Lactose permease

Ovchinnikov 234 237) has shown that bovine rhodopsin, although quite different in amino acid sequence (348 residues), also forms seven transmembrane helices. This structural similarity between bacterial and mammalian light activated membrane proteins is remarkable. Since the two amino acid sequences have little in common it would appear that the necessary requirement is seven transmembrane helices to form a channel which is specific for proton migration. For example it has been suggested that a similar arrangement and function is performed by the lactose permease of E. coli237). 

Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H.R. and Iwata, S. (2003). Structure and mechanism of the lactose permease of Escherichia coli. Science, 301, 610-615... 

Voss, J., Hubbell, W. L., and Kaback, H. R. (1995) Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling application to the lactose permease of Escherichia coli. Proc. Natl. Acad. Sci. USA 92, 12300-12303. 

Kaback H. R. (1998). Structure/function studies on the lactose permease of Escherichia coli, Acta Physiol. Scand. Suppl. 643, 163, 21-33. 

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 bacterial lactose-transport protein (lactose permease) transports j3-galactosides, such as lactose, o-nitrophenyl-jS-galactoside, and isopropyl-jS-thiogalactoside. It does not transport galactosides with an a-glycosidic linkage. 

S. F. Permuth, and R. J. Brooker, Isolation and characterization of lactose permease mutants with an enhanced recognition of maltose and diminished recognition of cellobiose, J. Biol. Chem. 264 14698, 1989.)... 

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. 

Biichel, D.E., Gronebom, B Miiller-Hill, B. (1980). Sequence of the lactose permease gene. Nature (London)283, 541-545. 

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. ... 

Relatively few membrane transport proteins have been structurally characterized. Some of the best understood examples to date are the lactose permease and glycerol-3-phosphate transporter and the Ca + P-type ATPase (which is a primary ion pump). Other structurally well-characterized transport proteins include the bacterial porins and siderophore receptor proteins. In addition structures have been determined for several ion channels and additional bacterial transporters that are either directly relevant to or models for proteins important in drug transport. The following web sites maintained by Hartmut Michel and Stephen White respectively, contain exceptionally useful listings of these and other solved membrane protein structures and are frequently updated ... 

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 13.11. Action of Lactose Permease. Lactose permease pumps lactose into bacterial cells by drawing on the proton-motive force. The binding sites evert when a lactose molecule (L) and a proton (H+) are bound to external sites. After these species are released inside the cell, the binding sites again evert to complete the transport cycle. Lactose permease is an example of a symporter.

Pumping protons. Design an experiment to show that lactose permease can be reversed in vitro to pump protons. See answer... 

A.L. Green, E. J. Anderson, and R.J. Brooker. 2000. A revised model for the structure and function of the lactose permease Evidence that a face on transmembrane segment 2 is important for conformational changes J. Biol. Chem. 

The outward-facing conformation of E. coli lactose permease has a ... 

We labeled the lactose permease with a yellow fluorescent protein. The two specific phenotypes under discussion are first, above a certain threshold number of the permease, a cell has a fluorescent membrane and is capable of lactose metabolism second below this threshold, a cell is non-fluorescent and is incapable of lactose metabolism. The two phenotypes coexist in a population of cells (Fig. 22.7B) and show a bimodal distribution (Fig. 22.7C). This threshold is determined to be 300, corresponding to a big burst of permease production. 

Other secreted or membrane proteins that lack a cleaved signal sequence include the . coli signal peptidase, lactose permease, and NADH... 

---