J. Biochem. 83, 117-128 (1978)
Cytoplasmic Membrane Vesicles of Escherichia coli II.
Orientation of the Vesicles Studied by Localization of Enzymes 1 Ichiro YAMATO, Masamitsu FUTAI, Yasuhiro ANRAKU, 1 and Yoshiaki NONOMURA* Department of Botany, Faculty of Science, The University of Tokyo, and 'Department of Pharmacology, Faculty of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113 Received for publication, July 29, 1977
Orientation of cytoplasmic membrane vesicles (IM vesicles) of Escherichia coli K12 prepared by the method of Yamato, Anraku and Hirosawa ( (1975) / . Biochem. 77, 705-718) was studied by determining uptake activities for calcium ion and proline and by estimating localization of certain membrane marker enzymes. The IM vesicles showed respiration-driven uptake activities of Ca 1+ and proline. Calcium ion was taken up by the vesicles with N A D H but the NADH-dependent Ca 1+ uptake was not inhibited by D-lactate. Effects of NADH oxidation on the active, D-Iactate-dependent proline uptake was found to be additive. These findings suggested strongly that orientation of IM vesicles was heterogeneous and that the IM preparation contained right-side out and inverted vesicles but no mosaic vesicles. About 60% of the D-lactate and glycerol-3-phosphate oxidase activities were inhibited by the antibody against D-lactate dehydrogenase [EC 1.1.1.28] and ferricyanide, respectively. About 70% of the glycerol-3-phosphate-ferricyanide reductase was detectable without toluenization. D-Lactate- and glycerol-3-phosphate-dependent proline uptakes were inhibited by 40%, by the antibody and ferricyanide, respectively. From these data and the available criteria for orientation, the populations of IM vesicles with different orientations were estimated: 70% of the total population were right-side out closed vesicles and 30% were inverted vesicles or unsealed membrane fragments. In addition, about 40% of the D-lactate dehydrogenase of the right-side out closed vesicles was suggested to be dislocated from the original location and found on the outside.
1
This work was supported in part by grants, 048095 and 148319, from the Ministry of Education, Science and Culture, Japan. 1 To whom correspondence should be addressed. Abbreviations: IM vesicles, cytoplasmic membrane vesicles prepared by the method of Yamato, Anraku, and Hirosawa ((1975) /. Biochem. 17, 705); EDTA-lysozyme vesicles, membrane vesicles prepared by the method of Kaback ((1971) Methods in Enzymol. 22, 99); French press vesicles, membrane vesicles prepared by passing cell suspension through a French press as described by Futai ( (1974)/. Membrane Biol. 15, 15); EDTA, ethylenediamine tetraacetate; ATPase, adenosine triphosphatase. Vol. 83, No. 1, 1978
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I. YAMATO, M. FUTAI, Y. ANRAKU, and Y. NONOMURA Morphological appearances of IM vesicles and ATPase [EC 3.6.1.3] were studied electronmicroscopically using negative staining with uranylformate. These studies also suggested heterogeneity of the vesicles in orientation. On the basis of our findings, advantages of the use of a French press cell for preparing the cytoplasmic membrane vesicles with high purity and with major population of the vesicles as right-side out are discussed.
In the previous paper (7), we described a new procedure for preparing a large quantity of the cytoplasmic membrane of E. coli K12 which was not contaminated with the outer membrane. This preparation consisted of closed vesicles as judged from electron microscopy (7), and had higher activity in the concentrative uptake of isoleucine and proline (2) than EDTA-lysozyme vesicles prepared by the method of Kaback (3). There have been several other papers describing the preparations of cytoplasmic membranes from gramnegative bacteria (4-6). These authors, however, did not report orientation and uptake activities of amino acids and sugars of their preparations except that Mizushima (7) reported uptake of proline in the vesicles prepared by his procedure. As reviewed by several investigators (8-10), studies on active transport in bacteria using EDTAlysozyme vesicles prepared by the method of Kaback (3) have promoted general understandings in its mechanism, especially in the sources of energy and coupling of the energy to transport carriers. Since IM vesicles have much higher uptake activities for various amino acids and sugars than EDTA-lysozyme vesicles (2), we are naturally interested in their structure and orientation because these as well as the purity of the vesicles are important for elucidating the mechanism of the transport reaction. Orientation of bacterial membrane vesicles prepared by different procedures has been studied by several investigators (11-18). EDTA-lysozyme vesicles (3) were shown to be essentially rightside out (12-14) and French press vesicles (77) were completely inverted (77, 15), on the basis of experiments estimating the location of membrane marker enzymes. The fact that orientation of membrane vesicles determines strictly the direction of transport for various ions has been demonstrated. French press vesicles take up proton (79) and Ca1+ (15, 20)
driven by respiration or hydrolysis of ATP, by mechanisms comparable to the active extrusion of them in intact cells. However, these French press vesicles can not take up proline and other amino acids (77, 75), because the polarity of a proton motive force generated by respiration in these vesicles is opposite to that in intact cells, inside acid (70). Membrane vesicles prepared by sonic oscillation of intact cells were shown to be inverted and had no uptake activity for proline (77). Recently, Amanuma, Itoh, and Anraku (21) confirmed this and further showed that proline carrier in the membrane is protonated by local H + and the resulting active carrier binds proline with a high affinity, indicating that orientation of membrane vesicles is directly related to the energization of carrier mediating transmembrane diffusion. Right-side out vesicles such as EDTA-lysozyme vesicles take up proline and other solutes depending on respiration, although efficiencies of respiratory substrates as energy donors in supporting active uptake are different from each other. These observations were argued in relation to coupling efficiencies of carriers (22, 23) or changes in localization of primary dehydrogenase in the respiratory chain during preparation of the membranes (77, 72). In this connection, recent reports describing reconstitution of certain oxidase activities in membrane vesicles and of respiration-driven proline uptake were interesting. For instance, Futai (24) reconstituted r>lactate or glyceroI-3-phosphate oxidase activity by incubating right-side out vesicles defective in the respective dehydrogenase activity with purified dehydrogenase. It was shown that the activities of D-lactate dehydrogenase and glycerol-3-phosphate dehydrogenase bound to the membranes were completely inhibitable by specific antibody (13, 14) and ferricyanide (24), respectively, and the rates of proline uptake by the reconstituted vesicles were nearly equal to control membrane vesicles (24). There have been several
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ORIENTATION OF CYTOPLASMIC MEMBRANE VESICLES papers reporting that changes in the location of certain membrane enzymes occurred during preparation of EDTA-lysozyme vesicles and these changes were interpreted by a mechanism of dislocation (12, 25) or mosaicism (16). These findings suggest that changes in the location of enzymes were inevitable during preparation of membrane vesicles and that one must be careful in estimating the orientation of vesicles by studying the location of certain enzymes. Recently, Adler and Rosen stated that EDTAlysozyme vesicles consist of one homogeneous population of mosaic vesicles and do not consist of right-side out vesicles (16). They estimated the direction of the proton motive force generated by NADH oxidation and ATP hydrolysis indirectly by measuring uptake of Ca' + and inhibition of this Ca1+ uptake by D-lactate. Their results were consistent with the interpretation that external NADH dehydrogenase or ATPase in these vesicles could establish a proton motive force of the opposite polarity from that established in the same population through D-lactate oxidation. On the basis of this and other lines of evidence they claimed that the vesicles are mosaic and not hybrid since they observed that NADH dehydrogenase and ATPase distributed randomly on both sides of the membranes (16) while D-lactate dehydrogenase did not (12-14). It should be noted that direct measurement of the direction of the proton motive force should be done to obtain conclusive evidence for functional mosaicism. In this connection, studies on the topology of the whole electron transport chain are required for differentiating between mosaic vesicles and right-side out vesicles having dislocated membrane enzymes. In this paper we attempted to estimate the orientation of IM vesicles on the basis of these previous findings and arguments mentioned above. The primary criterion taken into account was NADH-dependent Ca1+ uptake by IM vesicles and its inhibition by D-lactate. The second was studies of enzyme location on both sides of IM vesicles using specific antibody and ferricyanide, an impermeant electron acceptor. The third was morphological examination of the vesicles by means of electron-microscopy using the negative staining technique. The results suggested that IM vesicles consisted of heterogeneous vesicles showing different orientation. The majority of the vesicles Vol. 83, No. 1, 1978
was right-side out closed vesicles with certain marker enzymes dislocated. MATERIALS AND METHODS Materials—Purified ATPase (specific activity, 80 units per mg protein) was prepared and stored as described previously (26). Anti-serum against purified ATPase (11) and antibody (immunoglobulin G) against D-lactate dehydrogenase (12) used were prepared as previously reported. Chemicals were obtained as follows: [14C]-proline (231 mCi/ mmol), Dai-ichi Chem. Co., Tokyo; "CaCl,(600 mCi/mmol), Radiochemical Centre, Amersham; D,L-glycerol-3-phosphate, Nakarai Chem. Co., Kyoto; D-lactate and 3-(4,5-dimethyl(thiazolyl-2>2,5-diphenyl tetrazolium bromide, Sigma Chem. Co., St. Louis. Other reagents used were of the highest grade commercially available. Strain and Culture Conditions—E. coli K12 strain W3092 (27) was grown aerobically as described previously (28). Preparation of Membrane Vesicles—IM vesicles were prepared by the published procedure (1) except that the pressure of a French press used was 200 kg/cm1. French press vesicles were prepared by the published procedure (11) using a French press cell (Ohtake Co., Tokyo) at 200 kg/cm1 and suspended in a solution of 0.05 M Tris-HCl, pH 8.0, containing 10 ITIM MgCl,. Treatment with RNase was omitted in this study. Assays of Proline and Cat+ Uptake—Assay of proline uptake was carried out as described previously (29) with a slight modification. The incubation mixture contained 50 ITIM Tris-maleate, pH 8.0, 2 ITIM MgSO4, 8 (M "C-proline, membrane vesicles (10-100 ftg of membrane protein) and respiratory substrate (20 ITIM D-lactate (Li) or 20 ITIM glycerol-3-phosphate(Na)). Assay of Ca1+ uptake was carried out as described by Tsuchiya and Rosen (20) with 2.5 nun "CaCl, as substrate. Measurement of Ferricyanide Reductase—The incubation mixture contained 0.7 mM potassium ferricyanide, 0.08 M Tris-HCl, pH 7.3, 25 mM glycerol-3-phosphate, 9 mM KCN, and membrane vesicles (100 fi% of membrane protein) in a total volume of 1.0 ml. After incubation at 30°C for 5 min, 0.15 ml of 1.0 M trichloroacetic acid was added. The supernatant obtained after centrifugation was assayed for ferrocyanide formed (30).
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The supernatant (0.3 ml) was mixed with 0.7 ml of 2.0 M sodium formate, pH 3.0, 0.3 ml of 3.0 ITIM
bathophenanthroline sulfonate and 0.3 ml of 4.0 mM Fe(NO3)3-5.6 IHM EDTA. The mixture was incubated for 10 min at room temperature and the absorbancy at 535 nm was measured. Ferrocyanide formed was estimated from standard ferrocyanide treated as above. Other Assays—D-Lactate dehydrogenase (31), ATPase (26), and protein (32) were assayed by the published procedures. Oxygen consumption was measured as described previously (28) in the buffer used for the uptake assay. Electron Microscopy—IM vesicles and French press vesicles were collected and suspended in 0.15 M sodium phosphate, pH 7.2, before use. For negative staining a drop of the membrane suspension on a carbon-coated collodion grid (400 mesh) was mixed with 2 % uranylformate (pH 7.0) repeatedly. Then the excess fluid was removed and the specimen was dried by touching it with a piece of filter paper at room temperature. Samples were examined with a JEM-100B electron microscope at 80 kV. Negative staining of ATPase was carried out similarly as described above using the sample solution prepared as mentioned. RESULTS 1) Heterogeneity of IM Vesicles in Orientation—Since IM vesicles were prepared by disrupting spheroplasts in 20% sucrose-3.0 ITIM EDTA, pH 7.2, in a French press (1), the preparation may contain membrane vesicles of different orientations. Heterogeneous orientation of membranes may be categorized into three major groups: 1) right-side out vesicles, 2) inverted vesicles, and 3) mosaic vesicles. We are able to estimate the presence or absence of these three types of vesicles in the IM preparation by measuring the uptake of Ca l+ and proline driven by D-lactate and/or NADH oxidation, as suggested by Adler and Rosen (16). Table I shows that IM vesicles took up Ca I+ with NADH as respiratory substrate. This result suggests that NADH generated the proton motive force of the direction inside positive in the inverted or mosaic vesicles (75, 16). D-Lactate did not inhibit the NADH-dependent Ca1+ uptake. This finding
TABLE I. Respiration-driven uptake of calcium ions and proline in IM vesicles. Assays were carried out under standard conditions (see "MATERIALS AND METHODS ") at 25°C for 1 min. Respiratory substrate D-Lactate D-Lactate+NADH NADH
CaI+ uptake1 Proline uptake11 (nmol/min/mg protein) -0.6 3.9 4.0
0.60 0.69 0.14
» Uptake of CaI+ by 2.8 nmol/min/mg protein was observed in the absence of respiratory substrate and this value was used for correction of the respiration driven Ca*+ uptake. Endogenous Ca*+ uptake was also observed in EDTA-lysozyme vesicles (16). b Concentration of proline used was 2 /JM. suggests that the IM preparation contained no mosaic vesicles, in contrast to the result of EDTAlysozyme vesicles studied by Adler and Rosen (16). Furthermore, we could not detect D-lactate-dependent Ca1+ uptake, suggesting that the IM preparation contained inverted vesicles as a minor population. The IM vesicles took up proline actively depending on D-lactate. NADH supported the proline uptake (Table I) but its coupling efficiency was as low as the isoleucine uptake (2). We also found that the effect of NADH on the D-lactatedependent proline uptake was somehow additive but not inhibitory. On the basis of these findings we concluded that IM vesicles consist of membranes with different orientations; the preparation may contain right-side out and inverted vesicles but not mosaic vesicles. Unsealed membrane fragments which have no uptake activities may also be present in the preparation. 2) Orientation of IM Vesicles Studied by Localization of Membrane Enzymes—As D-lactate dehydrogenase and glycerol-3-phosphate-ferricyanide reductase are located normally on the inside surface of cytoplasmic membrane (11-14), we are able to take these activities as marker enzymes for further estimation of the orientation of IM vesicles. According to the argument described above it is possible that the enzymes in IM vesicles are located in three ways as shown in Fig. 1: (A) The enzymes are located in the original orientation in right-side out vesicles, (B) the enzymes are rearranged during preparation and / . Biochem.
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IM vesicles "Right-side out" vesicles (capable of respirationdriven proline uptake)
—-. "Unsealed" or "Inverted" vesicles (incapable of respiration-driven proline uptake)
(70%)
(30*)
inner surface outer surface
D-LDH on Tnner surface
(401)
D-LDH on outer surface
(30*) B
Fig. 1. Location of D-lactate dehydrogenase in the preparation of IM vesicles. Populations of D-lactate dehydrogenase of different locations in the vesicles estimated in this paper, are summarized schematically: Enzymes located in the original orientation in right-side out vesicles (A), 40%; enzymes located on the outside of the right-side out vesicles (B), 30%; enzymes located in unsealed or inverted vesicles (C), 30%. D-Lactate dehydrogenase (D-LDH) ( • ) is drawn schematically in the figure. For further details, see text.
located on the outside of the right-side out vesicles, ( Q the enzymes are located in inverted vesicles or unsealed membrane fragments. The enzymes located on the inner surface of inverted vesicles may be minor, because all the inner surface enzymes so far studied were located on the outside of such vesicles prepared by a French press (11, 12). Since specific antibody against D-lactate dehydrogenase was accessible to the enzyme located in reconstituted vesicles (24) or inverted vesicles (12), we could estimate the population of the enzyme located differently as mentioned above. However, we cannot estimate the orientation of IM vesicles by only determining the location of enzymes. Since IM vesicles may contain inverted vesicles or unsealed membrane fragments (Table I and Fig. 1), inhibition study on the D-lactateVol. 83, No. 1, 1978
dependent proline uptake by specific antibody against D-lactate dehydrogenase is required. Through such studies we can estimate the proportions of right-side out vesicles and inverted vesicles in the preparation. Figure 2 shows that the antibody against D-lactate dehydrogenase inhibited 60% of the Dlactate dehydrogenase activity and D-lactate oxidation in IM vesicles. This indicates that 60% of the total D-lactate dehydrogenase of the membranes were accessible by the antibody. In other words, the activity inhibited by the antibody was due to the enzyme located outside the vesicles as described in (B) and ( Q and the rest (40% of the total) located inside as in (A) (see Fig. 1). Therefore, the ratio of the enzyme located on the inner surface of right-side out vesicles ( A / A + B + Q should be 0.4.
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0
0.2 0.4 0.6 0.8 1.0 ANTIBODY(mg)/D-LACTATE DEHYDROGENASE(unit)
Fig. 2. Effect of antibody against D-lactate dehydrogenase on D-lactate dehydrogenase and oxygen consumption dependent on D-lactate in IM vesicles. IM vesicles (2.1 mg protein/ml) were incubated with varying concentrations of antibody (immunoglobulin G fraction from immunized rabbits) in 0.05 M Tris-maleate, pH 8.0, containing 2 ITIM MgSCV D-Lactate dehydrogenase (O) and oxygen consumption dependent on D-lactate (•) were assayed after three hours of incubation. Results of assay of D-lactate dehydrogenase are expressed as relative rates, % of control. Control rate was 1.47 /umol/min/mg. On the other hand, the antibody inhibited 40% of the proline uptake in IM vesicles (Fig. 3). If we assume that the dehydrogenase located as in (A) and (B) can support transport equally, this suggests that 40% of the D-lactate dehydrogenase in right-side out vesicles was on the outer surface of the vesicles (Location (B)), and the rest of the dehydrogenase was on the inner surface (Location (A)). Thus we can estimate the portion of the enzyme located on the inner surface of the rightside out vesicles (A): A/A+B=0.6 We can calculate the population of the rightside out vesicles from the ratio of the enzyme located as in (A) and (B); the ratio of right-side out vesicles to the total population was 0.67 ( A + B / A + B + Q , suggesting that 67% of the IM vesicles were right-side out. The rest (33%) may be inverted vesicles or unsealed membrane fragments, because the possibility that the IM preparation contained mosaic vesicles was slight (Table
0
0.2 0.4 0.6 0.8 1.0
ANTIBODY(mg)/D-LACTATE DEHYDROGENASE(unit)
Fig. 3. Effect of antibody against D-lactate dehydrogenase on proline uptake stimulated by D-lactate in IM vesicles. IM vesicles were incubated with antibody against D-lactate dehydrogenase as described in the legend of Fig. 2. After incubation for 2 h, proline uptake was assayed as described in the text. TABLE II. Ferricyanide reductase activity in IM vesicles prepared from E. coli W3092. IM vesicles (8.2 mg protein/ml) were incubated in 10% sucrose containing 1% toluene at 37°C for 10 min. Aliquots of 10 to 50 ft\ of toluene-treated and control membranes were assayed for glycerol 3-phosphate-ferricyanide reductase with the indicated concentration of ferricyanide in the presence of 9 mM KCN. Other procedures are described in the text. Concentration of femcyanide 0.7 mM 1.8 mM
Ferricyanide reduction (/imol reduced/mg/min) Control (untreated)
Treated with toluene
0.18
0.26 0.17
0.12
ferricyanide accepts electrons from the respiratory chain are located on the inner surface of the cytoplasmic membrane. Thus, a ferricyanide reductase activity was not measurable in spheroplasts and intact cells, unless permeability barriers were destroyed with toluene or detergents (11, 33). Based on this and other evidence, Futai suggested that measurement of ferricyanide reductase activity under proper experimental conditions proD. vides useful information on the orientation of 3) Location of Ferricyanide Reductase in IM membrane vesicles (11). Vesicles—As shown previously (77), the sites where J. Biochem.
ORIENTATION OF CYTOPLASMIC MEMBRANE VESICLES
B Fig. 4. Electronmicrographs of IM vesicles and purified ATPase showing typical morphological appearances. A) IM vesicles obtained by the published procedure (7) were stained with 2% uranylformate (see " MATERIALS AND METHODS "). Typical morphological appearances of the vesicles are seen in this picture. Globular particles with stalks are seen on the inner edge of the membrane (shown by arrows). These globular particles were considered to be ATPase as discussed in the text. Final magnification x 269,000. B) Electronmicrograph of an IM vesicle having globular particles on its outer surface. Final magnification x 243,000. C) Electronmicrograph of purified ATPase. ATPase purified by the published procedure (26) was stained with 2% uranylformate. Final magnification x 263,000.
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Fig. 5. Electronmicrograph of an inverted vesicle prepared by disrupting cells with a French press. Typical morphological appearance of an inverted vesicle is shown. Globular particles are seen on the outer surface of the vesicle. Larger particles seen around the vesicles were considered to be ribosomes from their sizes (41) (shown by arrows). Final magnification x 269,000.
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TABLE III. Effects of ferricyanide on glycerol-3-phosphate-dependent oxidation and proline uptake in IM vesicles. Oxygen consumption and proline uptake stimulated by glycerol-3-phosphate were assayed in the presence and absence of ferricyanide. Assay procedures are described in " MATERIALS AND METHODS." Inhibition of oxygen consumption by ferricyanide was measured after adding a small volume (34 //!) of ferricyanide solutions of different concentrations to the reaction mixture incubated with glycerol-3-phosphate. Oxygen consumption and proline uptake are expressed as /Jmoles of O t consumed and nmoles proline taken up per min per mg protein, respectively. Activity Oxygen consumption Proline uptake
Activity before addion of ferricyanide 0.31 0.42
Table II shows the ferricyanide reductase activity of IM vesicles with glycerol-3-phosphate as substrate. Although ferricyanide affected the apparent activity in higher concentration, about 70% of the total sites in IM vesicles were shown to be available for electron transfer under the experimental conditions. The above results suggest that about 70% of the ferricyanide reactive sites were either on the outside of the right-side out vesicles or inverted vesicles and 30% of the sites on the inner surface of the right-side out vesicles. These values are comparable with the results obtained using antibody and suggest that random distribution of membrane marker enzymes had occurred independently during the preparation of IM vesicles. Although details of the calculation are not shown here, essentially similar values for the location of ferricyanide reactive sites were obtained from the following results. It has been established that glycerol-3-phosphate stimulates proline uptake in membrane vesicles and that ferricyanide inhibits oxidation of glycerol-3-phosphate by vesicles as well as the respiration dependent uptake of amino acids (24). Table III summarizes the inhibition of glycerol-3-phosphate oxidation and glycerol-3phosphate dependent-proline uptake in IM vesicles by ferricyanide. The ferricyanide inhibited 38% of the proline uptake and 60% of the oxygen consumption and this result was consistent with the conclusion for orientations of IM vesicles, obtained using antibody (see Fig. 1). 4) Electron Micrograph of IM Vesicles—IM vesicles retained about 20% of the total ATPase activity of the crude membrane fraction when Vol. 83, No. 1, 1978
Activity in the presence of ferricyanide (2 ) 0.12 0.26
Inhibition by ferricyanide (%) 60 38
prepared by the published method (1). Localization of this activity in these vesicles was examined using antiserum against ATPase. Table IV shows that only 18% of the ATPase activity in the IM vesicles was measurable without toluene treatment and inhibitable almost completely by antiserum. This indicates that about 20% of the ATPase activity of IM vesicles seems to be on the outer surface of the closed vesicles or on the unsealed membrane fragments. The molecular organization (34-36) and morphology (34, 37, 38) of bacterial ATPase (F^ coupling factor of oxidative phosphorylation) have been reported, which are essentially identical with those of mitochondria! Fx (see reviews, Refs. 39,40). Therefore, we examined the structure of IM vesicles and localization of ATPase on the membranes electron-microscopically using negative staining with uranylformate. TABLE IV. ATPase activity of IM vesicles. IM vesicles (40-80 ftg protein/ml) were incubated in 10% sucrose containing 1% toluene at 37°C for 10 min. Samples of 10 to 50 fi\ of toluene-treated and control membranes were assayed for ATPase activity as described previously (26). Antiserum against purified ATPase (3 fi\) was added to the incubation mixture. Serum from preimmune rabbits had no effect on ATPase activity.
Addition
None Antiserum
ATPase activity (ftmo\ Pi released/min/mg protein) No treatment
Treated with toluene
0.20 0.03
1.10 0.09
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Figure 4C shows the morphological appearance of ATPase. The globular particles of about 10 nm in diameter are seen to be the major population. Morphologically, the particles show a polyhedral view and have one or a few holes and grooves on the surface of the particle. The morphology of these particles is similar to ATPases from other sources (34-36). In the present study, we stained the samples with phosphotungstate, silicontungstate, uranylacetate as well as uranylformate and found that the latter stain gives better contrast than the former three, although all these reagents showed essentially the same structure of ATPase and membranes. Figure 4A shows typical morphological appearances of IM vesicles. The vesicles were heterogeneous in sizes in the range of 50-200 nm in diameter. However, most of the population consisted of closed vesicles surrounded by the membrane of about 10 nm thickness. It was also found that the vesicles contained a number of globular particles on the membranes, some with stalks as shown by arrows (Fig. 4A). From the morphological appearance and dimension of the particles, it was suggested that these particles were ATPase and located on the inside surface of the closed '-ssicles. Sometimes the globular particles were seen more clearly on a few IM vesicles than those in Fig. 4A. Figure 4B shows a typical example; this IM vesicle shows clusters of globular particles, the fine structure of which are identical with those of ATPase in Fig. 4C. In addition, this particular vesicle has globular particles on its edge (shown by arrows) but the edge of the vesicle does not show the membraneous structure seen in Fig. 4A. These characteristics suggest that this vesicle was inverted, thereby exposing the ATPase on the outer surface of the membrane. As mentioned already, French press vesicles are known to be completely inverted (11). Moreover, since dislocation of ATPase did not take place during the inversion of the cytoplasmic membrane by this procedure, we are interested in examining the morphological structure of French press vesicles as a control for the inversion of IM vesicles. Figure 5 shows an electronmicrograph of an inverted vesicle. This particular vesicle was larger than the IM vesicles (see Fig. 4A) and had a number
of polyhedral particles on the membrane with dimension identical to that of ATPase (see Fig. 4C). The morphological appearance of the particles is clear and resembles those observed in Fig. 4B. These observations suggest that ATPase molecules, when located on the outer surface of the inverted vesicles, were stained in clearer contrast by uranylformate under the conditions used. There are a number of larger particles than ATPase around the vesicles (Fig. 5). These particles are considered to be ribosomes based on their dimension and morphological appearance (41). We think that a minor population of IM vesicles consisted of inverted vesicles, on the basis of these morphological studies. In the preparation of IM vesicles so far examined, less than 5 to 10% of the total population were vesicles having the morphological characteristics as shown in Fig. 4B. The observed value was close to and consistent with those obtained by estimation of enzyme location (Table IV), indicating heterogeneity of IM vesicles in orientation. DISCUSSION We have presented evidence that IM vesicles prepared by our new procedure (1) consisted of heterogeneous populations in their orientation of membranes. It was possible to estimate that about 70% of the IM vesicles were right-side out assuming the following: 1) Dislocated D-lactate dehydrogenase on the right-side out closed vesicles can energize proline uptake as efficiently as that localized normally (14, 24), although the actual coupling efficiency could not be estimated. 2) No dislocation of the enzymes occurs in the inverted vesicles. All the D-lactate dehydrogenase or ferricyanide reactive sites are located on the outer surface of inverted vesicles prepared by a French press (11, 12), though the method of membrane preparation is different from ours. 3) D-Lactatedependent respiration is the rate limiting step of proline uptake. This was suggested by the finding that the ratios of stimulation of oxygen consumption and proline uptake were essentially the same for various respiratory substrates except NADH (2). 4) Right-side out vesicles with Dlactate dehydrogenase dislocated and/or located normally, inverted vesicles with all the D-lactate dehydrogenase on the outer surface and unsealed
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ORIENTATION OF CYTOPLASMIC MEMBRANE VESICLES membrane fragments are major populations in the preparation. We found that IM vesicles took up Ca I+ with NADH as respiratory substrate but that the NADHdependent Ca I+ uptake was not inhibited by simultaneous oxidation of D-lactate (Table I). This finding strongly suggested that the IM preparation did not contain mosaic vesicles and that Ca1+ was taken up by inverted vesicles. D-Lactate did not stimulate Ca1+ uptake. This implicated that the inverted vesicles were a minor population. The presence of inverted vesicles, the population being less than 30%, was suggested by enzyme location (Fig. 3 and Tables HI and IV) and the morphology of membranes (Fig. 5B). On the basis of these findings and the criteria stated by Adler and Rosen (16), we concluded that the IM vesicles may not contain mosaic vesicles. We also showed that IM vesicles were heterogeneous with respect to the localization of membrane proteins. As estimated (see Fig. 1), a significant portion of the right-side out vesicles were found to have marker enzymes dislocated. Changes in the location or dislocation of D-lactate dehydrogenase and ferricyanide reactive sites seemed to occur randomly during the preparation of IM vesicles because the apparent extent of inhibition of these activities was essentially the same (Fig. 2 and Table II). It should be noted that random distribution of membrane proteins may take place through the fusion between rightside out and inverted vesicles. In order to distinguish enzyme dislocation from membrane fusion, it would be desirable to have marker proteins originally located on the outside of the cytoplasmic membrane. More important would be studies on the entire topology of the electron transport chain and its changes during the preparation of membrane vesicles by different procedures. We prefer to interprete our data by dislocation of enzymes according to the observations at present available (11, 12, 21, 24), although the mechanism of dislocation is not known yet. Several investigators have discussed dislocation or non-random distribution of membrane marker enzymes in EDTAlysozyme vesicles (12, 16, 25). It is important to note that use of a French press to disrupt spheroplasts in our procedure did not give totally inverted vesicles as shown above. This may have resulted from disrupting spheroVol. 83, No. 1, 1978
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plasts in 20% sucrose-3 ITIM EDTA, which is not included in the medium to make inverted vesicles by a French press from intact cells (11, 15, 19). It should also be noted that IM vesicles are smaller than EDTA-lysozyme vesicles in size (3) and much smaller than French press vesicles (cf. Figs. 4A and 5). These and the apparent differences of the location of D-lactate dehydrogenase in IM vesicles from EDTA-lysozyme vesicles (12) may be due to the use of French press for the preparation of IM vesicles. In conclusion, the major population of IM vesicles consists of right-side out vesicles with sizes ranging from 50 to 200 nm in diameter. We suggest that these structural properties and the high purity (7) of the membranes support high transport activities for various amino acids and sugars (2). We are grateful to Dr. L.A. Heppel for his instruction of ferricyanide reductase assay, and to Drs. B.P. Rosen and F.M. Harold for showing us their manuscripts before publication. We also thank Dr. Y. Tanaka for his help in preparing the antibody against purified r> lactate dehydrogenase. REFERENCES 1. Yamato, I., Anraku, Y., & Hirosawa, K. (1975) /. Biochem. 11, 705-718 2. Yamato, I. & Anraku, Y. (1977) J. Biochem. 81, 1517-1523 3. Kaback, H.R. (1971) Methods in Enzymology 22, 99-120 4. Miura, T. & Mizushima, S. (1968) Biochim. Biophys. Acta 150, 159-161 5. Osborn, M.J., Gander, J.E., Parise, E., & Carson, J. (1972) / . Biol. Chem. 247, 3962-3972 6. Schnaitman, C.A. (1970) J. Bacleriol. 104, 890-901 7. Mizushima, S. (1976) Biochim. Biophys. Acta 419, 261-270 8. Kaback, H.R. (1974) Science 186, 882-892 9. Simoni, R.D. & Postma, P.W. (1975) Annual Rev. Biochem. 44, 523-554 10. Harold, F.M. (1977) in Current Topics in Bioenergetics (Sanadi, D.R., ed.) Vol. 6, pp. 84-151, Academic Press, New York & London 11. Futai, M. (1974) / . Membrane Biol. 15, 15-28 12. Futai, M. & Tanaka, Y. (1975) J. Bacterial. 124, 470-475 13. Short, S.A., Kaback, H.R., Kaczorowski, G., Fisher, J., Walsh, C.T., & Silverstein, S.C. (1974) Proc. Nail. Acad. Sci. U.S. 71, 5032-5036
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14. Short, S.A., Kaback, H.R., & Kohn, L.D. (1975) J. Biol. Chem. 250, 4291^(296 15. Rosen, B.P. & McClees, J.S. (1974) Proc. Nail. Acad. Sci. U.S. 71, 5042-5046 16. Adler, L.W. & Rosen, B.P. (1977) /. Bacteriol. 129, 959-966 17. Wickner, W. (1975) Proc. Nail. Acad. Sci. U.S. 72, 4749-4753 18. Wickner, W. (1976) Proc. Natl. Acad. Sci. U.S. 73, 1159-1163 19. Hertzberg, E.L. & Hinkle, P.C. (1974) Biochem. Biophys. Res. Commun. 58, 178-184 20. Tsuchiya, T. & Rosen, B.P. (1975) J. Biol. Chem. 250, 7687-7692 21. Amanuma, H., Itoh, J., & Anraku, Y. (1977) FEBS Lett. 78, 173-176 22. Barnes, E.M., Jr. & Kaback, H.R. (1971) / . Biol. Chem. 246, 5518-5522 23. Kaback, H.R. & Barnes, E.M., Jr. (1971) J. Biol. Chem. 246, 5523-5531 24. Futai, M. (1974) Biochemistry 13, 2327-2333 25. Altendorf, K.H. & Staehelin, L.A. (1974) / . Bacteriol. 117, 888-899 26. Futai, M., Sternweis, P.C, & Heppel, L.A. (1974) Proc. Natl. Acad. Sci. U.S. 71, 2725-2729 27. Anraku, Y. (1971) J. Biochem. 70, 855-866 28. Kasahara, M. & Anraku, Y. (1972) / . Biochem. 74, 777-781
29. Kasahara, M. & Anraku, Y. (1974) / . Biochem. 76, 977-983 30. Shoen, R. (1965) Anal. Biochem. 12, 413-^20 31. Futai, M. (1973) Biochemistry 12, 2468-2474 32. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 33. Weiner, J.H. (1974) /. Membrane Biol. 15, 1-14 34. Kagawa, Y., Sone, N., Yoshida, M., Hirata, H., & Okamoto, H. (1976) J. Biochem. 80, 141-151 35. Abrams, A., Jensen, C , & Morris, D.H. (1976) Biochem. Biophys. Res. Commun. 69, 804-811 36. Johansson, B.C. & Baltscheffsky, M. (1975) FEBS Lett. 53, 221-224 37. Gel'man, N.S., Lukoyanova, M.A., & Ostrovskii, D.N. (1975) Bacterial membranes and the respiratory chain, in Biomembranes 38. Schnebli, H., Vatter, A.E., & Abrams, A. (1970) J. Biol. Chem. 245, 1122-1127 39. Senior, A.E. (1973) Biochim. Biophys. Ada 301, 249-277 40. Kagawa, Y. (1972) Biochim. Biophys. Ada 265, 297-338 41. Lake.J.A., Sabatini, D., & Nonomura, Y. (1974) in Ribosomes (Nomura, M., ed.) pp. 543-557, Cold Spring Harbor Lab.
/ . Biochem.
Cytoplasmic Membrane Vesicles of Escherichia coli II.
Orientation of the Vesicles Studied by Localization of Enzymes 1 Ichiro YAMATO, Masamitsu FUTAI, Yasuhiro ANRAKU, 1 and Yoshiaki NONOMURA* Department of Botany, Faculty of Science, The University of Tokyo, and 'Department of Pharmacology, Faculty of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113 Received for publication, July 29, 1977
Orientation of cytoplasmic membrane vesicles (IM vesicles) of Escherichia coli K12 prepared by the method of Yamato, Anraku and Hirosawa ( (1975) / . Biochem. 77, 705-718) was studied by determining uptake activities for calcium ion and proline and by estimating localization of certain membrane marker enzymes. The IM vesicles showed respiration-driven uptake activities of Ca 1+ and proline. Calcium ion was taken up by the vesicles with N A D H but the NADH-dependent Ca 1+ uptake was not inhibited by D-lactate. Effects of NADH oxidation on the active, D-Iactate-dependent proline uptake was found to be additive. These findings suggested strongly that orientation of IM vesicles was heterogeneous and that the IM preparation contained right-side out and inverted vesicles but no mosaic vesicles. About 60% of the D-lactate and glycerol-3-phosphate oxidase activities were inhibited by the antibody against D-lactate dehydrogenase [EC 1.1.1.28] and ferricyanide, respectively. About 70% of the glycerol-3-phosphate-ferricyanide reductase was detectable without toluenization. D-Lactate- and glycerol-3-phosphate-dependent proline uptakes were inhibited by 40%, by the antibody and ferricyanide, respectively. From these data and the available criteria for orientation, the populations of IM vesicles with different orientations were estimated: 70% of the total population were right-side out closed vesicles and 30% were inverted vesicles or unsealed membrane fragments. In addition, about 40% of the D-lactate dehydrogenase of the right-side out closed vesicles was suggested to be dislocated from the original location and found on the outside.
1
This work was supported in part by grants, 048095 and 148319, from the Ministry of Education, Science and Culture, Japan. 1 To whom correspondence should be addressed. Abbreviations: IM vesicles, cytoplasmic membrane vesicles prepared by the method of Yamato, Anraku, and Hirosawa ((1975) /. Biochem. 17, 705); EDTA-lysozyme vesicles, membrane vesicles prepared by the method of Kaback ((1971) Methods in Enzymol. 22, 99); French press vesicles, membrane vesicles prepared by passing cell suspension through a French press as described by Futai ( (1974)/. Membrane Biol. 15, 15); EDTA, ethylenediamine tetraacetate; ATPase, adenosine triphosphatase. Vol. 83, No. 1, 1978
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I. YAMATO, M. FUTAI, Y. ANRAKU, and Y. NONOMURA Morphological appearances of IM vesicles and ATPase [EC 3.6.1.3] were studied electronmicroscopically using negative staining with uranylformate. These studies also suggested heterogeneity of the vesicles in orientation. On the basis of our findings, advantages of the use of a French press cell for preparing the cytoplasmic membrane vesicles with high purity and with major population of the vesicles as right-side out are discussed.
In the previous paper (7), we described a new procedure for preparing a large quantity of the cytoplasmic membrane of E. coli K12 which was not contaminated with the outer membrane. This preparation consisted of closed vesicles as judged from electron microscopy (7), and had higher activity in the concentrative uptake of isoleucine and proline (2) than EDTA-lysozyme vesicles prepared by the method of Kaback (3). There have been several other papers describing the preparations of cytoplasmic membranes from gramnegative bacteria (4-6). These authors, however, did not report orientation and uptake activities of amino acids and sugars of their preparations except that Mizushima (7) reported uptake of proline in the vesicles prepared by his procedure. As reviewed by several investigators (8-10), studies on active transport in bacteria using EDTAlysozyme vesicles prepared by the method of Kaback (3) have promoted general understandings in its mechanism, especially in the sources of energy and coupling of the energy to transport carriers. Since IM vesicles have much higher uptake activities for various amino acids and sugars than EDTA-lysozyme vesicles (2), we are naturally interested in their structure and orientation because these as well as the purity of the vesicles are important for elucidating the mechanism of the transport reaction. Orientation of bacterial membrane vesicles prepared by different procedures has been studied by several investigators (11-18). EDTA-lysozyme vesicles (3) were shown to be essentially rightside out (12-14) and French press vesicles (77) were completely inverted (77, 15), on the basis of experiments estimating the location of membrane marker enzymes. The fact that orientation of membrane vesicles determines strictly the direction of transport for various ions has been demonstrated. French press vesicles take up proton (79) and Ca1+ (15, 20)
driven by respiration or hydrolysis of ATP, by mechanisms comparable to the active extrusion of them in intact cells. However, these French press vesicles can not take up proline and other amino acids (77, 75), because the polarity of a proton motive force generated by respiration in these vesicles is opposite to that in intact cells, inside acid (70). Membrane vesicles prepared by sonic oscillation of intact cells were shown to be inverted and had no uptake activity for proline (77). Recently, Amanuma, Itoh, and Anraku (21) confirmed this and further showed that proline carrier in the membrane is protonated by local H + and the resulting active carrier binds proline with a high affinity, indicating that orientation of membrane vesicles is directly related to the energization of carrier mediating transmembrane diffusion. Right-side out vesicles such as EDTA-lysozyme vesicles take up proline and other solutes depending on respiration, although efficiencies of respiratory substrates as energy donors in supporting active uptake are different from each other. These observations were argued in relation to coupling efficiencies of carriers (22, 23) or changes in localization of primary dehydrogenase in the respiratory chain during preparation of the membranes (77, 72). In this connection, recent reports describing reconstitution of certain oxidase activities in membrane vesicles and of respiration-driven proline uptake were interesting. For instance, Futai (24) reconstituted r>lactate or glyceroI-3-phosphate oxidase activity by incubating right-side out vesicles defective in the respective dehydrogenase activity with purified dehydrogenase. It was shown that the activities of D-lactate dehydrogenase and glycerol-3-phosphate dehydrogenase bound to the membranes were completely inhibitable by specific antibody (13, 14) and ferricyanide (24), respectively, and the rates of proline uptake by the reconstituted vesicles were nearly equal to control membrane vesicles (24). There have been several
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ORIENTATION OF CYTOPLASMIC MEMBRANE VESICLES papers reporting that changes in the location of certain membrane enzymes occurred during preparation of EDTA-lysozyme vesicles and these changes were interpreted by a mechanism of dislocation (12, 25) or mosaicism (16). These findings suggest that changes in the location of enzymes were inevitable during preparation of membrane vesicles and that one must be careful in estimating the orientation of vesicles by studying the location of certain enzymes. Recently, Adler and Rosen stated that EDTAlysozyme vesicles consist of one homogeneous population of mosaic vesicles and do not consist of right-side out vesicles (16). They estimated the direction of the proton motive force generated by NADH oxidation and ATP hydrolysis indirectly by measuring uptake of Ca' + and inhibition of this Ca1+ uptake by D-lactate. Their results were consistent with the interpretation that external NADH dehydrogenase or ATPase in these vesicles could establish a proton motive force of the opposite polarity from that established in the same population through D-lactate oxidation. On the basis of this and other lines of evidence they claimed that the vesicles are mosaic and not hybrid since they observed that NADH dehydrogenase and ATPase distributed randomly on both sides of the membranes (16) while D-lactate dehydrogenase did not (12-14). It should be noted that direct measurement of the direction of the proton motive force should be done to obtain conclusive evidence for functional mosaicism. In this connection, studies on the topology of the whole electron transport chain are required for differentiating between mosaic vesicles and right-side out vesicles having dislocated membrane enzymes. In this paper we attempted to estimate the orientation of IM vesicles on the basis of these previous findings and arguments mentioned above. The primary criterion taken into account was NADH-dependent Ca1+ uptake by IM vesicles and its inhibition by D-lactate. The second was studies of enzyme location on both sides of IM vesicles using specific antibody and ferricyanide, an impermeant electron acceptor. The third was morphological examination of the vesicles by means of electron-microscopy using the negative staining technique. The results suggested that IM vesicles consisted of heterogeneous vesicles showing different orientation. The majority of the vesicles Vol. 83, No. 1, 1978
was right-side out closed vesicles with certain marker enzymes dislocated. MATERIALS AND METHODS Materials—Purified ATPase (specific activity, 80 units per mg protein) was prepared and stored as described previously (26). Anti-serum against purified ATPase (11) and antibody (immunoglobulin G) against D-lactate dehydrogenase (12) used were prepared as previously reported. Chemicals were obtained as follows: [14C]-proline (231 mCi/ mmol), Dai-ichi Chem. Co., Tokyo; "CaCl,(600 mCi/mmol), Radiochemical Centre, Amersham; D,L-glycerol-3-phosphate, Nakarai Chem. Co., Kyoto; D-lactate and 3-(4,5-dimethyl(thiazolyl-2>2,5-diphenyl tetrazolium bromide, Sigma Chem. Co., St. Louis. Other reagents used were of the highest grade commercially available. Strain and Culture Conditions—E. coli K12 strain W3092 (27) was grown aerobically as described previously (28). Preparation of Membrane Vesicles—IM vesicles were prepared by the published procedure (1) except that the pressure of a French press used was 200 kg/cm1. French press vesicles were prepared by the published procedure (11) using a French press cell (Ohtake Co., Tokyo) at 200 kg/cm1 and suspended in a solution of 0.05 M Tris-HCl, pH 8.0, containing 10 ITIM MgCl,. Treatment with RNase was omitted in this study. Assays of Proline and Cat+ Uptake—Assay of proline uptake was carried out as described previously (29) with a slight modification. The incubation mixture contained 50 ITIM Tris-maleate, pH 8.0, 2 ITIM MgSO4, 8 (M "C-proline, membrane vesicles (10-100 ftg of membrane protein) and respiratory substrate (20 ITIM D-lactate (Li) or 20 ITIM glycerol-3-phosphate(Na)). Assay of Ca1+ uptake was carried out as described by Tsuchiya and Rosen (20) with 2.5 nun "CaCl, as substrate. Measurement of Ferricyanide Reductase—The incubation mixture contained 0.7 mM potassium ferricyanide, 0.08 M Tris-HCl, pH 7.3, 25 mM glycerol-3-phosphate, 9 mM KCN, and membrane vesicles (100 fi% of membrane protein) in a total volume of 1.0 ml. After incubation at 30°C for 5 min, 0.15 ml of 1.0 M trichloroacetic acid was added. The supernatant obtained after centrifugation was assayed for ferrocyanide formed (30).
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The supernatant (0.3 ml) was mixed with 0.7 ml of 2.0 M sodium formate, pH 3.0, 0.3 ml of 3.0 ITIM
bathophenanthroline sulfonate and 0.3 ml of 4.0 mM Fe(NO3)3-5.6 IHM EDTA. The mixture was incubated for 10 min at room temperature and the absorbancy at 535 nm was measured. Ferrocyanide formed was estimated from standard ferrocyanide treated as above. Other Assays—D-Lactate dehydrogenase (31), ATPase (26), and protein (32) were assayed by the published procedures. Oxygen consumption was measured as described previously (28) in the buffer used for the uptake assay. Electron Microscopy—IM vesicles and French press vesicles were collected and suspended in 0.15 M sodium phosphate, pH 7.2, before use. For negative staining a drop of the membrane suspension on a carbon-coated collodion grid (400 mesh) was mixed with 2 % uranylformate (pH 7.0) repeatedly. Then the excess fluid was removed and the specimen was dried by touching it with a piece of filter paper at room temperature. Samples were examined with a JEM-100B electron microscope at 80 kV. Negative staining of ATPase was carried out similarly as described above using the sample solution prepared as mentioned. RESULTS 1) Heterogeneity of IM Vesicles in Orientation—Since IM vesicles were prepared by disrupting spheroplasts in 20% sucrose-3.0 ITIM EDTA, pH 7.2, in a French press (1), the preparation may contain membrane vesicles of different orientations. Heterogeneous orientation of membranes may be categorized into three major groups: 1) right-side out vesicles, 2) inverted vesicles, and 3) mosaic vesicles. We are able to estimate the presence or absence of these three types of vesicles in the IM preparation by measuring the uptake of Ca l+ and proline driven by D-lactate and/or NADH oxidation, as suggested by Adler and Rosen (16). Table I shows that IM vesicles took up Ca I+ with NADH as respiratory substrate. This result suggests that NADH generated the proton motive force of the direction inside positive in the inverted or mosaic vesicles (75, 16). D-Lactate did not inhibit the NADH-dependent Ca1+ uptake. This finding
TABLE I. Respiration-driven uptake of calcium ions and proline in IM vesicles. Assays were carried out under standard conditions (see "MATERIALS AND METHODS ") at 25°C for 1 min. Respiratory substrate D-Lactate D-Lactate+NADH NADH
CaI+ uptake1 Proline uptake11 (nmol/min/mg protein) -0.6 3.9 4.0
0.60 0.69 0.14
» Uptake of CaI+ by 2.8 nmol/min/mg protein was observed in the absence of respiratory substrate and this value was used for correction of the respiration driven Ca*+ uptake. Endogenous Ca*+ uptake was also observed in EDTA-lysozyme vesicles (16). b Concentration of proline used was 2 /JM. suggests that the IM preparation contained no mosaic vesicles, in contrast to the result of EDTAlysozyme vesicles studied by Adler and Rosen (16). Furthermore, we could not detect D-lactate-dependent Ca1+ uptake, suggesting that the IM preparation contained inverted vesicles as a minor population. The IM vesicles took up proline actively depending on D-lactate. NADH supported the proline uptake (Table I) but its coupling efficiency was as low as the isoleucine uptake (2). We also found that the effect of NADH on the D-lactatedependent proline uptake was somehow additive but not inhibitory. On the basis of these findings we concluded that IM vesicles consist of membranes with different orientations; the preparation may contain right-side out and inverted vesicles but not mosaic vesicles. Unsealed membrane fragments which have no uptake activities may also be present in the preparation. 2) Orientation of IM Vesicles Studied by Localization of Membrane Enzymes—As D-lactate dehydrogenase and glycerol-3-phosphate-ferricyanide reductase are located normally on the inside surface of cytoplasmic membrane (11-14), we are able to take these activities as marker enzymes for further estimation of the orientation of IM vesicles. According to the argument described above it is possible that the enzymes in IM vesicles are located in three ways as shown in Fig. 1: (A) The enzymes are located in the original orientation in right-side out vesicles, (B) the enzymes are rearranged during preparation and / . Biochem.
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IM vesicles "Right-side out" vesicles (capable of respirationdriven proline uptake)
—-. "Unsealed" or "Inverted" vesicles (incapable of respiration-driven proline uptake)
(70%)
(30*)
inner surface outer surface
D-LDH on Tnner surface
(401)
D-LDH on outer surface
(30*) B
Fig. 1. Location of D-lactate dehydrogenase in the preparation of IM vesicles. Populations of D-lactate dehydrogenase of different locations in the vesicles estimated in this paper, are summarized schematically: Enzymes located in the original orientation in right-side out vesicles (A), 40%; enzymes located on the outside of the right-side out vesicles (B), 30%; enzymes located in unsealed or inverted vesicles (C), 30%. D-Lactate dehydrogenase (D-LDH) ( • ) is drawn schematically in the figure. For further details, see text.
located on the outside of the right-side out vesicles, ( Q the enzymes are located in inverted vesicles or unsealed membrane fragments. The enzymes located on the inner surface of inverted vesicles may be minor, because all the inner surface enzymes so far studied were located on the outside of such vesicles prepared by a French press (11, 12). Since specific antibody against D-lactate dehydrogenase was accessible to the enzyme located in reconstituted vesicles (24) or inverted vesicles (12), we could estimate the population of the enzyme located differently as mentioned above. However, we cannot estimate the orientation of IM vesicles by only determining the location of enzymes. Since IM vesicles may contain inverted vesicles or unsealed membrane fragments (Table I and Fig. 1), inhibition study on the D-lactateVol. 83, No. 1, 1978
dependent proline uptake by specific antibody against D-lactate dehydrogenase is required. Through such studies we can estimate the proportions of right-side out vesicles and inverted vesicles in the preparation. Figure 2 shows that the antibody against D-lactate dehydrogenase inhibited 60% of the Dlactate dehydrogenase activity and D-lactate oxidation in IM vesicles. This indicates that 60% of the total D-lactate dehydrogenase of the membranes were accessible by the antibody. In other words, the activity inhibited by the antibody was due to the enzyme located outside the vesicles as described in (B) and ( Q and the rest (40% of the total) located inside as in (A) (see Fig. 1). Therefore, the ratio of the enzyme located on the inner surface of right-side out vesicles ( A / A + B + Q should be 0.4.
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0
0.2 0.4 0.6 0.8 1.0 ANTIBODY(mg)/D-LACTATE DEHYDROGENASE(unit)
Fig. 2. Effect of antibody against D-lactate dehydrogenase on D-lactate dehydrogenase and oxygen consumption dependent on D-lactate in IM vesicles. IM vesicles (2.1 mg protein/ml) were incubated with varying concentrations of antibody (immunoglobulin G fraction from immunized rabbits) in 0.05 M Tris-maleate, pH 8.0, containing 2 ITIM MgSCV D-Lactate dehydrogenase (O) and oxygen consumption dependent on D-lactate (•) were assayed after three hours of incubation. Results of assay of D-lactate dehydrogenase are expressed as relative rates, % of control. Control rate was 1.47 /umol/min/mg. On the other hand, the antibody inhibited 40% of the proline uptake in IM vesicles (Fig. 3). If we assume that the dehydrogenase located as in (A) and (B) can support transport equally, this suggests that 40% of the D-lactate dehydrogenase in right-side out vesicles was on the outer surface of the vesicles (Location (B)), and the rest of the dehydrogenase was on the inner surface (Location (A)). Thus we can estimate the portion of the enzyme located on the inner surface of the rightside out vesicles (A): A/A+B=0.6 We can calculate the population of the rightside out vesicles from the ratio of the enzyme located as in (A) and (B); the ratio of right-side out vesicles to the total population was 0.67 ( A + B / A + B + Q , suggesting that 67% of the IM vesicles were right-side out. The rest (33%) may be inverted vesicles or unsealed membrane fragments, because the possibility that the IM preparation contained mosaic vesicles was slight (Table
0
0.2 0.4 0.6 0.8 1.0
ANTIBODY(mg)/D-LACTATE DEHYDROGENASE(unit)
Fig. 3. Effect of antibody against D-lactate dehydrogenase on proline uptake stimulated by D-lactate in IM vesicles. IM vesicles were incubated with antibody against D-lactate dehydrogenase as described in the legend of Fig. 2. After incubation for 2 h, proline uptake was assayed as described in the text. TABLE II. Ferricyanide reductase activity in IM vesicles prepared from E. coli W3092. IM vesicles (8.2 mg protein/ml) were incubated in 10% sucrose containing 1% toluene at 37°C for 10 min. Aliquots of 10 to 50 ft\ of toluene-treated and control membranes were assayed for glycerol 3-phosphate-ferricyanide reductase with the indicated concentration of ferricyanide in the presence of 9 mM KCN. Other procedures are described in the text. Concentration of femcyanide 0.7 mM 1.8 mM
Ferricyanide reduction (/imol reduced/mg/min) Control (untreated)
Treated with toluene
0.18
0.26 0.17
0.12
ferricyanide accepts electrons from the respiratory chain are located on the inner surface of the cytoplasmic membrane. Thus, a ferricyanide reductase activity was not measurable in spheroplasts and intact cells, unless permeability barriers were destroyed with toluene or detergents (11, 33). Based on this and other evidence, Futai suggested that measurement of ferricyanide reductase activity under proper experimental conditions proD. vides useful information on the orientation of 3) Location of Ferricyanide Reductase in IM membrane vesicles (11). Vesicles—As shown previously (77), the sites where J. Biochem.
ORIENTATION OF CYTOPLASMIC MEMBRANE VESICLES
B Fig. 4. Electronmicrographs of IM vesicles and purified ATPase showing typical morphological appearances. A) IM vesicles obtained by the published procedure (7) were stained with 2% uranylformate (see " MATERIALS AND METHODS "). Typical morphological appearances of the vesicles are seen in this picture. Globular particles with stalks are seen on the inner edge of the membrane (shown by arrows). These globular particles were considered to be ATPase as discussed in the text. Final magnification x 269,000. B) Electronmicrograph of an IM vesicle having globular particles on its outer surface. Final magnification x 243,000. C) Electronmicrograph of purified ATPase. ATPase purified by the published procedure (26) was stained with 2% uranylformate. Final magnification x 263,000.
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Fig. 5. Electronmicrograph of an inverted vesicle prepared by disrupting cells with a French press. Typical morphological appearance of an inverted vesicle is shown. Globular particles are seen on the outer surface of the vesicle. Larger particles seen around the vesicles were considered to be ribosomes from their sizes (41) (shown by arrows). Final magnification x 269,000.
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TABLE III. Effects of ferricyanide on glycerol-3-phosphate-dependent oxidation and proline uptake in IM vesicles. Oxygen consumption and proline uptake stimulated by glycerol-3-phosphate were assayed in the presence and absence of ferricyanide. Assay procedures are described in " MATERIALS AND METHODS." Inhibition of oxygen consumption by ferricyanide was measured after adding a small volume (34 //!) of ferricyanide solutions of different concentrations to the reaction mixture incubated with glycerol-3-phosphate. Oxygen consumption and proline uptake are expressed as /Jmoles of O t consumed and nmoles proline taken up per min per mg protein, respectively. Activity Oxygen consumption Proline uptake
Activity before addion of ferricyanide 0.31 0.42
Table II shows the ferricyanide reductase activity of IM vesicles with glycerol-3-phosphate as substrate. Although ferricyanide affected the apparent activity in higher concentration, about 70% of the total sites in IM vesicles were shown to be available for electron transfer under the experimental conditions. The above results suggest that about 70% of the ferricyanide reactive sites were either on the outside of the right-side out vesicles or inverted vesicles and 30% of the sites on the inner surface of the right-side out vesicles. These values are comparable with the results obtained using antibody and suggest that random distribution of membrane marker enzymes had occurred independently during the preparation of IM vesicles. Although details of the calculation are not shown here, essentially similar values for the location of ferricyanide reactive sites were obtained from the following results. It has been established that glycerol-3-phosphate stimulates proline uptake in membrane vesicles and that ferricyanide inhibits oxidation of glycerol-3-phosphate by vesicles as well as the respiration dependent uptake of amino acids (24). Table III summarizes the inhibition of glycerol-3-phosphate oxidation and glycerol-3phosphate dependent-proline uptake in IM vesicles by ferricyanide. The ferricyanide inhibited 38% of the proline uptake and 60% of the oxygen consumption and this result was consistent with the conclusion for orientations of IM vesicles, obtained using antibody (see Fig. 1). 4) Electron Micrograph of IM Vesicles—IM vesicles retained about 20% of the total ATPase activity of the crude membrane fraction when Vol. 83, No. 1, 1978
Activity in the presence of ferricyanide (2 ) 0.12 0.26
Inhibition by ferricyanide (%) 60 38
prepared by the published method (1). Localization of this activity in these vesicles was examined using antiserum against ATPase. Table IV shows that only 18% of the ATPase activity in the IM vesicles was measurable without toluene treatment and inhibitable almost completely by antiserum. This indicates that about 20% of the ATPase activity of IM vesicles seems to be on the outer surface of the closed vesicles or on the unsealed membrane fragments. The molecular organization (34-36) and morphology (34, 37, 38) of bacterial ATPase (F^ coupling factor of oxidative phosphorylation) have been reported, which are essentially identical with those of mitochondria! Fx (see reviews, Refs. 39,40). Therefore, we examined the structure of IM vesicles and localization of ATPase on the membranes electron-microscopically using negative staining with uranylformate. TABLE IV. ATPase activity of IM vesicles. IM vesicles (40-80 ftg protein/ml) were incubated in 10% sucrose containing 1% toluene at 37°C for 10 min. Samples of 10 to 50 fi\ of toluene-treated and control membranes were assayed for ATPase activity as described previously (26). Antiserum against purified ATPase (3 fi\) was added to the incubation mixture. Serum from preimmune rabbits had no effect on ATPase activity.
Addition
None Antiserum
ATPase activity (ftmo\ Pi released/min/mg protein) No treatment
Treated with toluene
0.20 0.03
1.10 0.09
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Figure 4C shows the morphological appearance of ATPase. The globular particles of about 10 nm in diameter are seen to be the major population. Morphologically, the particles show a polyhedral view and have one or a few holes and grooves on the surface of the particle. The morphology of these particles is similar to ATPases from other sources (34-36). In the present study, we stained the samples with phosphotungstate, silicontungstate, uranylacetate as well as uranylformate and found that the latter stain gives better contrast than the former three, although all these reagents showed essentially the same structure of ATPase and membranes. Figure 4A shows typical morphological appearances of IM vesicles. The vesicles were heterogeneous in sizes in the range of 50-200 nm in diameter. However, most of the population consisted of closed vesicles surrounded by the membrane of about 10 nm thickness. It was also found that the vesicles contained a number of globular particles on the membranes, some with stalks as shown by arrows (Fig. 4A). From the morphological appearance and dimension of the particles, it was suggested that these particles were ATPase and located on the inside surface of the closed '-ssicles. Sometimes the globular particles were seen more clearly on a few IM vesicles than those in Fig. 4A. Figure 4B shows a typical example; this IM vesicle shows clusters of globular particles, the fine structure of which are identical with those of ATPase in Fig. 4C. In addition, this particular vesicle has globular particles on its edge (shown by arrows) but the edge of the vesicle does not show the membraneous structure seen in Fig. 4A. These characteristics suggest that this vesicle was inverted, thereby exposing the ATPase on the outer surface of the membrane. As mentioned already, French press vesicles are known to be completely inverted (11). Moreover, since dislocation of ATPase did not take place during the inversion of the cytoplasmic membrane by this procedure, we are interested in examining the morphological structure of French press vesicles as a control for the inversion of IM vesicles. Figure 5 shows an electronmicrograph of an inverted vesicle. This particular vesicle was larger than the IM vesicles (see Fig. 4A) and had a number
of polyhedral particles on the membrane with dimension identical to that of ATPase (see Fig. 4C). The morphological appearance of the particles is clear and resembles those observed in Fig. 4B. These observations suggest that ATPase molecules, when located on the outer surface of the inverted vesicles, were stained in clearer contrast by uranylformate under the conditions used. There are a number of larger particles than ATPase around the vesicles (Fig. 5). These particles are considered to be ribosomes based on their dimension and morphological appearance (41). We think that a minor population of IM vesicles consisted of inverted vesicles, on the basis of these morphological studies. In the preparation of IM vesicles so far examined, less than 5 to 10% of the total population were vesicles having the morphological characteristics as shown in Fig. 4B. The observed value was close to and consistent with those obtained by estimation of enzyme location (Table IV), indicating heterogeneity of IM vesicles in orientation. DISCUSSION We have presented evidence that IM vesicles prepared by our new procedure (1) consisted of heterogeneous populations in their orientation of membranes. It was possible to estimate that about 70% of the IM vesicles were right-side out assuming the following: 1) Dislocated D-lactate dehydrogenase on the right-side out closed vesicles can energize proline uptake as efficiently as that localized normally (14, 24), although the actual coupling efficiency could not be estimated. 2) No dislocation of the enzymes occurs in the inverted vesicles. All the D-lactate dehydrogenase or ferricyanide reactive sites are located on the outer surface of inverted vesicles prepared by a French press (11, 12), though the method of membrane preparation is different from ours. 3) D-Lactatedependent respiration is the rate limiting step of proline uptake. This was suggested by the finding that the ratios of stimulation of oxygen consumption and proline uptake were essentially the same for various respiratory substrates except NADH (2). 4) Right-side out vesicles with Dlactate dehydrogenase dislocated and/or located normally, inverted vesicles with all the D-lactate dehydrogenase on the outer surface and unsealed
/. Biochem.
ORIENTATION OF CYTOPLASMIC MEMBRANE VESICLES membrane fragments are major populations in the preparation. We found that IM vesicles took up Ca I+ with NADH as respiratory substrate but that the NADHdependent Ca I+ uptake was not inhibited by simultaneous oxidation of D-lactate (Table I). This finding strongly suggested that the IM preparation did not contain mosaic vesicles and that Ca1+ was taken up by inverted vesicles. D-Lactate did not stimulate Ca1+ uptake. This implicated that the inverted vesicles were a minor population. The presence of inverted vesicles, the population being less than 30%, was suggested by enzyme location (Fig. 3 and Tables HI and IV) and the morphology of membranes (Fig. 5B). On the basis of these findings and the criteria stated by Adler and Rosen (16), we concluded that the IM vesicles may not contain mosaic vesicles. We also showed that IM vesicles were heterogeneous with respect to the localization of membrane proteins. As estimated (see Fig. 1), a significant portion of the right-side out vesicles were found to have marker enzymes dislocated. Changes in the location or dislocation of D-lactate dehydrogenase and ferricyanide reactive sites seemed to occur randomly during the preparation of IM vesicles because the apparent extent of inhibition of these activities was essentially the same (Fig. 2 and Table II). It should be noted that random distribution of membrane proteins may take place through the fusion between rightside out and inverted vesicles. In order to distinguish enzyme dislocation from membrane fusion, it would be desirable to have marker proteins originally located on the outside of the cytoplasmic membrane. More important would be studies on the entire topology of the electron transport chain and its changes during the preparation of membrane vesicles by different procedures. We prefer to interprete our data by dislocation of enzymes according to the observations at present available (11, 12, 21, 24), although the mechanism of dislocation is not known yet. Several investigators have discussed dislocation or non-random distribution of membrane marker enzymes in EDTAlysozyme vesicles (12, 16, 25). It is important to note that use of a French press to disrupt spheroplasts in our procedure did not give totally inverted vesicles as shown above. This may have resulted from disrupting spheroVol. 83, No. 1, 1978
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plasts in 20% sucrose-3 ITIM EDTA, which is not included in the medium to make inverted vesicles by a French press from intact cells (11, 15, 19). It should also be noted that IM vesicles are smaller than EDTA-lysozyme vesicles in size (3) and much smaller than French press vesicles (cf. Figs. 4A and 5). These and the apparent differences of the location of D-lactate dehydrogenase in IM vesicles from EDTA-lysozyme vesicles (12) may be due to the use of French press for the preparation of IM vesicles. In conclusion, the major population of IM vesicles consists of right-side out vesicles with sizes ranging from 50 to 200 nm in diameter. We suggest that these structural properties and the high purity (7) of the membranes support high transport activities for various amino acids and sugars (2). We are grateful to Dr. L.A. Heppel for his instruction of ferricyanide reductase assay, and to Drs. B.P. Rosen and F.M. Harold for showing us their manuscripts before publication. We also thank Dr. Y. Tanaka for his help in preparing the antibody against purified r> lactate dehydrogenase. REFERENCES 1. Yamato, I., Anraku, Y., & Hirosawa, K. (1975) /. Biochem. 11, 705-718 2. Yamato, I. & Anraku, Y. (1977) J. Biochem. 81, 1517-1523 3. Kaback, H.R. (1971) Methods in Enzymology 22, 99-120 4. Miura, T. & Mizushima, S. (1968) Biochim. Biophys. Acta 150, 159-161 5. Osborn, M.J., Gander, J.E., Parise, E., & Carson, J. (1972) / . Biol. Chem. 247, 3962-3972 6. Schnaitman, C.A. (1970) J. Bacleriol. 104, 890-901 7. Mizushima, S. (1976) Biochim. Biophys. Acta 419, 261-270 8. Kaback, H.R. (1974) Science 186, 882-892 9. Simoni, R.D. & Postma, P.W. (1975) Annual Rev. Biochem. 44, 523-554 10. Harold, F.M. (1977) in Current Topics in Bioenergetics (Sanadi, D.R., ed.) Vol. 6, pp. 84-151, Academic Press, New York & London 11. Futai, M. (1974) / . Membrane Biol. 15, 15-28 12. Futai, M. & Tanaka, Y. (1975) J. Bacterial. 124, 470-475 13. Short, S.A., Kaback, H.R., Kaczorowski, G., Fisher, J., Walsh, C.T., & Silverstein, S.C. (1974) Proc. Nail. Acad. Sci. U.S. 71, 5032-5036
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14. Short, S.A., Kaback, H.R., & Kohn, L.D. (1975) J. Biol. Chem. 250, 4291^(296 15. Rosen, B.P. & McClees, J.S. (1974) Proc. Nail. Acad. Sci. U.S. 71, 5042-5046 16. Adler, L.W. & Rosen, B.P. (1977) /. Bacteriol. 129, 959-966 17. Wickner, W. (1975) Proc. Nail. Acad. Sci. U.S. 72, 4749-4753 18. Wickner, W. (1976) Proc. Natl. Acad. Sci. U.S. 73, 1159-1163 19. Hertzberg, E.L. & Hinkle, P.C. (1974) Biochem. Biophys. Res. Commun. 58, 178-184 20. Tsuchiya, T. & Rosen, B.P. (1975) J. Biol. Chem. 250, 7687-7692 21. Amanuma, H., Itoh, J., & Anraku, Y. (1977) FEBS Lett. 78, 173-176 22. Barnes, E.M., Jr. & Kaback, H.R. (1971) / . Biol. Chem. 246, 5518-5522 23. Kaback, H.R. & Barnes, E.M., Jr. (1971) J. Biol. Chem. 246, 5523-5531 24. Futai, M. (1974) Biochemistry 13, 2327-2333 25. Altendorf, K.H. & Staehelin, L.A. (1974) / . Bacteriol. 117, 888-899 26. Futai, M., Sternweis, P.C, & Heppel, L.A. (1974) Proc. Natl. Acad. Sci. U.S. 71, 2725-2729 27. Anraku, Y. (1971) J. Biochem. 70, 855-866 28. Kasahara, M. & Anraku, Y. (1972) / . Biochem. 74, 777-781
29. Kasahara, M. & Anraku, Y. (1974) / . Biochem. 76, 977-983 30. Shoen, R. (1965) Anal. Biochem. 12, 413-^20 31. Futai, M. (1973) Biochemistry 12, 2468-2474 32. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 33. Weiner, J.H. (1974) /. Membrane Biol. 15, 1-14 34. Kagawa, Y., Sone, N., Yoshida, M., Hirata, H., & Okamoto, H. (1976) J. Biochem. 80, 141-151 35. Abrams, A., Jensen, C , & Morris, D.H. (1976) Biochem. Biophys. Res. Commun. 69, 804-811 36. Johansson, B.C. & Baltscheffsky, M. (1975) FEBS Lett. 53, 221-224 37. Gel'man, N.S., Lukoyanova, M.A., & Ostrovskii, D.N. (1975) Bacterial membranes and the respiratory chain, in Biomembranes 38. Schnebli, H., Vatter, A.E., & Abrams, A. (1970) J. Biol. Chem. 245, 1122-1127 39. Senior, A.E. (1973) Biochim. Biophys. Ada 301, 249-277 40. Kagawa, Y. (1972) Biochim. Biophys. Ada 265, 297-338 41. Lake.J.A., Sabatini, D., & Nonomura, Y. (1974) in Ribosomes (Nomura, M., ed.) pp. 543-557, Cold Spring Harbor Lab.
/ . Biochem.
