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Interrelationships between protein phosphorylation and oligomerization in transport and chemotaxis via the Escherichia coli mannitol phosphotransferase system
Q INSTITUTPASTEUR/ELSEVIER Paris 1992
Res. Microbiol. 1992, 143, ll3-116
MINI-REVIEW
Interrelationships between protein phosphorylation and oligomerization in transport and chemotaxis via the Escherichia coli mannitol phosphotransferase system G. R. Jacobson
Department of Biology, Boston University, Boston, MA 02215 (USA) SUMMARY
The membrane-bound enzymes II of the bacterial carbohydrate phosphotransferase system (PTS) are multifunctional: they are required for the transport, phosphorylation and chemotactic sensing of their substrates. An oligomer (minimally a dimer) of at least one of these PTS permeases, the Escherichia coil mannitol permease, appears to be necessary for this protein to optir,lally carry out these functions. Much indirect evidence is consistent with this hypothesis, and recent experiments show that transport and phosphorylation of, and chemotaxis to, mannito| in E. coil involves an intersubunit phosphotransfer reaction, which can only occur in a protein oligomer. Membrane topological studies of the mannitol permease also argue in favour of an oligomeric structure in the membrane which may be necessary to form the hydrophilic channel through which mannitol must traverse the phospholipid bilayer. The possibility that the oligomerization state of the mannitol permease is a target for regulation of its activity in vivo is proposed, but has not yet been explored experimentally. Key'-words: Phosphotransferase, Permease, Mannitol, Oligomerization; System, Enzymes II, E. coil, Regulation, Membrane transport proteins.
Despite much recent work on the structures and functions of membrane transport proteins, in cases in which a single polypeptide comprises the transport protein it is often very difficult to determine its functional oligomeric structure (monomer, dimer, etc.) in the membrane. Molecular models of how such proteins carry out transport obviously depend critically on this knowledge. We and others have been studying the mannitol-specific enzyme II (E!I mtl or mannitol permease) of the Escherichia coil
Receiv,=.dJuly 20, 1991.
phosphoenolpyruvate(PEP)-dependent carbohydrate phosphotransferase system (PTS) (for reviews, cf. Postma and Lengeler, 1985; Meadow et al., 1990). This 68-kDa protein consists of two distinct domains of roughly equal size: an N-terminal, membrane-bound domain that traverses the cytoplasmic membrane at least 6 times, and a C-terminal cytoplasmic domain involved in the phosphorylation functions of the protein (reviewed in Robillard and Lolkema, 1988; Jacobson and Stephan, 1989).
G. R. J A C O B S O N
114
Recent work has established that the mannitol permease catalyses the concomitant transport and phosphorylation of mannitol via two sequentially phosphorylated sites in the Cterminal domain, His554 and Cys384 (Pas and Robillard, 1988 ; Grisafi et al., 1989; Mueller et al., 1990; Pas et al., 1991). These studies showed that His554 is phosphorylated by the general PTS phosphocarrier protein, phospho-HPr (which itself is phosphorylated by enzyme I of PTS and PEP), while Cys384 is phosphorylated by intraenzyme phosphotransfer from Cys384 which then donates the phosphogroup to mannitol as it is transported into the cell via the membrane-bound domain. Much work has established that, under physiological conditions at least, rapid substrate uptake by mannitol permease and other PTS permeases is tightly coupled to substrate phosphorylation (Postma and Lengeler, 1985 ; Meadow et al., 1990). Moreover, it has recently been shown that the mannitol transport/phosphorylation activity of mannitol permease is necessary for its ability to act as a chemotactic receptor for mannitol (Scholle et al., 1991), probably because it is the phosphorylation state of HPr that is sensed by the chemotactic machinery (Che proteins) io chemotactic responses to PTS substrates (Grfibl et al., 1990). Therefore, elucidation of the mechanism of phosphotransfer catalysed by PTS permeases is essential in understanding their roles in transport, catalysis and chemoreception. In the remainder of this article, I discuss the evidence that it is an oligomer of the mannitol permease that is necessary for (or is at least much more active in) carrying out these functions, and propose that the oligomerization state of the permease is a potential target for the regulation of its activity. That an oligomer of the mannitol permease might be important for activity was first shown by Leonard and Saier (1983) who showed a second-order dependence of its ability to catalyse mannitol :mannitol-l-phosphate phosphoex-
change on the concentration of purified protein in reconstituted membrane vesicles. Subsequently, other workers showed that mild detergent extraction of membranes led to solubilized enzyme II mtt that was at least partly oligomeric (possibly dimeric) (Roossien and Robillard, 1984; Stephan and Jacobson, 1986 ; Khandekar and Jacobson, 1989), and that PEPdependent phosphorylation of mannitol was also much more readily catalysed by an oligomer (Robillard and Blaauw, 1987). Recent experiments provide a rationale for these observations. It has now been unequivocally established that phosphotransfer between I4is554 and Cys384 can proceed in an intersubunit fashion, i.e. from His554 on one polypeptide to Cys384 on another. This was first observed in vitro using a full-length permease in which Cys384 had been inactivated, and a deletion protein in which His554 was missing: phosphotransfer from the former to the latter was directly demonstrated in mixtures containing these two proteins, 32p-PEP, enzyme I and HPr (Stephan et al., 1989). More recently, van Weeghel et al. (1991) also observed in vitro phosphotransfer between two permease proteins constructed by site-directed mutagenesis that lacked His554 and Cys384, respectively. Finally, we have extended this observation to the in vivo, membrane-bound, mannitol permease. When two inactive mutant proteins, His554--, Ala, and Cys384 --, His, were expressed in the same cell, mannitol permease activity was reconstituted to nearly the value expected from the presumed amount of heterodimer in the membrane (Weng, Q.P. and Jacobson, G.R., unpublished observations). The observations summarized above thus provide a rationale for the requirement of an oligomer for mannitol permease activities: an oligomer may be necessary for intersubunit phosphotransfer. Much less obvious is why this should be the case. It would seem to be a much simpler evolutionary solution to have designed .............
HPr PEP
:
hi,,lidine prolein. piiosphoenolpyruvate.
PTS
=
phosphotransferase system.
OLIGOMERIL4TION IN MANNITOL PERMEASE REGULATION AND FUNCTION
a protein that was, at most, phosphorylated at a single site. Indeed, m a n y PTS permeases are phosphorylated at only one site, but in these cases the enzyme 1I is smaller, and there is a separate, soluble enzyme III necessary for sugar transport which contains the phosphoacceptor site corresponding to His554 of the mannitol perincase (reviewed in Saier et al., 1988). At least one of these enzymes III, the one specific for glucose, has been shown to have i m p o r t a n t regulatory functions in the cell, which probably explains its structural and genetic independence (Saier, !989). One possibility for an active oligomer in the case of the m a n n i t o l permease could relate to regulation of its activity. It has been shown that the concentration o f mannitol, the phosphorylation state of the permease a n d the concentrations o f ions such as Pi and Mg 2+ , all influence the stability of the oligomer in vitro (Stephan and Jacobson, 1986; Khandekar and Jacobson, 1989; Lolkema and Robillard, 1990). If this is also the case in vivo, then a simple way of regulating the mannitol permease activity would be to destabilize or dissociate the oligomer for inhibition, a n d to stabilize or reassociate it for activation. For example, an energy-starved cell would have a relatively high intracellular concentration of Pi, which has been shown to promote oligomerization of the mannitol permease (Stephan and Jacobson, 1986; K h a n d e k a r and Jacobson, 1989). Under these conditions, the low concentrations of the permease in an uninduced cell would be optimally " p r i m e d " for mannitol transport, and thus for m a n n i t o l operon induction and mannitol taxis, should the cell encounter this hexitol in the medium. In contrast, in cells rapidly growing on glucose, for example, the small uninduced levels of the mannitol permease may be largely monomeric (because their activity is not needed) until the carbon source is depleted. It should be emphasized, however, that this hypothesis is almost entirely speculative at present, and needs to be tested experimentally. Finally, very recent studies on the topology of the mannitol permease using m t l A - p h o A gene fusions also provide a possible rationale for an oligomeric structure for this protein in the mem-
115
brane (Sugiyama et aL, 1991), These experiments gave strong evidence for 6 membrane-spanning regions in the N-terminal half of the protein. Only 2 of these regions (or possibly 3, depending on whether or not the N-terminal 20 residues span the m e m b r a n e ; Saier et al., 1989) are highly amphipathic and thus likely to comprise the hydrophilic channel through which mannitol must pass into the cell. Since simple geometric considerations argue that a bundle of at least 5, and more likely 6, transmembrane, amphipathic s-helices would be required to form a channel large enough to admit mannitol though the membrane (Jacobson and Stephan, 1989), an oligomer (minimally a dimer) might be necessary for such a structure. In this case, amphipathic helices from both subunits in a homodimer could contribute to a channel of svfficient size. Interestingly, the purified mannitol permease has been shown to bind tightly only one molecule of mannitol per dimeric equivalent (Pas et al., 1988), a result that would be predicted if the substrate/translocation site were composed of helices from both subunits of a dimer. It will therefore be fascinating in the near future to further explore the roles of oligomerization in the structure, activities and regulation of the bacterial PTS enzymes II.
References
Grisafi, P.L., Scholle, A., Sugiyama, J., Briggs, C., Jacobson, G.R. & Lengeler, J.W. 0989), Deletion n~-.ltants of the Escherichia coli Kl2 mannitol permease: dissection of transport-phosphorylation, phosphoexchange, and mannitol-binding activities. J. Bact., 171, 2719-2727. Gr/ibl, G., Vogler, A.P. & Lengeler, J.W. (1990), Involvement of the histidine protein (HPr) of the phosphotransferase system in chemotactic signaling of Escherichia coli K-12. J. Bact., 172, 5871-5876. Jacobson, G.R. & Stephan, M.M. (1989), Structural and functional domains of the mannitol-specific enzyme 11 of the E. coli phosphoenolpyruvate-dependent phosphotransferase system. FEMS Microbiol. Rev., 63, 25-34. Khandekar, S.S. & Jacobson, G.R. (1989), Evidence for two distinct conformations of the Escherichia coli lnannitol permease that are important for its t~ansport and phosphorylation functions. J. Cell Biochem., 39, 207-216. Leonard, J.E. & Saier, M.H. Jr. (1983), Mannitol-specific enzyme 1| of the bacterial phosphotransferasesystem.-
116
G. R. J A C O B S O N
II. Reconstitution of vectorial transphosphorylation in phospholipid vesicles. J. bioL Chem., 258, 10757-10760. Lolkema, J.S. & Robillard, G.T. (1990), Subunit structure and activity of the mannitol-specific enzyme I1 of the Eseherichia coil phosphoenolpyruvate-dependent phosphotransferase system solubilized in detergent. Biochemistry, 29, 10120-10125. Meadow, N.D., Fox, D.K. & Roseman, S. (1990), The bacterial phosphoenolpyruvate: glycose phosphotransfcrase system. Ann. Rev. Biochem., 59, 497542. MueUer, E.G., Khandekar, S.S., Knowles, J.R. & Jacobson, G.R. (1990), Stereochemical course of the reactions catalyzed by the bacterial phosphoenolpyruvate: mannitol phosphotransferase system. Biochemistry, 29, 6892-6896. Pas, H.H., Meyer, G.H., Kruizinga, W.H,, Tamminga, K.S., van Weeghel, R.P. & Robillard, G.T. (1991), s~Phospho-NM R demonstration of phosphocysteine as a catalytic intermediate on the Escherichia coil phosphotransferase system Ell TM. J. biol. Chem., 266, 6690-6692. Pas, H.H. & Robillard, G.T. (1988), S-phosphocysteine and phosphohistidine are the intermediates in the phosphoenolpyruvate-dependent mannitol transport catalyzed by the E. coil Ell ma. Biochemistry, 27, 5835-5839. Pas, H.H., ten Hoeve-Duurkens, R.H. & Robillard, G.T. (1988), Bacterial phosphoenolpyruvate-dependent phosphotransferase system: mannitol-specific Eli contains two phosphoryl-binding site per monomer and one high-affinity mannitol-binding site per dimer. Biochemistry, 27, 5520-5525. Postma, P.W. & Lengeler, J. (1985), Phosphoenolpyruvate: carbohydrate phosphotransferase system in bacteria. MicrobioL Rev., 49, 232-269. Robillard, G.T. & Blaauw, M. (1987), Enzyme II of the Escherichia coil phosphoenolpyruvate-dependent phosphotransferase system: protein-protein and protein-phospholipid interactions. Biochemistry, 26, 5796-5803. Robillard, G.T. & Lolkema, J.S. (1988), Enzymes 1I of the phosphoenolpyruvate-dependent sugar phosphotransferase system: a review of their structure and mechanism of sugar transport. Biochim. biophys. Acta (Amst.), 947, 493-519.
Roossien, F.F. & Robillard, G.T. (1984), Mannitol-specific carrier from Escherichia coil phosphoenolpyruvatedependent phosphotransferase system can be extracted as a dimer from the membrane. Biochemistry, 23, 5682-5685. Saier, M.H. Jr. (1989), Protein phosphorylation and allosteric control of inducer exclusion and catabolite repression by the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Microbiol. Rev., 53, 109-120. Saier, M.H. Jr., Werner, P.K. & Miiller, M. (1989), Insertion of proteins into bacterial membranes: mechanism, characteristics, and comparisons with the eucaryotic process. MicrobioL Rev., 53, 333-366. Saier, M.H. Jr., Yamada, M., Erni, B., Suda, K., Lengeler, J., Ebnei, R., Argos, P., Rak, B., Schnetz, K., Lee, C.A., Ste.vart, G.C., Breidt, F. Jr., Waygood, E.B., Peri, K.G. & Doolittle, R.F. (1988), Sugar permeases of the bacterial phosphoenolpyruvatedependent phosphotransferase system : sequence comparisons. FASEB J., 2, 199-208. Scholle, A., Elder, J., Weng, Q.P., Lengeler, J. & Jacobson, G. (1991), Coupling of mannitol chemotaxis to transport and phosphorylation by the mannitol permease of E. coil Abstr. Annam Meeting. Amer. Soc. MicrobioL, 142, 238. Stephan, M.M. & Jacobson, G.R. (1986), Subunit interactions of the Escherichia coil mannitol permease: correlation with enzymic activities. Biochemistry, 25, 4046-4051. Stephan, M.M., Khandekar, S.S. & Jacobson, G.R. (1989), Hydrophilic C-terminal domain of the Escherichia coil mannitol permease: phosphorylation, functional independence and evidence for intersubunit phosphotransfer. Biochemistry 28, 7941-7946. Sugiyama, J., Mahmoodian, S. & Jacobson, G. (1991), Membrane :.opologyanalysis of Escheriehia coil mannitol perntease by using a nested-deletion method to create mtlA-phoA fusions. Proc. nat. Acad. Sei. (Wash.), 88, 9603-9607. Van Weeghel, R.P., van der Hock, Y.Y., Pas, H.H., Elferink, M., Keck, W. & Robillard, G.T. (1991), Details of mannitol transport elucidated by site-specificmutagenesis and complementation of phosphorylation site mutants of the phosphoenolpyruvate-dependent mannitol-specific phosphotransferase system. Biochemistry, 30, 1768-1773.
Res. Microbiol. 1992, 143, ll3-116
MINI-REVIEW
Interrelationships between protein phosphorylation and oligomerization in transport and chemotaxis via the Escherichia coli mannitol phosphotransferase system G. R. Jacobson
Department of Biology, Boston University, Boston, MA 02215 (USA) SUMMARY
The membrane-bound enzymes II of the bacterial carbohydrate phosphotransferase system (PTS) are multifunctional: they are required for the transport, phosphorylation and chemotactic sensing of their substrates. An oligomer (minimally a dimer) of at least one of these PTS permeases, the Escherichia coil mannitol permease, appears to be necessary for this protein to optir,lally carry out these functions. Much indirect evidence is consistent with this hypothesis, and recent experiments show that transport and phosphorylation of, and chemotaxis to, mannito| in E. coil involves an intersubunit phosphotransfer reaction, which can only occur in a protein oligomer. Membrane topological studies of the mannitol permease also argue in favour of an oligomeric structure in the membrane which may be necessary to form the hydrophilic channel through which mannitol must traverse the phospholipid bilayer. The possibility that the oligomerization state of the mannitol permease is a target for regulation of its activity in vivo is proposed, but has not yet been explored experimentally. Key'-words: Phosphotransferase, Permease, Mannitol, Oligomerization; System, Enzymes II, E. coil, Regulation, Membrane transport proteins.
Despite much recent work on the structures and functions of membrane transport proteins, in cases in which a single polypeptide comprises the transport protein it is often very difficult to determine its functional oligomeric structure (monomer, dimer, etc.) in the membrane. Molecular models of how such proteins carry out transport obviously depend critically on this knowledge. We and others have been studying the mannitol-specific enzyme II (E!I mtl or mannitol permease) of the Escherichia coil
Receiv,=.dJuly 20, 1991.
phosphoenolpyruvate(PEP)-dependent carbohydrate phosphotransferase system (PTS) (for reviews, cf. Postma and Lengeler, 1985; Meadow et al., 1990). This 68-kDa protein consists of two distinct domains of roughly equal size: an N-terminal, membrane-bound domain that traverses the cytoplasmic membrane at least 6 times, and a C-terminal cytoplasmic domain involved in the phosphorylation functions of the protein (reviewed in Robillard and Lolkema, 1988; Jacobson and Stephan, 1989).
G. R. J A C O B S O N
114
Recent work has established that the mannitol permease catalyses the concomitant transport and phosphorylation of mannitol via two sequentially phosphorylated sites in the Cterminal domain, His554 and Cys384 (Pas and Robillard, 1988 ; Grisafi et al., 1989; Mueller et al., 1990; Pas et al., 1991). These studies showed that His554 is phosphorylated by the general PTS phosphocarrier protein, phospho-HPr (which itself is phosphorylated by enzyme I of PTS and PEP), while Cys384 is phosphorylated by intraenzyme phosphotransfer from Cys384 which then donates the phosphogroup to mannitol as it is transported into the cell via the membrane-bound domain. Much work has established that, under physiological conditions at least, rapid substrate uptake by mannitol permease and other PTS permeases is tightly coupled to substrate phosphorylation (Postma and Lengeler, 1985 ; Meadow et al., 1990). Moreover, it has recently been shown that the mannitol transport/phosphorylation activity of mannitol permease is necessary for its ability to act as a chemotactic receptor for mannitol (Scholle et al., 1991), probably because it is the phosphorylation state of HPr that is sensed by the chemotactic machinery (Che proteins) io chemotactic responses to PTS substrates (Grfibl et al., 1990). Therefore, elucidation of the mechanism of phosphotransfer catalysed by PTS permeases is essential in understanding their roles in transport, catalysis and chemoreception. In the remainder of this article, I discuss the evidence that it is an oligomer of the mannitol permease that is necessary for (or is at least much more active in) carrying out these functions, and propose that the oligomerization state of the permease is a potential target for the regulation of its activity. That an oligomer of the mannitol permease might be important for activity was first shown by Leonard and Saier (1983) who showed a second-order dependence of its ability to catalyse mannitol :mannitol-l-phosphate phosphoex-
change on the concentration of purified protein in reconstituted membrane vesicles. Subsequently, other workers showed that mild detergent extraction of membranes led to solubilized enzyme II mtt that was at least partly oligomeric (possibly dimeric) (Roossien and Robillard, 1984; Stephan and Jacobson, 1986 ; Khandekar and Jacobson, 1989), and that PEPdependent phosphorylation of mannitol was also much more readily catalysed by an oligomer (Robillard and Blaauw, 1987). Recent experiments provide a rationale for these observations. It has now been unequivocally established that phosphotransfer between I4is554 and Cys384 can proceed in an intersubunit fashion, i.e. from His554 on one polypeptide to Cys384 on another. This was first observed in vitro using a full-length permease in which Cys384 had been inactivated, and a deletion protein in which His554 was missing: phosphotransfer from the former to the latter was directly demonstrated in mixtures containing these two proteins, 32p-PEP, enzyme I and HPr (Stephan et al., 1989). More recently, van Weeghel et al. (1991) also observed in vitro phosphotransfer between two permease proteins constructed by site-directed mutagenesis that lacked His554 and Cys384, respectively. Finally, we have extended this observation to the in vivo, membrane-bound, mannitol permease. When two inactive mutant proteins, His554--, Ala, and Cys384 --, His, were expressed in the same cell, mannitol permease activity was reconstituted to nearly the value expected from the presumed amount of heterodimer in the membrane (Weng, Q.P. and Jacobson, G.R., unpublished observations). The observations summarized above thus provide a rationale for the requirement of an oligomer for mannitol permease activities: an oligomer may be necessary for intersubunit phosphotransfer. Much less obvious is why this should be the case. It would seem to be a much simpler evolutionary solution to have designed .............
HPr PEP
:
hi,,lidine prolein. piiosphoenolpyruvate.
PTS
=
phosphotransferase system.
OLIGOMERIL4TION IN MANNITOL PERMEASE REGULATION AND FUNCTION
a protein that was, at most, phosphorylated at a single site. Indeed, m a n y PTS permeases are phosphorylated at only one site, but in these cases the enzyme 1I is smaller, and there is a separate, soluble enzyme III necessary for sugar transport which contains the phosphoacceptor site corresponding to His554 of the mannitol perincase (reviewed in Saier et al., 1988). At least one of these enzymes III, the one specific for glucose, has been shown to have i m p o r t a n t regulatory functions in the cell, which probably explains its structural and genetic independence (Saier, !989). One possibility for an active oligomer in the case of the m a n n i t o l permease could relate to regulation of its activity. It has been shown that the concentration o f mannitol, the phosphorylation state of the permease a n d the concentrations o f ions such as Pi and Mg 2+ , all influence the stability of the oligomer in vitro (Stephan and Jacobson, 1986; Khandekar and Jacobson, 1989; Lolkema and Robillard, 1990). If this is also the case in vivo, then a simple way of regulating the mannitol permease activity would be to destabilize or dissociate the oligomer for inhibition, a n d to stabilize or reassociate it for activation. For example, an energy-starved cell would have a relatively high intracellular concentration of Pi, which has been shown to promote oligomerization of the mannitol permease (Stephan and Jacobson, 1986; K h a n d e k a r and Jacobson, 1989). Under these conditions, the low concentrations of the permease in an uninduced cell would be optimally " p r i m e d " for mannitol transport, and thus for m a n n i t o l operon induction and mannitol taxis, should the cell encounter this hexitol in the medium. In contrast, in cells rapidly growing on glucose, for example, the small uninduced levels of the mannitol permease may be largely monomeric (because their activity is not needed) until the carbon source is depleted. It should be emphasized, however, that this hypothesis is almost entirely speculative at present, and needs to be tested experimentally. Finally, very recent studies on the topology of the mannitol permease using m t l A - p h o A gene fusions also provide a possible rationale for an oligomeric structure for this protein in the mem-
115
brane (Sugiyama et aL, 1991), These experiments gave strong evidence for 6 membrane-spanning regions in the N-terminal half of the protein. Only 2 of these regions (or possibly 3, depending on whether or not the N-terminal 20 residues span the m e m b r a n e ; Saier et al., 1989) are highly amphipathic and thus likely to comprise the hydrophilic channel through which mannitol must pass into the cell. Since simple geometric considerations argue that a bundle of at least 5, and more likely 6, transmembrane, amphipathic s-helices would be required to form a channel large enough to admit mannitol though the membrane (Jacobson and Stephan, 1989), an oligomer (minimally a dimer) might be necessary for such a structure. In this case, amphipathic helices from both subunits in a homodimer could contribute to a channel of svfficient size. Interestingly, the purified mannitol permease has been shown to bind tightly only one molecule of mannitol per dimeric equivalent (Pas et al., 1988), a result that would be predicted if the substrate/translocation site were composed of helices from both subunits of a dimer. It will therefore be fascinating in the near future to further explore the roles of oligomerization in the structure, activities and regulation of the bacterial PTS enzymes II.
References
Grisafi, P.L., Scholle, A., Sugiyama, J., Briggs, C., Jacobson, G.R. & Lengeler, J.W. 0989), Deletion n~-.ltants of the Escherichia coli Kl2 mannitol permease: dissection of transport-phosphorylation, phosphoexchange, and mannitol-binding activities. J. Bact., 171, 2719-2727. Gr/ibl, G., Vogler, A.P. & Lengeler, J.W. (1990), Involvement of the histidine protein (HPr) of the phosphotransferase system in chemotactic signaling of Escherichia coli K-12. J. Bact., 172, 5871-5876. Jacobson, G.R. & Stephan, M.M. (1989), Structural and functional domains of the mannitol-specific enzyme 11 of the E. coli phosphoenolpyruvate-dependent phosphotransferase system. FEMS Microbiol. Rev., 63, 25-34. Khandekar, S.S. & Jacobson, G.R. (1989), Evidence for two distinct conformations of the Escherichia coli lnannitol permease that are important for its t~ansport and phosphorylation functions. J. Cell Biochem., 39, 207-216. Leonard, J.E. & Saier, M.H. Jr. (1983), Mannitol-specific enzyme 1| of the bacterial phosphotransferasesystem.-
116
G. R. J A C O B S O N
II. Reconstitution of vectorial transphosphorylation in phospholipid vesicles. J. bioL Chem., 258, 10757-10760. Lolkema, J.S. & Robillard, G.T. (1990), Subunit structure and activity of the mannitol-specific enzyme I1 of the Eseherichia coil phosphoenolpyruvate-dependent phosphotransferase system solubilized in detergent. Biochemistry, 29, 10120-10125. Meadow, N.D., Fox, D.K. & Roseman, S. (1990), The bacterial phosphoenolpyruvate: glycose phosphotransfcrase system. Ann. Rev. Biochem., 59, 497542. MueUer, E.G., Khandekar, S.S., Knowles, J.R. & Jacobson, G.R. (1990), Stereochemical course of the reactions catalyzed by the bacterial phosphoenolpyruvate: mannitol phosphotransferase system. Biochemistry, 29, 6892-6896. Pas, H.H., Meyer, G.H., Kruizinga, W.H,, Tamminga, K.S., van Weeghel, R.P. & Robillard, G.T. (1991), s~Phospho-NM R demonstration of phosphocysteine as a catalytic intermediate on the Escherichia coil phosphotransferase system Ell TM. J. biol. Chem., 266, 6690-6692. Pas, H.H. & Robillard, G.T. (1988), S-phosphocysteine and phosphohistidine are the intermediates in the phosphoenolpyruvate-dependent mannitol transport catalyzed by the E. coil Ell ma. Biochemistry, 27, 5835-5839. Pas, H.H., ten Hoeve-Duurkens, R.H. & Robillard, G.T. (1988), Bacterial phosphoenolpyruvate-dependent phosphotransferase system: mannitol-specific Eli contains two phosphoryl-binding site per monomer and one high-affinity mannitol-binding site per dimer. Biochemistry, 27, 5520-5525. Postma, P.W. & Lengeler, J. (1985), Phosphoenolpyruvate: carbohydrate phosphotransferase system in bacteria. MicrobioL Rev., 49, 232-269. Robillard, G.T. & Blaauw, M. (1987), Enzyme II of the Escherichia coil phosphoenolpyruvate-dependent phosphotransferase system: protein-protein and protein-phospholipid interactions. Biochemistry, 26, 5796-5803. Robillard, G.T. & Lolkema, J.S. (1988), Enzymes 1I of the phosphoenolpyruvate-dependent sugar phosphotransferase system: a review of their structure and mechanism of sugar transport. Biochim. biophys. Acta (Amst.), 947, 493-519.
Roossien, F.F. & Robillard, G.T. (1984), Mannitol-specific carrier from Escherichia coil phosphoenolpyruvatedependent phosphotransferase system can be extracted as a dimer from the membrane. Biochemistry, 23, 5682-5685. Saier, M.H. Jr. (1989), Protein phosphorylation and allosteric control of inducer exclusion and catabolite repression by the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Microbiol. Rev., 53, 109-120. Saier, M.H. Jr., Werner, P.K. & Miiller, M. (1989), Insertion of proteins into bacterial membranes: mechanism, characteristics, and comparisons with the eucaryotic process. MicrobioL Rev., 53, 333-366. Saier, M.H. Jr., Yamada, M., Erni, B., Suda, K., Lengeler, J., Ebnei, R., Argos, P., Rak, B., Schnetz, K., Lee, C.A., Ste.vart, G.C., Breidt, F. Jr., Waygood, E.B., Peri, K.G. & Doolittle, R.F. (1988), Sugar permeases of the bacterial phosphoenolpyruvatedependent phosphotransferase system : sequence comparisons. FASEB J., 2, 199-208. Scholle, A., Elder, J., Weng, Q.P., Lengeler, J. & Jacobson, G. (1991), Coupling of mannitol chemotaxis to transport and phosphorylation by the mannitol permease of E. coil Abstr. Annam Meeting. Amer. Soc. MicrobioL, 142, 238. Stephan, M.M. & Jacobson, G.R. (1986), Subunit interactions of the Escherichia coil mannitol permease: correlation with enzymic activities. Biochemistry, 25, 4046-4051. Stephan, M.M., Khandekar, S.S. & Jacobson, G.R. (1989), Hydrophilic C-terminal domain of the Escherichia coil mannitol permease: phosphorylation, functional independence and evidence for intersubunit phosphotransfer. Biochemistry 28, 7941-7946. Sugiyama, J., Mahmoodian, S. & Jacobson, G. (1991), Membrane :.opologyanalysis of Escheriehia coil mannitol perntease by using a nested-deletion method to create mtlA-phoA fusions. Proc. nat. Acad. Sei. (Wash.), 88, 9603-9607. Van Weeghel, R.P., van der Hock, Y.Y., Pas, H.H., Elferink, M., Keck, W. & Robillard, G.T. (1991), Details of mannitol transport elucidated by site-specificmutagenesis and complementation of phosphorylation site mutants of the phosphoenolpyruvate-dependent mannitol-specific phosphotransferase system. Biochemistry, 30, 1768-1773.