in Escherichia coli and Salmonella typhimurium revealed amino acid residues that are involved in the interaction with IIAGlc. It is concluded that there are multiple interaction sites for IIAGlc in these transporters. Introduction Since the glucose effect and diauxic growth were documented in 1940s (Monod, 1942; Gale, 1943), numerous studies have been performed to understand the molecular mechanisms behind those phenomena. It has been demonstrated that uptake of various sugars was inhibited in the presence of glucose in Escherichia coli. At the same time, it has been shown that cAMP and its receptor protein (CRP) are involved in the synthesis of many inducible carbohydrate catabolic enzymes (Pastan and Perlman, 1970). Gene expression of catabolic enzymes seems to be regulated by two distinct mechanisms: one is termed catabolite repression, which is at the level of gene expression via cAMP and CRP; the other is termed inducer exclusion, which is at the level of transport activity. It is now well known that phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is involved in the both mechanisms (Saier, 1989; Postma et al., 1993). In this review, the molecular mechanisms of inducer exclusion, regulation of activity of transporter, especially in the lactose transport system and the melibiose transport system, are discussed. Accepted September 29, 2000. *For correspondence. Email [email protected]; Tel. +81-86-251-7957; Fax. +81-86-2517957. © 2001 Horizon Scientific Press Figure 1. Proposed model for the regulation of melibiose utilization. IIAGlc of PTS is a central regulatory protein. Its phosphorylated form activates adenylate cyclase whereas non-phosphorylated form inhibits the melibiose transporter. aMG; methyl-a-glucoside, CRP; cAMP receptor protein, 2DG; 2-deoxyglucose, mel; melibiose. Further Reading Caister Academic Press is a leading academic publisher of advanced texts in microbiology, molecular biology and medical research. 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Since phosphorylated IIAGlc did not bind to the lactose transporter, it was concluded that only non-phosphorylated IIAGlc could bind to the transporter and inhibit the activity. It has been estimated that there were 1 to 1.5 molecules of IIAGlc bound per a molecule of the lactose transporter. Among four non-PTS sugar transport systems, direct binding of IIAGlc to the transporter has been shown except in the melibiose system. However, since it has been shown that inclusion of IIAGlc in membrane vesicles inhibited the melibiose transporter, interaction between melibiose transport protein and IIAGlc is expected to be similar to that in the other three systems. Identification of Mutations Which Tolerate the Regulation by IIAGlc Figure 2. Vertical view of a putative a-helix of the carboxyl-terminal region of the melibiose transporter of S. typhimurium. A region from Gly-437 to Thr-454 is shown. The region from Gly-437 to Arg-452 is predicted to form an a-helix structure. Amino acid residues which are involved in the interaction with IIAGlc are circled. Direct Evidence for the Interaction Between Sugar Transporter and IIAGlc Component of Glucose-PTS The role of IIAGlc in the inducer exclusion is now well established based on various experimental observations. In the study of the lactose transport system, considerable biochemical evidence for the interaction between the transport protein and IIAGlc have been obtained. The first evidence for the involvement of IIAGlc in the regulation of the lactose transport system has been reported by Dills et al. (1982). They demonstrated that the partially purified preparation of IIAGlc loaded into E. coli membrane vesicles possessing high activity of lactose transport inhibited the lactose uptake. In addition, in the presence of enzyme I and HPr, intravesicular PEP effaced the inhibition. The results have been later confirmed by Misko et al. (1987). It has been reported that in the presence of methyl-aglucoside, a non-metabolizable analog of glucose, the activity of lactose transport decreased in membrane vesicles. When the vesicles were prepared from ptsI-, ptsH, or crr-negative strain, methyl-a-glucoside did not inhibit the lactose transport. In this study, it has been also shown that melibiose and galactose transport activities reduced in the presence of intravesicular IIAGlc. Further evidence for the involvement of IIAGlc in the transport regulation of non-PTS sugar has been obtained in the lactose transport system by Osumi and Saier (1982; 1982). They used lacY gene cloned into a multicopy plasmid, overproduced the lactose transporter and studied the binding of purified IIAGlc to the membrane fragment from this strain. They demonstrated that IIAGlc directly bound to the membrane containing the overproduced lactose transporter, and the binding activity was enhanced Although inducer exclusion is observed in wild-type ptsI+, ptsH+ strains, the inhibition is much stronger in ptsI-leaky strain. Using such inducer exclusion-hypersensitive strain, Saier and coworkers isolated mutants of E. coli and S. typhimurium in which individual non-PTS sugar transport system was no longer sensitive to inducer exclusion (Saier et al., 1978). These mutations were mapped in or closely linked to the gene(s) responsible for the metabolism of each non-PTS sugar. This report touched off the extensive studies on the domains or amino acid residues of the transporters that interact with IIAGlc. With respect to the lactose transport system, Nelson et al. (1983) first reported that a mutant lactose carrier with triple amino acid substitutions in N-terminal region exhibited no binding to IIAGlc. They concluded that the N-terminal cytoplasmic portion of the LacY was probably a binding site of IIAGlc . Wilson et al. reported the amino acid substitutions in lactose transport mutants of E. coli resistant to inducer exclusion (Wilson et al. , 1990). It was demonstrated that Ala-198 and Ser-209 were replaced in these mutants. The results suggested that cytoplasmic central loop of the lactose carrier protein was also a binding site of IIAGlc. Hoischen et al. (1996) later confirmed the results of Wilson et al. by using direct binding assay. Hoischen et al. reported that Pro-192 which is located in cytoplasmic central loop of the protein was also important for the regulation by the PTS. In contrast to the results of Nelson et al. some deletion mutations in N-terminal region had little effect on the binding. It might be due to the difference between point mutation and deletion. Seok et al. (1997) examined the binding activity of LacY mutants in which 6 contiguous His residues were inserted into each loop or N-terminus, or C-terminal deletion to IIAGlc. Insertion mutations of cytoplasmic loops IV/V and VI/VII (which is called cytoplasmic central loop as indicated above) resulted in retention of sugar transport (i.e. binding) activity, but loss of IIAGlc binding. Interestingly, a mutant with insertion in periplasmic loop VII/VIII also exhibited similar properties. Since IIAGlc is a cytoplasmic soluble protein and binds to the LacY from cytoplasmic side, involvement of this periplasmic loop in IIAGlc binding remains to be examined. Sondej et al. (1999) further examined the cytoplasmic loops IV/V and VI/VII by Cys- PTS Regulation of Galactoside Transport 383 scanning mutagenesis. It has been shown that replacement of either Val-132, Arg-135, or Arg-142 in or adjacent to loop IV/V by Cys resulted in decreased binding with no effect on the transport activity. In loop VI/VII, mutation of either Thr-196, Val-197, Asn-199, Gly-202, Asn-204 or Ser-206 had similar effects. With respect to the melibiose transport system, we isolated inducer exclusion resistant mutants in MelB of S. typhimurium and identified the following substitutions of amino acid residues: Asp-438 with Tyr, Arg-441 with Ser, and Ile-445 with Asn (Kuroda et al., 1992). Recently, we have identified different amino acid substitution (Arg-441 with Gly) in another mutant (unpublished result). The replacement of Ile-445 by Asn was identified in more than half of independently isolated mutants, suggesting that Ile445 was the most important residue for interaction with the IIAGlc. Amino acid residues substituted in the mutants were located in carboxyl-terminal cytoplasmic region and the region containing these residues was predicted to form an α-helix structure. According to the helical wheel analysis, these residues were on the same side of the α-helix (Figure 2) To further evaluate the role of the C-terminal region of the melibiose transporter, site-directed mutants were constructed and characterized. Mutants of Asp-449 and Arg-452, which are on the same side of the α-helix, exhibited the transport activity fairly resistant to inducer exclusion (unpublished result). Surprisingly, any amino acid replacements of Ile-445 (including replacements by Val or Leu residues) tested resulted in resistance to the PTS regulation. Since prediction of the secondary structure by the method of Chou and Fasman revealed that these substitutions did not significantly affect the secondary structure of the C-terminal region, this result suggests that side chain of Ile-445 is critical for the binding of IIAGlc (unpublished result). We introduced a series of C-terminal truncations in the E. coli MelB (each mutants lacked 16, 19, 20, 21, 22, 23, or 24 amino acid residues from the C-terminus) and examined regulation by the PTS. The inhibition was 65, 65, 63, 46, and 19% in the MelB mutants lacking 16, 19, 20, 21, 22 amino acid residues, respectively, whereas inhibition in wild type was 84 %. Mutants lacking 23 residues and lacking 24 residues exhibited very little transport activity. These results suggest that the C-terminal region of MelB is essential not only for the regulation of activity by the IIA Glc but also for the transport activity itself (unpublished result). Deduction of Interaction Sites in Target Protein of IIAGlc In the maltose transport system, analysis of inducer exclusion resistant mutants has identified amino acid residues in MalK, which were involved in IIAGlc interaction (Dean et al., 1990; Kühnau et al., 1991). Additionally, analysis of three-dimensional structure of E. coli glycerol kinase-IIAGlc complex revealed amino acid residues at the contact sites (Hurley et al., 1993). Based on studies summarized above, some reports postulated the consensus sequences of proteins that interact with IIAGlc (Dean et al., 1990; Titgemeyer et al., 1994; Sondej et al., 1999). Especially, REGION I and II proposed by Sondej et al. are also present in HPr, which contacts with IIAGlc in phospho-transfer reaction of the PTS. Among four proteins (LacY, MelB, MalK, and GlpK) that interact with IIAGlc, MelB is considered to be a little different one, since some of the conserved residues in REGION II are absent. In fact, our recent experiments demonstrated that deletion of Arg-215 to Leu-220, which corresponds to the latter half of the REGION II, did not affect the PTS regulation and transport activity in the melibiose carrier of S. typhimurium. In agreement with this result, REGION II is not conserved well in four MelBs of enteric bacteria, sequences of which have been reported so far. Furthermore, E. coli PTS can regulate MelBs from S. typhimurium and K. pneumoniae (unpublished result). Therefore, we proposed that C-terminal region of the MelB protein is at least one of the interaction sites with IIAGlc, instead of the REGION II. Discussion Although several reports revealed amino acid residues that were involved in IIAGlc binding, consensus sequences with complete match seems not to be completely deduced so far. As shown in these studies, several portions of target proteins are considered to be required to form regulatory complex with IIAGlc. To solve the contact sites, threedimensional structure of target protein-IIAGlc complex remains to be studied. Through these studies, it has been demonstrated that IIAGlc is one of the most important proteins that regulates various events occurring in Enterobacteriae. References Dean, D. A., Reizer, J., Nikaido, H. and Saier, M. H. Jr. 1990. Regulation of maltose transport system of Escherichia coli by glucose-specific enzyme III of the phosphoenolpyruvate-sugar phosphotransferase system. Characterization of inducer exclusion-resistant mutants and reconstitution of inducer exclusion in proteoliposomes. J. Biol. Chem. 265: 21005-21010 Dills, S. S., Schmidt, M. R., and Saier, M. H. Jr. 1982. Regulation of lactose transport by phosphoenolpyruvate-sugar phosphotransferase system in membrane vesicles of Escherichia coli. J. Cell Biochem. 18: 239-244 Gale, E. F. 1943. Factors influencing the enzymatic activities of bacteria. Bacteriol. Rev. 7: 139-173 Hoischen, C., Levin, J., Pitaknarongphorn, S., Reizer, J., and Saier , M. H. Jr. 1996. J. Bacteriol. Involvement of the central loop of the lactose permease of Escherichia coli in its allosteris regulation by the glucosespecific enzyme IIA of the phosphoenolpyruvate-dependent phosphotransferase system. J. Bacteriol. 178: 6082-6086 Hurley, J. H., Faber, H. R., Worthylake, D., Meadow, N. D., Roseman, S. and Remington, S. J. 1993. Structure of the regulatory complex of Escherichia coli IIIGlc with glycerol kinase. Science. 259: 673-677 Kühnau, S., Reyes, M., Sievertsen, A., Shuman, H. A., and Boos, W. 1991. The activities of the Escherichia coli MalK protein in maltose transport, regulation, and inducer exclusion can be separated by mutations. J. Bacteriol. 173: 2180-2186 Kundig, W., Sudhamoy, G., and Roseman, S. 1964. Phosphate bound to histidine in a protein as an intermediate in a novel phosphotransferase system. Proc. Natl. Acad. Sci. USA 52: 1067-1074 Kuroda, M., de Waard, S., Mizushima, K., Tsuda, M., Postma, P., and Tsuchiya, T. 1992. Resistance of the melibiose carrier to inhibition by the phosphotransferase system due to substitutions of amino acid residues in the carrier of Salmonella typhimurium. J. Biol. Chem. 267: 18336-18341 Misko, T. P., Mitchell, W. J., Meadow, N. 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Coordinate regulation of adenylate cyclase and carbohydrate permeases by the phosphoenolpyruvate: sugar phosphotransferase system in Salmonella typhimurium. J. Biol. Chem. 250: 7078-7080 Saier, M. H. Jr. and Stiles, C. D. 1975. Molecular dynamics in membrane biology. Springer-Verlag, New York. Saier, M. H. Jr., Straud, H., Massman, L. S., Judice, J. J., Newman,M. J., and Feucht, B. U. 1978. Permease-specific mutations in Salmonella typhimurium and Escherichia coli that release the glycerol, maltose, melibiose and lactose transport systems from regulation by the phosphoenolpyruvate: sugar phosphotransferase system. J. Bacteriol. 133: 1358-1367 Soek, Y. –J., Sun, J., Kaback, H. R., and Peterkofsky, A. 1997. Topology of allosteric regulation of lactose permease. Proc. Natl. Acad. Sci. USA 94: 13515-13519 Sondej, M., Sun, J., Seok, Y. –J., Kaback, H. R. and Peterkofsky, A. 1999. 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