The lactose permease meets Frankenstein.
The lactose permease (lac) of Escherichia coli is a paradigm for membrane transport proteins. Encoded by the lacY gene, the permease has been solubilized, purified to homogeneity, reconstituted into phospholipid vesicles and shown to catalyse the coupled translocation of beta-galactosides and H+ with a stoichiometry of unity. Circular dichroism and other spectroscopic approaches demonstrate that the purified permease is about 80% helical. Based on hydropathy analysis of the primary amino-acid sequence, a secondary structure has been proposed in which the protein has 12 hydrophobic domains in alpha-helical conformation that traverse the membrane in zigzag fashion connected by hydrophilic loops. A variety of other approaches are consistent with the model and demonstrate that both the N and C termini are on the inner surface of the membrane, and studies on an extensive series of lac permease/alkaline phosphatase fusion proteins provide exclusive support for the topological predictions of the 12-helix motif. This presentation concentrates on the use of site-directed fluorescence spectroscopy to study structure-function relationships in the permease.
Kaback
H R
HR
Howard Hughes Medical Institute, Department of Physiology, University of California Los Angeles 90024-1662.
Frillingos
S
S
Jung
H
H
Jung
K
K
Privé
G G
GG
Ujwal
M L
ML
Weitzman
C
C
Wu
J
J
Zen
K
K
D000595:Amino Acid Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D008961:Models, Structural; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D017433:Protein Structure, Secondary; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease