Deletion mutants of the Escherichia coli K-12 mannitol permease: dissection of transport-phosphorylation, phospho-exchange, and mannitol-binding activities

Abstract

We have constructed a series of deletion mutations of the cloned Escherichia coli K-12 mtUA gene, which encodes the mannitol-specffic enzyme II of the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system. This membrane-bound permease consists of 637 amino acid residues and is responsible for the concomitant transport and phosphorylation of D-mannitol in E. coli. Deletions into the 3' end of mtL4 were constructed by exonuclease III digestion. Restriction mapping of the resultant plasmids identified several classes of deletions that lacked approximately 5% to more than 75% of the gene. Immunoblotting experiments revealed that many of these plasmids expressed proteins within the size range predicted by the restriction analyses, and all of these proteins were membrane localized, which demonstrated that none of the C-terminal half of the permease is required for membrane insertion. Functional analyses of the deletion proteins, expressed in an E. coli strain deleted for the chromosomal copy of mtlA, showed that all but one of the strains containing confirmed deletions were inactive in transport and PEP-dependent phosphorylation of mannitol, but deletions removing up to at least 117 amino acid residues from the C terminus of the permease were still active in catalyzing phospho exchange between mannitol 1-phosphate and mannitol. A deletion protein that lacked 240 residues from the C terminus of the permease was inactive in phospho exchange but still bound mannitol with high affinity. These experiments localize sites important for transport and PEP-dependent phosphorylation to the extreme C terminus of the mannitol permease, sites important for phospho exchange to between residues 377 and 519, and sites necessary for mannitol binding to the N-terminal 60% of the molecule. The results are discussed with respect to the fact that the mannitol permease consists of structurally independent N-and C-terminal domains. This phospho-exchange reaction (transphosphorylation) has been shown to occur vectorially in whole cells and membrane vesicles (35,36).The mannitol permease has been purified (11) and extensively characterized (7,10,30,32) been cloned and sequenced (17). These studies have revealed that the protein is made up of a single kind of polypeptide chain consisting of 637 amino acid residues (11, 17) that is highly specific for D-mannitol (12, 19) and may function as a dimer in the membrane (24,30,33,41). The topography of the mannitol permease with respect to the membrane has also been studied, and it is clear that this protein consists of a hydrophobic, membrane-bound Nterminal domain (residues 1 to 334) and a hydrophilic Cterminal domain (residues 335 to 637) that is exposed on the cytoplasmic surface of the inner membrane (9,17,42). In addition to carrying out the transport and phosphorylation functions described above, the mannitol permease functions as the primary chemotactic receptor for D-mannitol in E. coli (20).The domain structure of the mannitol permease and its multiple functions suggest...

Details of mannitol transport in Escherichia coli elucidated by site-specific mutagenesis and complementation of phosphorylation site mutants of the phosphoenolpyruvate-dependent mannitol-specific phosphotransferase system

“…The increase in specific activity to 12.0 nmol min-' nmol-I, compared to the 9.7 nmol min-' nmol-I wild-type Ell"', indicates that the domain carrying His-554 interacts with and influences the activity of the domain carrying the Cys-384. Grisafi et al (1989) observed increased activities in more than half of the deletion mutants in which segments extending from the C-terminus (637) into residue 520 had been deleted; however, since the Ell concentration of their mutants was not determined, quantitative statements could not be made.…”

“…Figure 2 presents a schematic representation of the dimer protein and shows the residues identified as the two phosphorylated intermediates. His-554, which is located in van Weeghel et al an EIII-like domain (Pas et al, 1988;Grisafi et al, 1989;van Weeghel et al, 1991), receives its phosphoryl group from phospho-HPr and passes it on to the second phosphorylation site, Cys-384. From there, the phosphoryl group is coupled to mannitol.…”

“…These data, together with the results of the construction and characterization of E . coli EII"' deletion mutants reported by Grisafi et al (1989), the complementation experiments between a truncated EII"' and a partially purified C-terminal domain , or an expressed large C-terminal fragment (White & Jacobson, I Abbreviations: PTS, phosphotransferase system; PEP, phosphoenolpyruvate; El, enzyme I; E I I m l '~n a~~~l c~b~' , mannitol-, N-acetylglucosamine-, glucose-, and P-glucoside-specific enzyme 11's of the PTS; Elllg'c, glucose-specific enzyme 111 of the PTS; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; decyl-PEG, decylpoly(ethy1ene glycol) 300; ISO, inside-out. 1990), confirm the location of P1 (His-554) on a fused EIII-like domain.…”

Expression and characterization of a structural and functional domain of the mannitol-specific transport protein involved in the coupling of mannitol transport and phosphorylation in the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli

“…EII"' can be divided into three structural and functional domains, which can be stably expressed, and biochemically and kinetically studied (Stephan & Jacobson, 1986;Stephan et al, 1989;Grisafi et al, 1989;White &Jacobson, 1990;van Weeghelet al, 1991a,c;Lolkemaet al, 1990;Pasetal., 1991). Primary sequences of the E11 single species or EII/EIII pairs indicate that several of these proteins are structurally and evolutionarily related (Ebner & Lengeler, 1988;Saier et al, 1988) and that they all consist of a hydrophobic transmembrane domain (IIC), a hydrophilic EIII-like domain (IIA) containing the P-HPr phosphoryl group accepting site, and another hydrophilic domain (IIB) containing the second phosphorylation site.…”

The membrane-bound domain of the phosphotransferase enzyme IImtl of Escherichia coli constitutes a mannitol translocating unit

“…Data have been presented showing that the site is localized in the Nterminal half of the protein (Grisafi et al, 1989). The present study focuses on the orientation of the binding site on enzyme 11"" with respect to the two sides of the membrane.…”