328
6 Ih F O R U M I N M I C R O B I O L O G Y
TuorFns, B., SarKar, H.K., KABACK,H.R. & LorJIsH,H.F. (1987), Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney and [3-pancreatic islet cells. Cell. 55, 281-290. Von HEJJnE, G. (1988), Transcending the impenetrable: how proteins come to terms with membranes. Biochim. biophys. Acta (Amst.), 947, 307-333. Von HEIJr~E,G. (1987), Sequence analysis in molecular biology - - treasure trove or trivial pursuit. Academic Press, London. Watstst ev, A.R. (1988), The dynamics of the glucose transporter. Trends in Biochem. Sci., 13, 226-231. WarFrs, S.H., Roc;owsKv,J., GRINSTEAD0J., ALTENBUCHNER,J. • ScHMrl'T,R. (1983), The tetracycline resistance determinants of RPI and Tn1721: nucleotide sequenceanalysis. Nucl. Acids Res., II, 6089-6140. YazvlJ, H., S,,OTa-NllVA,S., SmmaMOTO,T., KanazAwa,H., FUTa,,M. & TSUCmVA,T. (1984), Nucleotide sequence of the melB gene and characteristics of deduced amino acid sequence of the melibiose carrier in Escherichia coll. J. biol. Chem., 259, 4320.4326. Za,sn(~, C.-C., Durano, M.-C., JL~nJEan, R. & JosEt, F. (1989), Molecular and genetical analysis of the fructose-glucosetransport system in the cyanobacterium Synechoeyslis PCC6803. Mot. Mierobiol., 3, 1221-1229. Zua~rs'rEin, D. & DWVER, D.M. (1985), Protonmotive force-driven active transport of D-glucose and L-prnline in the protozoan parasite Leishmania donovani. Proe. hal. Acad. Sci. (Wash.), 82, 1716-1720. ZII.aEgSTE,N, D., DWVER, D.M., MaTTHEI, S. & HORUK, R. (1986), Identification and biochemical characterization of the plasma membrane glucose transporter o~" Leishmania donovani. J. biol. Chem., 261, 15053-15057. This researchis supported by SERC, MRC, The SmithKlineFoundation and The WellcomeTrust.
T H E MELIBIOSE CARRIER OF E S C H E R I C H I A COLI
M.C. Botfield, D.M. Wilson and T. Hastings Wilson Department o f Cellular and Molecular Physiology, Harvard Medical School Boston, M A 02115 (USA)
Substrate specificity and kinetics. The melibiose carrier of Escherichia colt is an example of a substrate-cation cotransport system. It has an unusually broad cation specificity, being able to transport ~t- and [3-galactosides with H +, Na ÷ or Li + (Tsuchiya and Wilson, 1978). Unexpectedly, direct measurements of cation uptake into cells upon the addition of sugar have shown different cation specificities for different sugars: ~-galactosides can utilize H +, Na + or Li + for cotransport while [~-galactosides can utilize only
Na + or Li + (Wilson and Wilson, 1988). For example, lactose accumulation in the above study was stimulated 40-fold by the addition of Na + and 100-fold by the addition o f Li +. However, no accumulation occurred in the absence of Na + or Li +. Direct measurement of H + movement indicated that the melibiose carrier was unable to eatalyse H +-coupled cotransport with any of the tested [3-galaetosides. These observations lead to the conclusion that there is a functional interaction (direct or allosteric) between the sugar and the cation-binding sites.
BACTERIAL
Leblanc and his colleagues have studied the sugar binding and transport characteristics of the melibiose carrier in the presence of Na +, Li + and H ~ (Bassilana et al., 1987) and have provided evidence in favour of the model shown in figure 1. They found that Na + and Li + increased the binding affinity of ~-galactosides (a-p-nitrophenylgalactoside) for the carrier in deenergized membrane vesicles. In addition, they have shown that imposing a membrane potential (inside negative) greatly increases the Vma~ of entry for melibiose in the presence of Na * and Li + . Mutants.
Mutants of the melibiose carrier with altered cation and/or sugar specificity have been isolated by several laboratories. One mutant of particular importance was isolated by Tsuchiya and coworkers based on the ability to grow on melibiose in the presence of normally inhibitory (10 mM) concentrations of Li ÷ (Niiya et al., 1982). As a result of a single point mutation (Pro-122 to serine), the carrier lost the ability to cotransport melibiose with protons and showed an absolute requirement for Na + or Li + for cotransport and growth on melibiose (Yazyu et aL, 1985).
TRANSPORT
329
A number of mutants have also been constructed by site-specific mutagenesis (table I). Sarkar and Kaback (see Kabaek, 1988) have changed Glu-361 to glycine or aspartate. With both substitutions transport was reduced to 2-6 % of normal. Subsequently an additional mutation of Glu-361 to alanine was also reported (Pourcher et aL, 1989). An interesting observation in these mutants was that the K, for TMG transport was normal or subnormal and the binding of ~-pNPG was as tight or tighter than normal. This suggests that the Vrua~ for transport was affected by the site change. Rece.~tly sugar recognition mutants were isolated (Botfield and Wilson, 1988) by growing cells on melibiose in the presence of a high concentration of L.on-metabolizable inhibitor (thiomethylgalactoside). Twenty-three unique amino acid substitutions resulting in pheeotypes that could grow in the presence of this inhibitor were isolated. These mutations were found to be clustered into 4 distinct regions (see fig. 2). An interesting observation was that most of these sugar recognition mutants also showed changes in cation specificity (Li + produced distinctly less inhibition of growth on melibiose compared with the parent), it was concluded that there is an intimate relationship between the cation-recognition site and sugar-recognition site in the protein.
OUT
IN C 'l
Nn+C Mel - ~
Mel Na+C FIG.
I. --
--C
C No + ~
Mel
~, C N+ Mel
Scheltlalic representalion o f reaclions in volved in Na +-melibiose colratTsporl,
Modified from Pourcher et al., 1989.
330
6 th F O R U M I N M I C R O B I O L O G Y
TABLE I. - - S i t e - s p e c i f i c m u t a n t s o f t h e mdibios¢ carrier. Amino acid substitution
T M G transport (% normal)
Ref.
6 2 10 10 0 (**) 0 (**) 0 0 0 0 60-100
Kaback, 1988 Kaback, 1988 Pourcher el al., 1989 (*) (*) (*) (*) (*) (*) Pourcher et al., 1989 ........
Glu-361 --, Gly Glu-361 --, Asp Glu-361 --, Ala Deletion o f Thr-2 Deletion of Thr-2-Str-6 Deletion of Thr-2-Ala-10 Asp-31 ~ Lys Lys-373 ~ Asp Asp-31 --, Lys: Lys-373 --, Asp His-94 --, Arg His- 198,213,318,357,442,456 ~ Arg
(*) Botfield and Wilson (unpublished results). I**) Little or no protein could be detected in the membrane, indicating failure of insertion or increased proleolytie scavenging.
Pourcher et aL, 1989 converted each of the 7 histidines to arginines using sitespecific mutagenesis. In most cases no changes were observed in the transport rates. However, conversion of His-94 to arginine dramatically interfered with transport activity and the cell was unable to catalyse H + - , N a ÷- or Li+-coupled facilitated diffusion reactions in de-energized membrane vesicles. This indicates that 6 o f the 7 histidine residues are not essential for transport,
while His-94 is critical for normal carrier function. The possible role of the N-terminal portion of the melibiose carrier was investigated by introducing short deletions in the first 10 amino acids (Botfi¢ld and Wilson, unpublished observations). Using site-specific mutagenesis, 3 different deletions were introduced. A deletion o f Thr-2 resulted in loss of 80-90 % of transport activity. Deletion of Thr-2 through Ser-6 (a deletion of
PERIPLASM
FIG. 2. - - Topo/ogica! model of the melibiose carrier protein showing the posidon o f the TMG-resistant mutants.
From Botfield, PhD Thesis, Harvard University, i989.
BACTERIAL
5 amino acids) resulted in complete loss of activity. Similarly, a deletion of Thr-2 through A l a - l l (a deletion of 10 amino acids) resulted in an inactive carrier. When assayed for the presence of carrier in the membrane using an antibody specific for the C-terminal portion of the molecule, it was found that only a small amount o f carrier could be detected in the membrane when 5 or 10 amino acids were deleted f r o m the N-terminal region o f the carrier. It was concluded that the N-terminal region of the melibiose carrier may be essential for normal insertion into the membrane. One might suggest that charged amino acids play an important role in
TRANSPORT
331
anchoring the polypeptide in the membrane, ahhough this role has never been defined. Changing a charged amino acid to one of the opposite charge (Asp-31 to Lys; Lys-373 to Asp) resulted in the exclusion of the carrier from the membrane and loss of biological activity (Botfield and Wilson, unpublished observations). Similarly, the simultaneous introduction o f both changes in the same carrier protein also resulted in failure of membrane insertion. More work is required to understand the role o f these charged amino acids in the membrane insertion process and subsequent stability o f the inserted protein.
References.
BASSILANA,M., POURCHER,T. & LEBLANC,G. (1987), Facilitated diffusion properties of melibiose permease of E. coli membrane vesicles. J. biol. Chem., 262, 16865-16879. BOT~IELD,M.C. & WILSON,T.H. (1988), Mutations that simultaneously alter both sugar and cation specificity in the melibiose of Escherichia coll. J. biol. Chem., 263, 12909-12915. KAeAcg,H.R. (1988), Site-directed mutagenesis and ion-gradient driven active transport: on the path of the proton. Ann. Rev. Physiol., 50, 243-256. KAWAXAM T. AXlZAWAY., ISmKAWAT. SmMAMO'¢O,T., TsuoA, M. &TsucmvA, T. (1988), Amino acid substitut ons and alterat on in cation specificity in the melibiose carrier of Escherichia coll. J. biol. Ct2em., 263, 14276-14280. NIIYA, S., YAMASAKI,K., WILSON,T.H. & TSUCHIYA,T. (1982), Altered cation coupling to melibiose transport in mutants of Escherichia coll. J. biol. Chem.. 257, 8902-8906. POURCI'IER, T., BASSILANA,M., SARKAR, H.K., KAnACr, R.H. & LEBLANC,G. (1989), Na ÷/melibiose symport mechanism of Escherichia cell: kinetic and molecular properties. Phil. Trans. royal Soc. London, 1989 (in press). Tsocmv^, T. & WILSON,T.H. (1978), Cation-sugar cotransport m the melibiose transport system of Escherichia coll. Membrane Biochem., 2, 63-79. WILSON D.M. & W:LSON,T.H. (1987), Cation specificity for sugar substrates of the melibiose carrier in Escherichia coll. Biochim. biophys. Acta (Amst.), 904, 191-200. Y^zvv H , SH O'rA,$., FUTAhM. & TSUCHIVA T. (1985), Alteration in cation specificity of the melib ose transport cart er of Escberichia cell due to replacement of proline 122 with serine. J. Bart., 162, 933-937.
6 Ih F O R U M I N M I C R O B I O L O G Y
TuorFns, B., SarKar, H.K., KABACK,H.R. & LorJIsH,H.F. (1987), Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney and [3-pancreatic islet cells. Cell. 55, 281-290. Von HEJJnE, G. (1988), Transcending the impenetrable: how proteins come to terms with membranes. Biochim. biophys. Acta (Amst.), 947, 307-333. Von HEIJr~E,G. (1987), Sequence analysis in molecular biology - - treasure trove or trivial pursuit. Academic Press, London. Watstst ev, A.R. (1988), The dynamics of the glucose transporter. Trends in Biochem. Sci., 13, 226-231. WarFrs, S.H., Roc;owsKv,J., GRINSTEAD0J., ALTENBUCHNER,J. • ScHMrl'T,R. (1983), The tetracycline resistance determinants of RPI and Tn1721: nucleotide sequenceanalysis. Nucl. Acids Res., II, 6089-6140. YazvlJ, H., S,,OTa-NllVA,S., SmmaMOTO,T., KanazAwa,H., FUTa,,M. & TSUCmVA,T. (1984), Nucleotide sequence of the melB gene and characteristics of deduced amino acid sequence of the melibiose carrier in Escherichia coll. J. biol. Chem., 259, 4320.4326. Za,sn(~, C.-C., Durano, M.-C., JL~nJEan, R. & JosEt, F. (1989), Molecular and genetical analysis of the fructose-glucosetransport system in the cyanobacterium Synechoeyslis PCC6803. Mot. Mierobiol., 3, 1221-1229. Zua~rs'rEin, D. & DWVER, D.M. (1985), Protonmotive force-driven active transport of D-glucose and L-prnline in the protozoan parasite Leishmania donovani. Proe. hal. Acad. Sci. (Wash.), 82, 1716-1720. ZII.aEgSTE,N, D., DWVER, D.M., MaTTHEI, S. & HORUK, R. (1986), Identification and biochemical characterization of the plasma membrane glucose transporter o~" Leishmania donovani. J. biol. Chem., 261, 15053-15057. This researchis supported by SERC, MRC, The SmithKlineFoundation and The WellcomeTrust.
T H E MELIBIOSE CARRIER OF E S C H E R I C H I A COLI
M.C. Botfield, D.M. Wilson and T. Hastings Wilson Department o f Cellular and Molecular Physiology, Harvard Medical School Boston, M A 02115 (USA)
Substrate specificity and kinetics. The melibiose carrier of Escherichia colt is an example of a substrate-cation cotransport system. It has an unusually broad cation specificity, being able to transport ~t- and [3-galactosides with H +, Na ÷ or Li + (Tsuchiya and Wilson, 1978). Unexpectedly, direct measurements of cation uptake into cells upon the addition of sugar have shown different cation specificities for different sugars: ~-galactosides can utilize H +, Na + or Li + for cotransport while [~-galactosides can utilize only
Na + or Li + (Wilson and Wilson, 1988). For example, lactose accumulation in the above study was stimulated 40-fold by the addition of Na + and 100-fold by the addition o f Li +. However, no accumulation occurred in the absence of Na + or Li +. Direct measurement of H + movement indicated that the melibiose carrier was unable to eatalyse H +-coupled cotransport with any of the tested [3-galaetosides. These observations lead to the conclusion that there is a functional interaction (direct or allosteric) between the sugar and the cation-binding sites.
BACTERIAL
Leblanc and his colleagues have studied the sugar binding and transport characteristics of the melibiose carrier in the presence of Na +, Li + and H ~ (Bassilana et al., 1987) and have provided evidence in favour of the model shown in figure 1. They found that Na + and Li + increased the binding affinity of ~-galactosides (a-p-nitrophenylgalactoside) for the carrier in deenergized membrane vesicles. In addition, they have shown that imposing a membrane potential (inside negative) greatly increases the Vma~ of entry for melibiose in the presence of Na * and Li + . Mutants.
Mutants of the melibiose carrier with altered cation and/or sugar specificity have been isolated by several laboratories. One mutant of particular importance was isolated by Tsuchiya and coworkers based on the ability to grow on melibiose in the presence of normally inhibitory (10 mM) concentrations of Li ÷ (Niiya et al., 1982). As a result of a single point mutation (Pro-122 to serine), the carrier lost the ability to cotransport melibiose with protons and showed an absolute requirement for Na + or Li + for cotransport and growth on melibiose (Yazyu et aL, 1985).
TRANSPORT
329
A number of mutants have also been constructed by site-specific mutagenesis (table I). Sarkar and Kaback (see Kabaek, 1988) have changed Glu-361 to glycine or aspartate. With both substitutions transport was reduced to 2-6 % of normal. Subsequently an additional mutation of Glu-361 to alanine was also reported (Pourcher et aL, 1989). An interesting observation in these mutants was that the K, for TMG transport was normal or subnormal and the binding of ~-pNPG was as tight or tighter than normal. This suggests that the Vrua~ for transport was affected by the site change. Rece.~tly sugar recognition mutants were isolated (Botfield and Wilson, 1988) by growing cells on melibiose in the presence of a high concentration of L.on-metabolizable inhibitor (thiomethylgalactoside). Twenty-three unique amino acid substitutions resulting in pheeotypes that could grow in the presence of this inhibitor were isolated. These mutations were found to be clustered into 4 distinct regions (see fig. 2). An interesting observation was that most of these sugar recognition mutants also showed changes in cation specificity (Li + produced distinctly less inhibition of growth on melibiose compared with the parent), it was concluded that there is an intimate relationship between the cation-recognition site and sugar-recognition site in the protein.
OUT
IN C 'l
Nn+C Mel - ~
Mel Na+C FIG.
I. --
--C
C No + ~
Mel
~, C N+ Mel
Scheltlalic representalion o f reaclions in volved in Na +-melibiose colratTsporl,
Modified from Pourcher et al., 1989.
330
6 th F O R U M I N M I C R O B I O L O G Y
TABLE I. - - S i t e - s p e c i f i c m u t a n t s o f t h e mdibios¢ carrier. Amino acid substitution
T M G transport (% normal)
Ref.
6 2 10 10 0 (**) 0 (**) 0 0 0 0 60-100
Kaback, 1988 Kaback, 1988 Pourcher el al., 1989 (*) (*) (*) (*) (*) (*) Pourcher et al., 1989 ........
Glu-361 --, Gly Glu-361 --, Asp Glu-361 --, Ala Deletion o f Thr-2 Deletion of Thr-2-Str-6 Deletion of Thr-2-Ala-10 Asp-31 ~ Lys Lys-373 ~ Asp Asp-31 --, Lys: Lys-373 --, Asp His-94 --, Arg His- 198,213,318,357,442,456 ~ Arg
(*) Botfield and Wilson (unpublished results). I**) Little or no protein could be detected in the membrane, indicating failure of insertion or increased proleolytie scavenging.
Pourcher et aL, 1989 converted each of the 7 histidines to arginines using sitespecific mutagenesis. In most cases no changes were observed in the transport rates. However, conversion of His-94 to arginine dramatically interfered with transport activity and the cell was unable to catalyse H + - , N a ÷- or Li+-coupled facilitated diffusion reactions in de-energized membrane vesicles. This indicates that 6 o f the 7 histidine residues are not essential for transport,
while His-94 is critical for normal carrier function. The possible role of the N-terminal portion of the melibiose carrier was investigated by introducing short deletions in the first 10 amino acids (Botfi¢ld and Wilson, unpublished observations). Using site-specific mutagenesis, 3 different deletions were introduced. A deletion o f Thr-2 resulted in loss of 80-90 % of transport activity. Deletion of Thr-2 through Ser-6 (a deletion of
PERIPLASM
FIG. 2. - - Topo/ogica! model of the melibiose carrier protein showing the posidon o f the TMG-resistant mutants.
From Botfield, PhD Thesis, Harvard University, i989.
BACTERIAL
5 amino acids) resulted in complete loss of activity. Similarly, a deletion of Thr-2 through A l a - l l (a deletion of 10 amino acids) resulted in an inactive carrier. When assayed for the presence of carrier in the membrane using an antibody specific for the C-terminal portion of the molecule, it was found that only a small amount o f carrier could be detected in the membrane when 5 or 10 amino acids were deleted f r o m the N-terminal region o f the carrier. It was concluded that the N-terminal region of the melibiose carrier may be essential for normal insertion into the membrane. One might suggest that charged amino acids play an important role in
TRANSPORT
331
anchoring the polypeptide in the membrane, ahhough this role has never been defined. Changing a charged amino acid to one of the opposite charge (Asp-31 to Lys; Lys-373 to Asp) resulted in the exclusion of the carrier from the membrane and loss of biological activity (Botfield and Wilson, unpublished observations). Similarly, the simultaneous introduction o f both changes in the same carrier protein also resulted in failure of membrane insertion. More work is required to understand the role o f these charged amino acids in the membrane insertion process and subsequent stability o f the inserted protein.
References.
BASSILANA,M., POURCHER,T. & LEBLANC,G. (1987), Facilitated diffusion properties of melibiose permease of E. coli membrane vesicles. J. biol. Chem., 262, 16865-16879. BOT~IELD,M.C. & WILSON,T.H. (1988), Mutations that simultaneously alter both sugar and cation specificity in the melibiose of Escherichia coll. J. biol. Chem., 263, 12909-12915. KAeAcg,H.R. (1988), Site-directed mutagenesis and ion-gradient driven active transport: on the path of the proton. Ann. Rev. Physiol., 50, 243-256. KAWAXAM T. AXlZAWAY., ISmKAWAT. SmMAMO'¢O,T., TsuoA, M. &TsucmvA, T. (1988), Amino acid substitut ons and alterat on in cation specificity in the melibiose carrier of Escherichia coll. J. biol. Ct2em., 263, 14276-14280. NIIYA, S., YAMASAKI,K., WILSON,T.H. & TSUCHIYA,T. (1982), Altered cation coupling to melibiose transport in mutants of Escherichia coll. J. biol. Chem.. 257, 8902-8906. POURCI'IER, T., BASSILANA,M., SARKAR, H.K., KAnACr, R.H. & LEBLANC,G. (1989), Na ÷/melibiose symport mechanism of Escherichia cell: kinetic and molecular properties. Phil. Trans. royal Soc. London, 1989 (in press). Tsocmv^, T. & WILSON,T.H. (1978), Cation-sugar cotransport m the melibiose transport system of Escherichia coll. Membrane Biochem., 2, 63-79. WILSON D.M. & W:LSON,T.H. (1987), Cation specificity for sugar substrates of the melibiose carrier in Escherichia coll. Biochim. biophys. Acta (Amst.), 904, 191-200. Y^zvv H , SH O'rA,$., FUTAhM. & TSUCHIVA T. (1985), Alteration in cation specificity of the melib ose transport cart er of Escberichia cell due to replacement of proline 122 with serine. J. Bart., 162, 933-937.
