Effect of the lipid phase transition on the lactose permease from Escherichia coli.
The temperature dependence of lactose active transport, efflux down a concentration gradient, and equilibrium exchange were analyzed in right-side-out membrane vesicles from Escherichia coli containing wild-type lactose permease and mutant Glu325 --> Ala. With respect to uphill transport and efflux down a concentration gradient, both of which involve H(+) symport, Arrhenius plots with wild-type permease exhibit a discontinuity at 18-19 degrees C with a 7-8-fold decrease in activation energy above the phase transition. For equilibrium exchange, which does not involve H(+) symport, the change in activation energy is much less pronounced (2-3-fold) than that observed for active transport or efflux. Strikingly, mutant Glu325 --> Ala, which catalyzes equilibrium exchange as well as wild-type permease but is defective in all translocation reactions that involve net H(+) translocation, exhibits no change whatsoever in activation energy. The findings are consistent with the conclusion that the primary effect of the lipid phase transition is to alter coupling between substrate and H(+) translocation rather than the conformational change(s) responsible for translocation across the membrane.
Zhang
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Howard Hughes Medical Institute, Departments of Physiology and Microbiology & Molecular Genetics, Molecular Biology Institute, University of California Los Angeles, Los Angeles, California 90095-1662, USA.
Kaback
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D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001693:Biological Transport, Active / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004563:Electrochemistry; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D018698:Glutamic Acid / Q000235:genetics; D007785:Lactose / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D011522:Protons; D013379:Substrate Specificity; D027981:Symporters
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008563:Membrane Lipids; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011522:Protons; D027981:Symporters; D018698:Glutamic Acid; C021499:lactose permease; D007785:Lactose; D000409:Alanine