The lactose transporter of E. coli

The lactose transporter (lac permease) of E. coli is without doubt the most intensively studied and best understood of the bacterial proton-linked sugar transporters. Since its sequence was reported in 1980 [233] prodigious efforts have been made to elucidate its molecular mechanism by site-directed mutagenesis and other means. These studies have recently been reviewed elsewhere [234,235] and so will not be discussed in detail here. The important question for the present Chapter is whether the protein is related to the sugar-transporter family and so has lessons to teach us about their mechanisms. The permease is a 417-residue protein, and, like the other 

Studies of other members of the family have also added support to the topological model shown in the Fig. 3. In particular, chemical labelling of the native and mutated tetracycline transporter has confirmed the cytoplasmic location of the N-terminus and the loop connecting transmembrane helices 2 and 3 [231,232]. Protease digestion experiments on this protein have also provided preliminary evidence for the cyto- 

The information obtained from the experiments described above can be used to construct models for the three-dimensional arrangement of the membrane-spanning helices within the transport proteins. One such model, which takes the diameter of an a-helix as 1.1 nm and seems to fit the measured dimensions of lac permease quite nicely, is illustrated in Fig. 5, although it must be emphasized that this is only one of 

In summary, studies on the human erythrocyte glucose transporter and other members of a large family of prokaryotic and eukaryotic sugar transporters have yielded 

Research in the author s laboratory is supported by the SERC, the MRC and the Wellcome Trust. The author is indebted to Dr. M.T. Cairns, Dr. A. Davies, Dr. A.F. Davies and Dr. P.J.F. Henderson for many helpful discussions and access to unpublished information. Mr. R.A.J. Preston and Mrs. J. Baldwin provided invaluable help in the preparation of the figures. 

---

Includes the newly solved structure of the lactose transporter of E. coli. 

Water soluble carbodiimides inhibit the transcription of supercoiled PM2 DNA with E. coli B RNA polymerase. " Modification of the lactose permease of E. Coli with carbodiimides shows a preference for hydrophobic carbodiimides (DCC) over hydrophilic carbodiimides. In carbodiimide modification of EmrE, a small multidrug transporter in E. Coli, DIPCD modification indicates that Glu-14 is the target of the reaction. Polynucleotides react with positively charged water soluble carbodiimides much faster than do the monomers, owing to their electrostatic effect. ... 

Secondary transporters are ancient molecular machines, common today in bacteria and archaea as well as in eukaryotes. For example, approximately 160 (of approximately 4000) proteins encoded by the E. coli genome appear to be secondary transporters. Sequence comparison and hydropathy analysis suggest that members of the largest family have 12 transmemhrane helices that appear to have arisen by duplication and fusion of a membrane protein with 6 transmemhrane helices. Included in this family is the lactose permease of E. coli. This symporter uses the H+ gradient... 

FIGURE 11-43 Structure of the lactose transporter (lactose permease) of E. coli. (a) Ribbon representation viewed parallel to the plane of the membrane shows the 12 transmembrane helices arranged in two nearly symmetrical domains shown in different shades of blue. In the form of the protein for which the crystal structure was determined, the substrate sugar (red) is bound near the middle of the membrane where it is exposed to the cytoplasm (derived from PDB ID 1 PV7). (b) The structural changes postulated to take place during one transport... 

The rate-limiting step in lactose transport (in E. coli) is A. Binding of a H+ outside the cell... 

Many active-transport systems couple the uphill flow of one ion or molecule to the downhill flow of another. These membrane proteins, called secondary transporters or cotransporters, can be classified as antiporters, symporters, and uni porters. Antiporters couple the downhill flow of one type of ion in one direction to the uphill flow of another in the opposite direction. Symporters move both ions in the same direction. Uniporters transport a substrate in either direction, determined by the concentration differences. Studies of the lactose permease from E. coli have been a source of insight into both the structures and the mechanisms of secondary transporters. 

A surprisingly high number of carriers are present constitutively in the cytoplasmic membrane. One may speculate about the advantages for bacteria to possess always carriers for several solutes. Possibly the possession of constitute carriers would enable an organism to scavenge intermediates leaked passively out of the cell and/or allow the organism to react rapidly to changes in the external medium. Besides constitutive transport systems inducible transport systems are also found in bacteria such as those for lactose transport in E. coli or for citrate transport in B. subtilis. 

Forster DL, Garcia ML, Newman ML, Patel L and Kaback HR (1982) Lactose-proton symport by purified lac carrier protein, Biochem. 21, 3634-3638. Ghazi A and Shechter E (1981) Lactose transport in E. coli cells. Dependence of kinetic parameters on the transmembrane electrical potential difference, Biochim.Biophys.Acta 645, 305-315. 

The gradients of H, Na, and other cations and anions established by ATPases and other energy sources can be used for secondary active transport of various substrates. The best-understood systems use Na or gradients to transport amino acids and sugars in certain cells. Many of these systems operate as symports, with the ion and the transported amino acid or sugar moving in the same direction (that is, into the cell). In antiport processes, the ion and the other transported species move in opposite directions. (For example, the anion transporter of erythrocytes is an antiport.) Proton symport proteins are used by E. coU and other bacteria to accumulate lactose, arabinose, ribose, and a variety of amino acids. E. coli also possesses Na -symport systems for melibiose as well as for glutamate and other amino acids. 

Surface Density of a Membrane Protein E. coli can be induced to make about 10,000 copies of the lactose transporter (Mr 31,000) per cell. Assume that E. coli is a cylinder 1 /am in diameter and 2 /am long. What fraction of the plasma membrane surface is occupied by the lactose transporter molecules Explain how you arrived at this conclusion. 

I The second purification procedure we examine illus- Vri trates an unusual approach to the purification of a membrane-bound protein. The lactose carrier protein of E. coli is normally tightly bound to the plasma membrane. This protein is involved in the active transport of the dissaccharide lactose across the cytoplasmic membrane. When lactose carrier protein is present, the intracellular concentration of lactose can achieve levels 1,000-fold higher than those found in the external medium. Ron Kaback devised a simple yet elegant procedure for the purification of this protein. 

Active transport of a solute against a concentration gradient also can be driven by a flow of an ion down its concentration gradient. Table 17.6 lists some of the active-transport systems that operate in this way. In some cases, the ion moves across the membrane in the opposite direction to the primary substrate (antiport) in others, the two species move in the same direction (symport). Many eukaryotic cells take up neutral amino acids by coupling this uptake to the inward movement of Na+ (see fig. 17.26c). As we discussed previously, Na+ influx is downhill thermodynamically because the Na+-K+ pump keeps the intracellular concentration of Na+ lower than the extracellular concentration and sets up a favorable electric potential difference across the membrane. Another example is the /3-galactosidc transport system of E. coli, which couples uptake of lactose to the inward flow of protons (see fig. 17.26Proton influx is downhill because electron-transfer reactions (or,... 

Membrane vesicles of E. coli that possess the lactose permease are preloaded with KC1 and are suspended in an equal concentration of NaCl. It is observed that these vesicles actively, although transiently, accumulate lactose if valinomycin is added to the vesicle suspension. No such active uptake is observed if KC1 replaces NaCl in the suspending medium. Explain these results in light of what you know about the mechanism of lactose transport and the properties of valinomycin. 

FIGURE 11-42 Lactose uptake in E. coli. (a) The primary transport of out of the cell, driven by the oxidation of a variety of fuels, establishes both a proton gradient and an electrical potential (inside negative) across the membrane. Secondary active transport of lactose into the cell involves symport of and lactose by the lactose transporter. The uptake of lactose against its concentration gradient is entirely dependent on this inflow of H", driven by the electrochemical gradient. 

Studies of the lactose transport activity of cytoplasmic membrane vesicles from E. coli have demonstrated the functional symmetry of the jS-D-galacto-sidase carrier in the organism. ... 

Becker et al. specifically inactivated the biotin transport system of E. coli using biotin p-nitrophenyl ester. The lactose transport protein of E. coli was labeled by AT-bromoacetyl-/8-D-galactopyranosylamine. In the only reported attempt to isolate an affinity labeled protein, glucose 6-isothiocyanate, which is an affinity label for the glucose transport system in human erythrocytes, gave enough nonspecific labeling of other membrane proteins to render identification of the transport protein difficult. 

To investigate the importance of this type of regulation of solute transport for other (non-phototrophic) bacteria, Rps. sphaeroides was provided with the ability to transport lactose via transformation with a plasmid containing the lactose operon from Escherichia coli and subsequent expression of the plasmid DNA with the aid of the antibiotic kanamycine (F.E. Nano, S. Kaplan, unpublished). The initial rate of lactose transport in Rps. sphaeroides was studied as a function of the light intensity and the magnitude of the proton motive force. 

---