JOURNAL OF BACTERIOLOGY, Sept. 1990, p. 4870-4876 0021-9193/90/094870-07$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 172, No. 9
Electrochemical Potential Releases a Membrane-Bound Secretion Intermediate of Maltose-Binding Protein in Escherichia colit BRUCE L. GELLER
Department of Microbiology and Center for Gene Research and Biotechnology, Oregon State University, Corvallis, Oregon 97331-3804 Received 29 August 1989/Accepted 28 April 1990
A secretionary intermediate of the Escherichia coli maltose-binding protein accumulated in the inner membrane when the membrane electrochemical potential was reduced and the cytosolic ATP concentration was normal. The intermediate was mature in size, but maintained a conformation similar to the cytosolic precursor form, and not the mature periplasmic protein, as measured by differences in susceptibility to proteinase K in vitro. The intermediate was located on the periplasmic side of the inner membrane. Restoration of the membrane electrochemical potential resulted in the movement of the intermediate from the inner membrane to the periplasm. In other experiments in which the ATP concentration was reduced by 96% and the electrochemical potential remained normal, no intermediate accumulated. Thus, the final step in the export of maltose-binding protein requires the electrochemical potential of the inner membrane and does not require ATP.
The mechanism of protein export from the cytosol of Escherichia coli is a complex sequence of reactions. One of the early steps in export for many nascent proteins requires that a significant amount of tertiary structure be attained before insertion through the inner membrane can be initiated (32). This insertion must occur before signal sequences can be proteolytically removed (43, 46). The events following the proteolytic processing are not as well defined. For the periplasmic protein ,-lactamase, there exists an intermediate step between processing and release from the inner membrane (17, 25, 29). In the case of another periplasmic protein, maltose-binding protein (MBP), at least some of the molecules span the inner membrane after removal of the signal sequence, because the carboxyl termini are still being synthesized (32, 34, 39). These processed molecules continue translocation before finally reaching the periplasm. In both examples, a mature-length, membrane-bound intermediate that faces the periplasmic side of the membrane precedes the final, soluble, periplasmic form. The energy requirements for protein export from the cytosol include the electrochemical potential (proton motive force [PMF]) and ATP (3, 6, 7, 13-16, 20, 44-47). The use of two different forms of energy may indicate that more than one step in protein export requires energy. Recently, we have demonstrated that pro-OmpA translocates across inner membrane vesicles in vitro through two consecutive steps, each with distinct energy requirements (19). The insertion of the amino terminus of pro-OmpA requires only ATP, whereas a subsequent step that moves mature-length OmpA across the membrane can be driven by the PMF alone. If the results from in vitro experiments with OmpA are applicable to other exported proteins, then mature-length periplasmic proteins should be found in the inner membrane of cells with disrupted electrochemical potentials. Recently, the accumulation of an intermediate of MBP in the inner membrane of cells with a disrupted PMF has been demonstrated (39). In this report, evidence is presented that shows t Oregon State University Agricultural Experimental Station communication no. 9002. 4870
an accumulation of a mature-length MBP in the inner membrane when the PMF was disrupted, even while cytosolic ATP concentrations remained near normal. Also, the accumulated export intermediate had a different conformation than the periplasmic form, was exposed to the periplasmic side of the membrane, and was released from the membrane to the periplasm only after the PMF was restored. In other experiments in which the cytosolic ATP level was reduced by about 96%, and the PMF remained near normal, the intermediate did not accumulate.
MATERIALS AND METHODS Bacterial strains. E. coli HG48 [MC4100 A(uncB-uncC) ilv: :TnJO] was constructed by bacteriophage P1 vir transduction as described before (28, 35). The donor strain was DK8 [bglR thi-1, hfrPOJ A(uncB-uncC) ilv::TnJO] (24), and the recipient strain was MC4100 (F- AlacU169 araD139 rpsL150 thi flbB5301 deoC7 ptsF25 relAI). The transductants were selected on Luria broth plates with 15 p.g of tetracycline per ml. Transductants were picked at random and screened for inability to grow on M9 minimal medium with 0.4% glycerol, isoleucine and valine (20 p.g/ml each), thiamine (1 p.g/ml), and tetracycline (15 ,ug/ml). The lack of F1F0-ATPase was confirmed by measurements that found normal levels of ATP in HG48 treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) and normal uptake of proline in cells treated with arsenate (see Table 1). Also, the Coomassie bluestained gel electrophoretic pattern showed a loss of all three readily detectible F1 subunits in HG48 as compared with MC4100, using a purified F1 standard. Cell labeling and subcellular fractionation. Cells were grown in M9 minimal medium with 0.4% maltose, 19 amino acids (40 ,ug/ml each, no methionine), thiamine (1 ,ug/ml), and tetracycline (15 ,ug/ml) at 37°C with shaking until the optical density at 600 nm was about 0.23. The culture was cooled to 30°C, and 1-ml aliquots were removed for the ATP concentration and protein concentration measurements. Labeling was done by adding 10 ,uCi (0.2 mg/Ci) of [35S] methionine (cell-labeling grade; Du Pont-New England Nuclear) per ml for 15 s, followed by 40 ,uM CCCP. Incubation continued for 30 s in the presence of CCCP; then
VOL. 172, 1990
unlabeled methionine (4 ,ugIml) and chloramphenicol (30 ,ug/ml) were added. Immediately following the chloramphenicol, 10 ml was transferred to a prechilled tube on ice. The remainder of the culture was transferred as quickly as possible to flasks that contained 2-mercaptoethanol (3.5 RI/ml), CCCP (40 puM), or a volume of medium equal to the volume of the added poisons. In the experiments in which the ATP levels were reduced, a culture was labeled as above, during which time 11-ml aliquots were transferred to separate flasks. After 45 s, 0.1 M sodium arsenate (pH 7.0), 0.1 M sodium phosphate (pH 7.0), or both arsenate and CCCP were added. After another 45 s in the presence of the poisons, unlabeled methionine and chloramphenicol were added, and the "no chase" sample was immediately transferred to a prechilled (0°C) tube. The incubation continued for a total of 6.5 min, after which all samples were rapidly cooled to 0°C by transferring to prechilled tubes. All subsequent work was done at 0 to 4°C. The samples were centrifuged at 5,000 x g for 10 min, and the cells were suspended in 0.5 ml of 0.1 M Tris acetate-0.5 M sucrose-5 mM EDTA, pH 8.2. A freshly prepared solution (40 pul) of lysozyme (2 mg/ml in H20) was then added, followed immediately by 0.5 ml of H20. The cells were gently mixed and allowed to stand for 5 min. An 18-,ul aliquot of 1 M MgSO4 was added, and the spheroplasts were pelleted at 12,000 x g for 40 s. The supernatant (periplasm) was removed and saved, and the pellet was gently washed without resuspension in 1.0 ml of 50 mM Tris acetate (pH 8.2)-0.25 M sucrose-10 mM MgSO4. The spheroplasts were centrifuged as in the previous step, and the wash was discarded. The pellet was vigorously suspended in 1.0 ml of 50 mM Tris acetate (pH 8.2)-2.5 mM EDTA to lyse the spheroplasts. The suspension was allowed to stand for 10 min, then 20 of 1 M MgSO4 was added. The membranes (pellet) were separated from the cytosol (supernatant) by centrifugation at 100,000 x g for 30 min. Resuspension of the pellet in 1.0 ml of 50 mM Tris acetate (pH 8.2)-10 mM MgSO4 was achieved with a water-bath-type sonicator (model 220; Branson) for 10 min. Proteolysis and immunoprecipitation. Aliquots of 100, 300, and 450 RIl of periplasm, cytosol, and membranes, respectively, were mixed with proteinase K (EC 3.4.21.14; Boehringer Mannheim Biochemicals) to a final concentration of 25 ,ug/ml, or without proteinase in the case of the controls. In the experiment shown in Fig. 3, 450 p.l of cytosol was analyzed. Proteolysis occurred for 15 min at 0C; then freshly prepared phenylmethylsulfonyl fluoride in ethanol was added to a final concentration of 2 mM and 2% ethanol. The mixtures were allowed to stand at 0°C for 10 min before a 0.1 volume of 100% (wt/vol) trichloroacetic acid in water was added. After 30 min, the samples were collected by centrifugation at 12,000 x g for 10 min. The supernatants were discarded and the precipitates were washed with 1.0 ml of ice-cold acetone. The washed precipitates were collected as in the previous step and then dried at 100°C for 5 min and suspended by boiling in 50 of 50 mM Tris hydrochloride (pH 8.9)-2% sodium dodecyl sulfate. The samples were diluted, immunoprecipitated with anti-MBP (12), and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (30) and fluorography (26). In some experiments, the samples were not proteolyzed, but instead analyzed for total protein before precipitation by trichloroacetic acid (27), total protein by silver-stained sodium dodecyl sulfate-polyacrylamide electrophoretic gels (1), and OmpA and MBP by immunodetection on protein blots (40), using peroxidase-conjugated goat anti-rabbit sec-
PMF RELEASES MBP
4871
ondary antibody and 4-chloro-1-naphthol according to the instructions of the manufacturer (Boehringer Mannheim). In these cases, 0.9 ml of each subcellular fraction was precipitated with trichloroacetic acid, and the dried pellet was dissolved in 90 ,ul as described above. In Fig. 2A, 5 RI of periplasm, 3 RId of cytosol, and 5 ,ul of membranes were loaded on the gel. In Fig. 2B and C, 10 and 2 ,u, respectively, of all fractions were loaded. Antiserum. Purified MBP was a gift from William Wickner, University of California, Los Angeles. Antibodies were raised in New Zealand White rabbits by intradermal injections of MBP in Freund adjuvant. The monospecificity of the immune serum was verified by comparing its reactivity with that of preimmune serum (i) on Ouchterlony plates, using the pure MBP as antigen; and (ii) by immunoprecipitation and visualization by electrophoresis and fluorography of 35Slabeled cell extracts from maltose-induced and uninduced cultures. Assays. ATP was measured in a luciferin/luciferase-coupled assay as described previously (22) and modified (2). Protein was measured with bovine serum albumin as the standard (27). The PMF was measured indirectly as a function of proline uptake (5, 21, 23, 36, 37) by growing the cells in the same medium described above, except without proline. The assay was initiated by adding 50 p.g of chloramphenicol per ml for 1 min prior to the addition of the same concentrations of CCCP and arsenate as used for the 35Slabeled MBP experiments described above. After 30 s, 1.0 mCi of [3H]proline (1.7 mmollCi; Du Pont-New England Nuclear) per liter was added, and 1-ml aliquots were removed at 1-min intervals for 7 min and quickly cooled to 0°C. The aliquots were filtered through 0.45-pum-pore filters (Gelman Sciences), washed with 10 ml of ice-cold M9 medium, dried, and counted in liquid scintillation fluid. The proline uptake is expressed as picomoles of proline per milligram of protein per minute, as averaged over the time interval between the second and the fifth minutes after the proline was added. Fluorograms were quantified as described previously (38) or by scanning laser densitometry (Biomed Instruments model SL-DNA). RESULTS PMF and ATP can be independently manipulated in strain HG48. To study the effects of PMF and ATP independently on protein export, strain HG48 was used in all experiments. HG48 is genetically deleted for the F1FO-ATPase operon. Changes in either the PMF or the ATP concentration have very little effect on the other in HG48. In wild-type cells, the PMF is transduced to ATP by the F1FO-ATPase. Reduction of the PMF by CCCP usually results in a large decrease in ATP concentration, as the cell hydrolyzes the nucleotide in an attempt to restore the PMF. Table 1 shows the results of PMF and ATP measurements from the same samples used in the experiments on MBP export shown in the following sections or from samples taken from parallel cultures under identical conditions. The PMF was measured indirectly by proline uptake, a cellular function that requires only the PMF and not ATP (5, 21, 23, 36, 37). While CCCP reduced the PMF in the cells containing the F1FO-ATPase (strain MC4100) by 98%, it also reduced the ATP by 84%. This was not the case in HG48. Treatment with CCCP lowered the PMF to undetectable levels, but did not reduce the ATP. The effect of CCCP in reducing the PMF was reversed 79% by 2-mercaptoethanol in HG48, but only 21% in MC4100.
4872
GELLER
J. BACTERIOL. TABLE 1. Measurements of PMF and [ATP]
Strain
MC4100 HG48 a
PMF-dependent proline uptake
[ATP]
(pmol/mg of protein per min)
(nmollmg of protein)
FTPas ATPase Control
CCCP
CCCP + 2-MEa
ASO4
2-ME
Control
CCCP
CCCP + 2-ME
ASO4
2-ME
42 86
1 0
9 68
0 79
26 82
15 7.0
2.3 7.0
8.9 10
2.9 0.3
11 8.8
+ -
2-ME, 2-Mercaptoethanol.
In other experiments, arsenate reduced the cellular ATP concentration 96% in HG48, but reduced the PMF only 8% (Table 1). In comparison, arsenate reduced ATP and the PMF in MC4100 by 80 and 100%, respectively. These measurements demonstrate that the PMF and the ATP concentration in HG48 can be independently manipulated. Accumulation and release of an export intermediate. The PMF was eliminated by adding CCCP to a culture of E. coli HG48 that had been pulse-labeled with [35S]methionine. Chloramphenicol and unlabeled methionine were added to stop the radiolabeling. The amounts of radiolabeled preMBP and MBP found in the periplasm, cytosol, and membranes immediately after this treatment are shown in Fig. 1A. Most (87%) of the radiolabeled MBP was found in the periplasm (lane 1), although significant amounts were found in the cytosol (6%, lane 4) and the membranes (6%, lane 7). All of the pre-MBP was found about evenly distributed between the cytosol and membranes. Further incubation of the CCCP-treated culture had no effect on the amounts or distribution of pre-MBP and MBP in all fractions (lanes 3, 6, and 9). However, when 2-mercaptoethanol was added to counteract the effect of the CCCP and restore the PMF, the membrane-associated pre-MBP and MBP were removed from the membrane fraction (lane 8), and a concurrent increase in the periplasmic MBP was found (lane 2). A decrease in the total amount of cytosolic forms was also observed only in cells treated with 2mercaptoethanol (lane 5). This demonstrates that the maA
.J
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ture-length MBP associated with the membrane is a bona fide export intermediate and is released from the membrane to the periplasm in a step that requires the PMF. The membrane-associated pre-MBP is also an export intermediate that has been observed previously (4, 39), but has a different membrane topology compared with the MBP (see below). As a control for systematic errors, all fractions were analyzed for total protein (Fig. 2A), total MBP (Fig. 2B), and the outer membrane marker OmpA (Fig. 2C). Without exception, there was no significant variability between equivalent fractions. No MBP was detected in any of cytosolic or membrane fractions, and no OmpA was detected in any of the periplasmic or cytosolic fractions by immunodetection (data not shown). This demonstrates that the observed changes in the amounts of radiolabeled MBP in the other figures are not due to differences in the amounts of total protein in equivalent fractions. Conformational differences between the export intermediate and mature MBP. The membrane-associated MBP was different from the periplasmic MBP, as determined by their susceptibilities to proteinase K. When the subcellular fractions were mixed with proteinase, the membrane-associated MBP was proteolyzed (Fig. 1, cf. lanes 7 to 9 in panels A and B), whereas the periplasmic MBP was completely resistant (cf. lanes 1 to 3 in panels A and B). Proteolytic resistance is a known characteristic of many periplasmic proteins, including MBP. The results suggest that the two differ in confor-
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Vol. 172, No. 9
Electrochemical Potential Releases a Membrane-Bound Secretion Intermediate of Maltose-Binding Protein in Escherichia colit BRUCE L. GELLER
Department of Microbiology and Center for Gene Research and Biotechnology, Oregon State University, Corvallis, Oregon 97331-3804 Received 29 August 1989/Accepted 28 April 1990
A secretionary intermediate of the Escherichia coli maltose-binding protein accumulated in the inner membrane when the membrane electrochemical potential was reduced and the cytosolic ATP concentration was normal. The intermediate was mature in size, but maintained a conformation similar to the cytosolic precursor form, and not the mature periplasmic protein, as measured by differences in susceptibility to proteinase K in vitro. The intermediate was located on the periplasmic side of the inner membrane. Restoration of the membrane electrochemical potential resulted in the movement of the intermediate from the inner membrane to the periplasm. In other experiments in which the ATP concentration was reduced by 96% and the electrochemical potential remained normal, no intermediate accumulated. Thus, the final step in the export of maltose-binding protein requires the electrochemical potential of the inner membrane and does not require ATP.
The mechanism of protein export from the cytosol of Escherichia coli is a complex sequence of reactions. One of the early steps in export for many nascent proteins requires that a significant amount of tertiary structure be attained before insertion through the inner membrane can be initiated (32). This insertion must occur before signal sequences can be proteolytically removed (43, 46). The events following the proteolytic processing are not as well defined. For the periplasmic protein ,-lactamase, there exists an intermediate step between processing and release from the inner membrane (17, 25, 29). In the case of another periplasmic protein, maltose-binding protein (MBP), at least some of the molecules span the inner membrane after removal of the signal sequence, because the carboxyl termini are still being synthesized (32, 34, 39). These processed molecules continue translocation before finally reaching the periplasm. In both examples, a mature-length, membrane-bound intermediate that faces the periplasmic side of the membrane precedes the final, soluble, periplasmic form. The energy requirements for protein export from the cytosol include the electrochemical potential (proton motive force [PMF]) and ATP (3, 6, 7, 13-16, 20, 44-47). The use of two different forms of energy may indicate that more than one step in protein export requires energy. Recently, we have demonstrated that pro-OmpA translocates across inner membrane vesicles in vitro through two consecutive steps, each with distinct energy requirements (19). The insertion of the amino terminus of pro-OmpA requires only ATP, whereas a subsequent step that moves mature-length OmpA across the membrane can be driven by the PMF alone. If the results from in vitro experiments with OmpA are applicable to other exported proteins, then mature-length periplasmic proteins should be found in the inner membrane of cells with disrupted electrochemical potentials. Recently, the accumulation of an intermediate of MBP in the inner membrane of cells with a disrupted PMF has been demonstrated (39). In this report, evidence is presented that shows t Oregon State University Agricultural Experimental Station communication no. 9002. 4870
an accumulation of a mature-length MBP in the inner membrane when the PMF was disrupted, even while cytosolic ATP concentrations remained near normal. Also, the accumulated export intermediate had a different conformation than the periplasmic form, was exposed to the periplasmic side of the membrane, and was released from the membrane to the periplasm only after the PMF was restored. In other experiments in which the cytosolic ATP level was reduced by about 96%, and the PMF remained near normal, the intermediate did not accumulate.
MATERIALS AND METHODS Bacterial strains. E. coli HG48 [MC4100 A(uncB-uncC) ilv: :TnJO] was constructed by bacteriophage P1 vir transduction as described before (28, 35). The donor strain was DK8 [bglR thi-1, hfrPOJ A(uncB-uncC) ilv::TnJO] (24), and the recipient strain was MC4100 (F- AlacU169 araD139 rpsL150 thi flbB5301 deoC7 ptsF25 relAI). The transductants were selected on Luria broth plates with 15 p.g of tetracycline per ml. Transductants were picked at random and screened for inability to grow on M9 minimal medium with 0.4% glycerol, isoleucine and valine (20 p.g/ml each), thiamine (1 p.g/ml), and tetracycline (15 ,ug/ml). The lack of F1F0-ATPase was confirmed by measurements that found normal levels of ATP in HG48 treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) and normal uptake of proline in cells treated with arsenate (see Table 1). Also, the Coomassie bluestained gel electrophoretic pattern showed a loss of all three readily detectible F1 subunits in HG48 as compared with MC4100, using a purified F1 standard. Cell labeling and subcellular fractionation. Cells were grown in M9 minimal medium with 0.4% maltose, 19 amino acids (40 ,ug/ml each, no methionine), thiamine (1 ,ug/ml), and tetracycline (15 ,ug/ml) at 37°C with shaking until the optical density at 600 nm was about 0.23. The culture was cooled to 30°C, and 1-ml aliquots were removed for the ATP concentration and protein concentration measurements. Labeling was done by adding 10 ,uCi (0.2 mg/Ci) of [35S] methionine (cell-labeling grade; Du Pont-New England Nuclear) per ml for 15 s, followed by 40 ,uM CCCP. Incubation continued for 30 s in the presence of CCCP; then
VOL. 172, 1990
unlabeled methionine (4 ,ugIml) and chloramphenicol (30 ,ug/ml) were added. Immediately following the chloramphenicol, 10 ml was transferred to a prechilled tube on ice. The remainder of the culture was transferred as quickly as possible to flasks that contained 2-mercaptoethanol (3.5 RI/ml), CCCP (40 puM), or a volume of medium equal to the volume of the added poisons. In the experiments in which the ATP levels were reduced, a culture was labeled as above, during which time 11-ml aliquots were transferred to separate flasks. After 45 s, 0.1 M sodium arsenate (pH 7.0), 0.1 M sodium phosphate (pH 7.0), or both arsenate and CCCP were added. After another 45 s in the presence of the poisons, unlabeled methionine and chloramphenicol were added, and the "no chase" sample was immediately transferred to a prechilled (0°C) tube. The incubation continued for a total of 6.5 min, after which all samples were rapidly cooled to 0°C by transferring to prechilled tubes. All subsequent work was done at 0 to 4°C. The samples were centrifuged at 5,000 x g for 10 min, and the cells were suspended in 0.5 ml of 0.1 M Tris acetate-0.5 M sucrose-5 mM EDTA, pH 8.2. A freshly prepared solution (40 pul) of lysozyme (2 mg/ml in H20) was then added, followed immediately by 0.5 ml of H20. The cells were gently mixed and allowed to stand for 5 min. An 18-,ul aliquot of 1 M MgSO4 was added, and the spheroplasts were pelleted at 12,000 x g for 40 s. The supernatant (periplasm) was removed and saved, and the pellet was gently washed without resuspension in 1.0 ml of 50 mM Tris acetate (pH 8.2)-0.25 M sucrose-10 mM MgSO4. The spheroplasts were centrifuged as in the previous step, and the wash was discarded. The pellet was vigorously suspended in 1.0 ml of 50 mM Tris acetate (pH 8.2)-2.5 mM EDTA to lyse the spheroplasts. The suspension was allowed to stand for 10 min, then 20 of 1 M MgSO4 was added. The membranes (pellet) were separated from the cytosol (supernatant) by centrifugation at 100,000 x g for 30 min. Resuspension of the pellet in 1.0 ml of 50 mM Tris acetate (pH 8.2)-10 mM MgSO4 was achieved with a water-bath-type sonicator (model 220; Branson) for 10 min. Proteolysis and immunoprecipitation. Aliquots of 100, 300, and 450 RIl of periplasm, cytosol, and membranes, respectively, were mixed with proteinase K (EC 3.4.21.14; Boehringer Mannheim Biochemicals) to a final concentration of 25 ,ug/ml, or without proteinase in the case of the controls. In the experiment shown in Fig. 3, 450 p.l of cytosol was analyzed. Proteolysis occurred for 15 min at 0C; then freshly prepared phenylmethylsulfonyl fluoride in ethanol was added to a final concentration of 2 mM and 2% ethanol. The mixtures were allowed to stand at 0°C for 10 min before a 0.1 volume of 100% (wt/vol) trichloroacetic acid in water was added. After 30 min, the samples were collected by centrifugation at 12,000 x g for 10 min. The supernatants were discarded and the precipitates were washed with 1.0 ml of ice-cold acetone. The washed precipitates were collected as in the previous step and then dried at 100°C for 5 min and suspended by boiling in 50 of 50 mM Tris hydrochloride (pH 8.9)-2% sodium dodecyl sulfate. The samples were diluted, immunoprecipitated with anti-MBP (12), and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (30) and fluorography (26). In some experiments, the samples were not proteolyzed, but instead analyzed for total protein before precipitation by trichloroacetic acid (27), total protein by silver-stained sodium dodecyl sulfate-polyacrylamide electrophoretic gels (1), and OmpA and MBP by immunodetection on protein blots (40), using peroxidase-conjugated goat anti-rabbit sec-
PMF RELEASES MBP
4871
ondary antibody and 4-chloro-1-naphthol according to the instructions of the manufacturer (Boehringer Mannheim). In these cases, 0.9 ml of each subcellular fraction was precipitated with trichloroacetic acid, and the dried pellet was dissolved in 90 ,ul as described above. In Fig. 2A, 5 RI of periplasm, 3 RId of cytosol, and 5 ,ul of membranes were loaded on the gel. In Fig. 2B and C, 10 and 2 ,u, respectively, of all fractions were loaded. Antiserum. Purified MBP was a gift from William Wickner, University of California, Los Angeles. Antibodies were raised in New Zealand White rabbits by intradermal injections of MBP in Freund adjuvant. The monospecificity of the immune serum was verified by comparing its reactivity with that of preimmune serum (i) on Ouchterlony plates, using the pure MBP as antigen; and (ii) by immunoprecipitation and visualization by electrophoresis and fluorography of 35Slabeled cell extracts from maltose-induced and uninduced cultures. Assays. ATP was measured in a luciferin/luciferase-coupled assay as described previously (22) and modified (2). Protein was measured with bovine serum albumin as the standard (27). The PMF was measured indirectly as a function of proline uptake (5, 21, 23, 36, 37) by growing the cells in the same medium described above, except without proline. The assay was initiated by adding 50 p.g of chloramphenicol per ml for 1 min prior to the addition of the same concentrations of CCCP and arsenate as used for the 35Slabeled MBP experiments described above. After 30 s, 1.0 mCi of [3H]proline (1.7 mmollCi; Du Pont-New England Nuclear) per liter was added, and 1-ml aliquots were removed at 1-min intervals for 7 min and quickly cooled to 0°C. The aliquots were filtered through 0.45-pum-pore filters (Gelman Sciences), washed with 10 ml of ice-cold M9 medium, dried, and counted in liquid scintillation fluid. The proline uptake is expressed as picomoles of proline per milligram of protein per minute, as averaged over the time interval between the second and the fifth minutes after the proline was added. Fluorograms were quantified as described previously (38) or by scanning laser densitometry (Biomed Instruments model SL-DNA). RESULTS PMF and ATP can be independently manipulated in strain HG48. To study the effects of PMF and ATP independently on protein export, strain HG48 was used in all experiments. HG48 is genetically deleted for the F1FO-ATPase operon. Changes in either the PMF or the ATP concentration have very little effect on the other in HG48. In wild-type cells, the PMF is transduced to ATP by the F1FO-ATPase. Reduction of the PMF by CCCP usually results in a large decrease in ATP concentration, as the cell hydrolyzes the nucleotide in an attempt to restore the PMF. Table 1 shows the results of PMF and ATP measurements from the same samples used in the experiments on MBP export shown in the following sections or from samples taken from parallel cultures under identical conditions. The PMF was measured indirectly by proline uptake, a cellular function that requires only the PMF and not ATP (5, 21, 23, 36, 37). While CCCP reduced the PMF in the cells containing the F1FO-ATPase (strain MC4100) by 98%, it also reduced the ATP by 84%. This was not the case in HG48. Treatment with CCCP lowered the PMF to undetectable levels, but did not reduce the ATP. The effect of CCCP in reducing the PMF was reversed 79% by 2-mercaptoethanol in HG48, but only 21% in MC4100.
4872
GELLER
J. BACTERIOL. TABLE 1. Measurements of PMF and [ATP]
Strain
MC4100 HG48 a
PMF-dependent proline uptake
[ATP]
(pmol/mg of protein per min)
(nmollmg of protein)
FTPas ATPase Control
CCCP
CCCP + 2-MEa
ASO4
2-ME
Control
CCCP
CCCP + 2-ME
ASO4
2-ME
42 86
1 0
9 68
0 79
26 82
15 7.0
2.3 7.0
8.9 10
2.9 0.3
11 8.8
+ -
2-ME, 2-Mercaptoethanol.
In other experiments, arsenate reduced the cellular ATP concentration 96% in HG48, but reduced the PMF only 8% (Table 1). In comparison, arsenate reduced ATP and the PMF in MC4100 by 80 and 100%, respectively. These measurements demonstrate that the PMF and the ATP concentration in HG48 can be independently manipulated. Accumulation and release of an export intermediate. The PMF was eliminated by adding CCCP to a culture of E. coli HG48 that had been pulse-labeled with [35S]methionine. Chloramphenicol and unlabeled methionine were added to stop the radiolabeling. The amounts of radiolabeled preMBP and MBP found in the periplasm, cytosol, and membranes immediately after this treatment are shown in Fig. 1A. Most (87%) of the radiolabeled MBP was found in the periplasm (lane 1), although significant amounts were found in the cytosol (6%, lane 4) and the membranes (6%, lane 7). All of the pre-MBP was found about evenly distributed between the cytosol and membranes. Further incubation of the CCCP-treated culture had no effect on the amounts or distribution of pre-MBP and MBP in all fractions (lanes 3, 6, and 9). However, when 2-mercaptoethanol was added to counteract the effect of the CCCP and restore the PMF, the membrane-associated pre-MBP and MBP were removed from the membrane fraction (lane 8), and a concurrent increase in the periplasmic MBP was found (lane 2). A decrease in the total amount of cytosolic forms was also observed only in cells treated with 2mercaptoethanol (lane 5). This demonstrates that the maA
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ture-length MBP associated with the membrane is a bona fide export intermediate and is released from the membrane to the periplasm in a step that requires the PMF. The membrane-associated pre-MBP is also an export intermediate that has been observed previously (4, 39), but has a different membrane topology compared with the MBP (see below). As a control for systematic errors, all fractions were analyzed for total protein (Fig. 2A), total MBP (Fig. 2B), and the outer membrane marker OmpA (Fig. 2C). Without exception, there was no significant variability between equivalent fractions. No MBP was detected in any of cytosolic or membrane fractions, and no OmpA was detected in any of the periplasmic or cytosolic fractions by immunodetection (data not shown). This demonstrates that the observed changes in the amounts of radiolabeled MBP in the other figures are not due to differences in the amounts of total protein in equivalent fractions. Conformational differences between the export intermediate and mature MBP. The membrane-associated MBP was different from the periplasmic MBP, as determined by their susceptibilities to proteinase K. When the subcellular fractions were mixed with proteinase, the membrane-associated MBP was proteolyzed (Fig. 1, cf. lanes 7 to 9 in panels A and B), whereas the periplasmic MBP was completely resistant (cf. lanes 1 to 3 in panels A and B). Proteolytic resistance is a known characteristic of many periplasmic proteins, including MBP. The results suggest that the two differ in confor-
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