Proline uptake by an isolated cytoplasmic membrane preparation of Escherichia coli. (1966)

Preliminary studies by Kaback and Kostellowl indicated that the inability of cer- tain bacterial mutants to catalyze the concentrative uptake2 of specific amino acids is attributable to defects in the cytoplasmic membrane.The present investigation is an extension of these earlier studies and provides more direct evidence that amino acid uptake is a functional characteristic of the cytoplasmic membrane.Data are presented showing that isolated membrane preparations of a proline auxotroph, strain W6, of Escherichia coli can catalyze the concentrative uptake of proline, whereas comparable membrane preparations of a mutant strain, Wi57, are unable to do so.The mutant strain, W157, which was isolated by Lubin et al.,3 differs from the parental strain, W6, in that it requires high external concentrations of proline for growth and is unable to catalyze concentrative uptake of proline at 370 or rapid exchange at O between the intracellular and extracellular proline pools.4The uptake of proline by isolated membrane preparations of strain W6 is stimu- lated by glucose, and is inhibited by anaerobiosis and by a variety of compounds known to uncouple oxidative phosphorylation or inhibit electron transport.These properties and the fact that membrane preparations from the proline uptake mu- tant, W157, are unable to catalyze concentrative proline uptake point to a physio- logically significant role of the membrane-catalyzed proline uptake.Methods.-Spheroplasts: E. coli W6 and W157 (obtained from Dr. R. W. Hendler) were grown at 370 in medium A5 supplemented with 500 ,g/ml L-proline to the end of the logarithmic phase (9-10 hr).Cultures were then diluted 1:3 with fresh medium and incubated at 370 for 90-120 min.Then they were diluted 1: 4 in Difco Penassay broth containing 20% sucrose, 0.2% MgSO4, 1000 units/ml penicillin, and 500 ug/ml Lproline, and were incubated for 2.5 hr at 370 with occa- sional stirring.6After spheroplast formation was confirmed by inspection with a phase contrast microscope, the suspensions were centrifuged at 16,000 X g for 30 min.Spheroplasts thus ob- tained were then gently resuspended in a solution of 0.1 M K phosphate (pH 6.6), 20% sucrose, and 0.2% MgSO4 and centrifuged at 16,000 X g for 15 min.Membrane preparations: To prepare membranes, 1 vol of spheroplasts was resuspended in approximately 70 vol of cold medium A containing 20 jug/ml pancreatic DNase (Worthington, 2 X cryst.)but without sucrose or MgSO4.The lysates were incubated 10 min at 370 with vigor- ous swirling, then potassium EDTA, pH 6.6, was added to 5 mM final concentration and the incubation was continued for 5 min, during which time the turbidity decreased and the viscosity increased.Finally, MgSO4 was added to 10 mM final concentration and the incubation was continued for another 5 min, during which time the viscosity decreased.The lysates were then centrifuged at 16,000 X g for 30 min.The pellets were washed twice by resuspending in a solution of 0.1 M K phosphate (pH 6.6) and 10 mM EDTA and centrifuging at 37,000 X g for 15-20 min.Prior to the last high-speed centrifugation,-the membrane preparations were centrifuged at 120 X g for 15 min.The sediment was discarded.After the second washing, the membranes were re- suspended in a solution of 0.5 M K phosphate (pH 6.6), 10 mM MgSO4, and 14 mM glucose.Electron microscopy: Electron microscopy was performed by Dr. Samuel Silverstein at the Rockefeller Institute, New York.Sedimented membranes prepared as described were fixed in 2% glutaraldehyde7 in 0.1 M Na phosphate buffer, 0.01 M MgCl2 at pH 7.2 for 30 min at 40C, washed three times in cold Michaelis buffer, pH 6.0, and then placed in 1% uranyl acetate in Michaelis buffer for 3 hr at room temperature.8The pellets were cut into thin strips, dehydrated in graded alcohols, and embedded in Epoxy resin.9Sections were cut on a Porter-Blum micro-