Phosphatidylethanolamine Is Required for in Vivo Function of the Membrane-associated Lactose Permease of Escherichia coli

Abstract

Experiments with mutant Escherichia coli cells lacking phosphatidylethanolamine (PE) as a membrane component (DeChavigny, A., Heacock, P. N., and Dowhan, W. (1991) J. Biol. Chem. 266, 5323-5332) were carried out to establish whether or not PE is necessary for full function of the lac permease in vivo. The Vmax for active transport of both lactose (in cells lacking beta-galactosidase, lacZ) and the unhydrolyzable lactose analog, methyl-beta-D-galactopyranoside (TMG), by mutant cells lacking PE was reduced 5-10-fold relative to cells containing PE, while the Km for the uptake of both substrates was the same in both types of cells. The low rate of TMG and lactose uptake by PE-deficient cells was unaffected by the presence of a protonophore (uncoupler) and for TMG uptake was on the order of the greatly reduced rate of uptake in uncoupler-treated cells containing PE. The rate of entry of lactose into lacZ+ derivatives of both types of cells, as a measure of facilitated diffusion, was nearly the same. The Km for lactose (lacZ cells) and TMG transport in PE-deficient cells was unaffected by the presence of an uncoupler which had a small effect on Vmax. In PE-containing cells these kinetic parameters for TMG transport were reduced by an uncoupler to the level found with PE-deficient cells while an uncoupler reduced lactose uptake by PE-containing (lacZ) cells to below measureable levels. Inverted membrane vesicles made from both types of cells could be loaded with TMG, but energizing TMG-loaded vesicles by ATP only induced rapid, uphill, permease-dependent efflux of TMG from PE-containing vesicles. The decrease in apparent active transport activity of cells with no PE was not due to a change in membrane permeability, to a reduced delta microH+ (proton electrochemical gradient) across the cell membrane, or to a reduced level of membrane-associated lac permease protein. These results suggest that in the absence of PE the lac permease cannot couple substrate uptake to delta microH+ in order to effect accumulation of substrate and as a result only carries out facilitated diffusion.