Proc. NatL Acad. Sci. USA Vol. 78, No. 6, pp. 3446-3449, June 1981 Biochemistry ATP-driven active transport in right-side-out bacterial membrane vesicles (Salmonella typhimurium/Escherichia coli/phosphoglycerate transport/electrochemical proton gradient/cloning) JEROEN HUGENHOLTZ*, JEN-SHIANG HONGt, AND H. RONALD KABACK*t *lAboratory of Membrane Biochemistry, Roche Institute of Molecular Biology, Nutley, New Jersey 07110; and tGraduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154 Communicated by Sidney Udenfrtend, March 2, 1981 ABSTRACT Membrane vesicles from Salmonella typhimurium phimurium LT-2 that catalyzes the uptake of2-phosphoglycer- induced for phosphoglycerate transport, were loaded with pyru- ate, 3-phosphoglycerate, and phosphoenolpyruvate, and, sub- vate kinase and ADP by lysing spheroplasts under appropriate sequently, they demonstrated that the transport system allows conditions. Vesicles so prepared catalyze active transport of pro- energy-depleted cells to use external phosphoenolpyruvate line and serine in the presence of phosphoenolpyruvate; this ac- more efficiently for vectorial phosphorylation of methyl a-D- tivity is abolished by the protonophore carbonyl cyanide-m-chlo- glucopyranoside (33). These workers also suggested the possi- rophenylhydrazone and by the HW-ATPase inhibitor NN' bility of using the phosphoglycerate transport system to affect dicyclohexylcarbodiimide but not by anoxia or cyanide. In con- the intravesicular generation ofATP from phosphoenolpyruvate trast, D-lactate-driven active transport is abolished by the hydra- added to the medium (32). zone and by anoxia or cyanide but not by the carbodiimide. More- The experiments presented here demonstrate that external over, phosphoenolpyruvate does not drive transport effectively in vesicles that lack the phosphoglycerate transport system. The re- phosphoenolpyruvate drives active transport in right-side-out sults are consistent with an overall mechanism in which phos- vesicles containing the phosphoglycerate transport system and phoenolpyruvate gains access to the interior of the vesicles by an ATP-generating system consisting of pyruvate kinase and means of the phosphoglycerate transporter and is then acted on ADP. They also provide evidence suggesting that transport ac- by pyruvate kinase to phosphorylate ADP. ATP formed inside of tivity under these conditions is due to the intravesicular for- the vesicles is then hydrolyzed by the H+-ATPase, leading to the mation of ATP that is subsequently hydrolyzed by the H+- generation of a proton electrochemical gradient that drives H+/ ATPase with generation of a &AfH+. In addition, the S. typhi- solute symport. By using pBR322 as vector and Escherichia coli. murium gene encoding for phosphoglycerate transport activity as host, a fragment of S. typhimurium DNA coding for the phos- has been cloned by transformation into E. coli, in which it is phoglycerate transport system has been cloned. E. coli membrane expressed functionally. vesicles containing the phosphoglycerate transport system also catalyze transport in the presence of phosphoenolpyruvate when MATERIALS AND METHODS they are loaded with pyruvate kinase and ADP. Growth of Cells and Preparation of Membrane Vesicles. S. typhimurium LT-2 was grown on medium A (34) with either According to the chemosmotic hypothesis of Mitchell (1-5), 0.5% sodium DL-lactate (uninduced) or 0.5% sodium 3-phos- energy derived from respiration, light, or ATP hydrolysis can phoglycerate (induced) as indicated. Cells were harvested in the be transformed into a transmembrane electrochemical gradient middle of the logarithmic growth phase (-=180 Klett units). E. of protons (IAAH+) that represents the immediate driving force coli/pBR322-pgt2 (see below) was grown on medium A con- for active transport and various other energy-dependent pro- taining 0.5% sodium 3-phosphoglycerate, methionine at 200 cesses. Cytoplasmic membrane vesicles from Escherichia coli pug/ml, thiamine-HCl at 20 ,ug/ml, and ampicillin at 20 ,Ag/ that have the same (right-side-out; refs. 6-11) or the opposite ml; cells were harvested at the end oflogarithmic growth (""200 (inverted; refs. 12-15) orientation as the membrane in the intact Klett units). Spheroplasts and membrane vesicles were pre- cell retain the capacity to convert respiratory energy into a pared as described from S. typhimurium (19) and E. coli K-12 IATH+, and studies with these preparations have provided vir- (18). Where indicated, ADP and pyruvate kinase were included tually unequivocal support for the central obligatory role ofche- in the lysis buffer at final concentrations of 5 mM and 50 Aug/ mosmotic phenomena in active transport (16-29). Similarly, ml, respectively. plasma membrane vesicles from Halobacterium halobium (30) Transport Assays. Respiration-driven transport of phos- and Rhodopseudomonas spheroides (31) generate a AgLH+ and phoenolpyruvate, proline, or serine was measured in the pres- catalyze active transport when exposed to light. Nonetheless, ence ofascorbate and phenazine methosulfate (PMS) or lithium one important aspect of the general chemosmotic hypothesis D-lactate as described (35). In the case ofphosphoenolpyruvate remains unresolved in the vesicle system. Although it is clear transport, the samples were washed twice after filtration to de- that ATP hydrolysis leads to the generation of a AfH+ in in- crease background activity further. Phosphoenolpyruvate-driven verted vesicles (21-29), this phenomenon has- not been eluci- proline or serine transport was measured under identical con- dated in right-side-out vesicles. despite numerous and varied ditions except that ascorbate, PMS, and D-lactate were omitted, attempts to make ATP accessible to the inner surface of the sodium phosphoenolpyruvate (final concentration, 10 mM) was vesicle membrane (7). added to the reaction mixtures, and the samples were incubated In 1975, Saier et al. (32) described and characterized an in- at 30°C for 20 min before addition of radioactive transport sub- ducible phosphoglycerate transport system in Salmonella ty- Abbreviations: &AH+, the proton electrochemical gradient; CCCP, car- The publication costs ofthis article were defrayed in part by page charge bonyl cyanide-m-chlorophenylhydrazone; DCCD, N,N'-dicyclohexyl- payment. This article must therefore be hereby marked "advertise- carbodiimide; PMS, phenazine methosulfate. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. t To whom reprint requests should be addressed. 3446 Downloaded by guest on September 30, 2021 Biochemistry: Hugenholtz et al. Proc. Natl. Acad. Sci. USA 78 (1981) 3447 strate. When carbonyl cyanide-m-chlorophenylhydrazone (CCCP) or N,N'-dicyclohexylcarbodiimide (DCCD) were used, small aliquots from concentrated ethanolic stock solutions were added such that the final concentration ofethanol in the reaction mixtures did not exceed 1% (vol/vol). Cloning of the Phosphoglycerate Transport System. The phosphoglycerate transporter of S. typhimurium LT-2 was 10 cloned by using the plasmid pBR322 and phenotypic comple- 4.130 mentation. Chromosomal DNA from S. typhimurium LT-2 was prepared by the method of Saito and Miura (36) and plasmid DNA was prepared by the method ofClewell and Helinski (37). pBR322 DNA (10 Ag) was linearized in 300 Al of 20 mM Tris HCl, pH 7.0/100 mM NaCl/7 mM MgCl2/2 mM 2-mer- 0 -captoethanol containing 10 units of BamHI at 37"C for 15 hr. Partially digested chromosomal DNA was prepared by incu- bating 15 ,ug ofDNA in 150 ,ul (final vol) of6 mM Tris-HCl, pH 7.5/50 mM NaCl/6 mM MgCl2 containing 1 unit of Sau3a for 4 min at 37°C. Ligation of BamHI-treated plasmid DNA with Sau3a partially digested DNA was carried out in 0.5 ml (final vol) of 66 mM Tris-HCl, pH 7.6/10 mM MgCl2/1 mM ATP/ 30 mM 2-mercaptoethanol containing 1 unit ofT4 ligase for 15 hr at 4°C. Transformation was carried out according to Mandel and Higa (38) using the E. coli K-12 strain MS401 (thi, R-M-, 0 1 2 3 4 5 endB, metC). Transformants able to use 3-phosphoglycerate as Time, min the sole carbon source for growth were selected on minimal medium (300 ,uM methionine/10 AM thiamine HCV0.5% so- FIG. 1. Transport ofphosphoenolpyruvate by isolated membrane dium 3-phosphoglycerate/1.5% agar; ref. 39). Six clones were vesicles. Vesicles were prepared from S. typhimurium LT-2 (o and obtained from =2 X 105 ampicillin-resistant transformants. *) and E. coli/pBR322-pgt2 (o and *) grown on 3-phosphoglycerate. One of these clones was E. Aliquots of membrane vesicles containing 0.08 mg of membrane pro- designated coli/pBR322-pgt2 and tein were diluted to 0.05 ml (final volume) containing (in final con- used for the experiments reported here. The hybrid plasmid centrations) 50 mM potassium phosphate (pH 6.6) and 10 mM mag- pBR322-pgt2 was shown to contain a 14.4-kilobase pair insert. nesium sulfate. The samples were incubated at 30°C and gassed with Protein Determinations. Protein was measured as described oxygen for 2-3 min before further additions and throughout the in- by Lowry et al. (40) using bovine serum albumin as standard. cubation as described (35). Where indicated (open symbols), ascorbate/ Materials. L-[U-'4C]Proline and L-[U-`4C]serine were pur- PMS was added to the reaction mixtures to final concentrations of 20 chased from New England Nuclear and phosphoenol[l-'4C]- mM and 0.1 mM, respectively, and 30 sec later, phosphoenol[1-'4C]- pyruvate (12.4 mCi/mmol; 1 Ci = 3.7 x 101s becquerels) was added to pyruvate was from Amersham/Searle. Pyruvate kinase (type II, a final concentration of0.15 mM. Closed symbols signify identical in- rabbit muscle) and ADP (grade III, yeast) were obtained from cubations carried out in the absence of ascorbate/PMS. After incu- Sigma. All other materials were reagent grade and obtained bation for given times, the reactions were terminated and the samples from commercial sources. were assayed as described (35). RESULTS and steady-state level of accumulation are approximately half those observed in the presence of D-lactate, the most effective Respiration-Driven Phosphoenolpyruvate Transport.