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Reassembly of Mycoplasma membranes disaggregated by detergents
ARCHIVES
OF
BIOCHEMISTRY
Reassembly
AND
BIOPHYSICS
46-56
126,
of Mycoplasma
(1968)
Membranes
Disaggregated
by
Detergents’ S. ROTTENI, Department
of
OLGA
STEIN,
AND
S. RAZIN
Clinical Microbiology, and Department of Experimental Medicine and The Hebrew University-Hadassah Medical School, Jerusalem, Israel Received
August
Cancer
Research,
28, 1967
Plasma membranes of Mycoplasma laidlawii and Mycoplasma gallisepticum were solubilized by several ionic and non-ionic detergents. Solubilization of the membranes by sodium dodecyl sulfate (SDS) separated membrane lipid from protein as demonstrated by polyacrylamide gel electrophoresis of the solubilized material. The solubilized membrane material reaggregated spontaneously on removal of the detergent by dialysis or by Sephadex G-25, and formed vesicles limited by a triplelayered membrane of about the same thickness as the original Mycoplasma membrane. A divalent cation (e.g., Mg2+) was essential for membrane reassembly. The ratio of lipid to protein in membrane reaggregates varied considerably according to the Mg*+ concentration. At a low Mg2+ concentration reaggregates contained a higher percentage of lipid. The present results seem to bear out the suggestion of Engelman et al. [Biochim. Biophys. Acta 136, 381 (1967)] that the SDS-solubilized membrane material does not. consist of homogeneous lipoprotein subunits but of separate SDS-lipid and SDS-protein complexes. The reassembly of solubilized membrane lipid and protein on remov.11 of the detergent indicates that these components contain sufficient structure-determining information to interact spontaneollsly in the presence of Mg2+ and produce membraneous structures.
centrifugally homogeneous lipoprotein subunits (2). If so, then in the det’ergent-solubilized material, membrane lipid should be bound to membrane protein. Alt,hough electrophoresis on cellulose acetate strips, and Sephadex G-200 filtration failed to separate the lipid from protein in the solubilized membrane material, the lipid peak did not coincide with the protein peak on centrifugation in a sucrose density gradient. Hence at least part of the lipid had separated from the protein (5). Engelman et al. (4) recently succeeded in separating the lipid from the protein in SDS-solubilized Mycoplasma laidlawii membranes by prolonged centrifugation in density gradients. Accordingly, the disaggregated membrane material does not consist of lipoprotein subunits. This report provides additional evidence for the separation of the lipid from protein
Mycoplasma are bounded by a single lipoprotein membrane, which can be readily isolated by osmotic lysis of the organisms (1). The mode of organization of protein and lipid in this membrane has been the subject of several recent reports (l-5). Disaggregation of Mycoplasma membranes by sodium dodecyl sulfate (SDS) resulted in a clear solution exhibiting a single symmetrical schlieren peak in the analytical ultracentrifuge. It was thought that this peak, which had an uncorrected sedimentation coefficient of about 3S, represented 1 This work was supported by grant FG-Is-174 from the United States Department of Agriculture under Public Law 480. 2 Taken in part from a dissertation to be submitted by the senior author in partial fulfillment of the requirements for the Ph.D. degree from the Hebrew University, Jerusalem, Israel. 46
REASSEMBLY
OF
illYCOPLASMA
in SDS - disaggregated Mycoplaslna membranes. Removal of the detergent from the disaggregated membrane material by dialysis against dilute buffer containing magnesium has resulted in the reassembly of the solubilized membrane components to a membraneous structure (2, 3). This spontaneous reassembly of disaggregated membrane material has been taken in support of the subunit hypot’hesis of membrane struct’ure (2, 6). The present investigation of the fat t,ors governing the self-assembly of membrane mat’erial was undertaken to elucidate the mechanism of this interesting phenomenon. K2TERIALS
AND
METHODS
Organisms and growth conditions. Mycoplasma Zaidlawii (oral strain) was isolated from the human oral cavity and was found to be related to Hycoplasma laidlawii strain A (7). Mycoplasma gallisepticutn strain A5969 was obtained from 11. E. Tourtellotte (Department of Animal Diseases, University of Connecticut, Storrs). The organisms were grown in 3- to g-liter volumes of a modified Edward medium (8) containing 2yo PPLO serum fraction (Difco). To label the membrane lipids, 1 PC of uniformly labeled sodium acetate-14C and 1 PC of oleic acid-l-‘% (The Radiochemical Centre, Amersham, England) were added to each liter of the growth medium. The organisms were harvested after 24-48 hours of incubation at. 37” by centrifugation at 13,000g for 10 minutes. The sedimented cells were washed twice in 0.25 hl NaCl. Isolation of cell membranes. Cell membranes were isolated by osmotic lysis of the organisms. For osmotic lysis of $2. Zaidlawii, the washed sedimented cells were resuspended in 250 ml of deionized water and incubated at 37” for 15 minutes. The more osmotically resistant Jf. galZisepticllm cells were lysed by a modification of the method devised by Robrish and Marr (9). The cells were resuspended in 5 ml of a 2 M glycerol solution and incubated at 37” for 10 minutes. The suspension was t,hen rapidly injected into 250 ml of deionized water and incubated at 37” for 15 The treated Mycoplasw~a suspensions minutes. were centrifuged at 8OOOg for 5 minutes to remove clumps of unbroken cells. The supernatant fluid was then cent,rifuged for 30 minutes at 34,OOOg to collect the membranes. The membranes were washed ten times alternatively with deionized m-ater and 0.05 M NaCl in 0.01 M phosphate buffer, pH 7..5. The washed membranes were re-
MEMBRANES
47
suspended in p-buffer (NaCl, 0.15 M; Tris, 0.05 31; 2-mercaptoethanol, 0.01 hf; in deionized water adjusted to pH 7.4 with HCl; Ref. lo), diluted 1:20 in deionized water (will be referred to as “dilute p-buffer”), and kept at -20” until used. The amount of protein in membrane suspensions was determined according to Lowry et 01. (11). Only traces of nucleic acids could be found in the membranes. Thin sections and negative staining of the membrane preparations with phosphotungstic acid displayed membraneous structures and showed little contamination with nonmembraneous material. Assessment of membrane solubilization by detergents. Various amounts of the tested detergents were added to 4 ml of membrane suspension in dilute p-buffer. The degree of membrane solubilization was estimated after incubation at 37” for 1,s minutes by measuring the decrease of the optical density of the membrane suspension at 500 m*. Removal of detergents by gel $ltration. Sephadex G-25 (Pharmacia AB, Uppsala, Sweden) was used to remove sodium dodecyl sulfate (SDS) or cetyltrimethylammonium bromide (CTAB) from membrane material, and Sephadex G-200 was used to remove Triton X-100. The columns (1.8 X 34 cm) were equilibrated with dilute p-buffer containing 5 X lo-” M EDTA. For the removal of SDS, onehalf ml of membrane suspension (containing 1.5-3 mg protein) solubilized in 0.02 M SDS containing 1 ~tc of 3%-SDS (The Radiochemical Centre, Amersham, England) was placed on the column. Elution was performed with dilute pbuffer containing 5 X 1OW M EDTA. Two-ml fractions were collected and assayed for protein and radioactivity. For the removal of Triton X-100, 0.2 ml of membranes (containing 2-3 mg protein) solubilized by Triton X-100 were placed on the column. Elution and fraction collection were performed as above. The fractions were assayed for protein and for Triton X-100. The presence of Triton X-100 in the fractions was determined by adding 0.5 ml of 2Oc/ Na&Os in I N NaOH to 1 ml of each fraction. Turbidity indicated the presence of Triton X-100. Isolation of membrane protein and lipid. Hydrophobic membrane proteins were separated from membrane lipids by solubilization of the membranes with a mixture of SDS and sodium deoxy cholate (DOC) and precipitation with ammonium sulfate (;i). Membrane lipids were extracted with chloroform-methanol (2: 1, v/v) and washed according to Folch et al. (12). The lipid solution in chloroform-methanol was brought to a volume of 0.5 ml by evaporation under nitrogen. Dilute p-buffer cont,aining 0.02 Y SDS (1.3 ml) was added
48
ROTTEM,
STEIN,
to the lipid solution. The resulting turbid emulsion cleared when the solvents were removed completely by a stream of nitrogen. Reaggregation of solubilized membranes. Labeled membranes solubilized by detergents were dialyzed for 34 days at 4” against 500-1000 volumes of dilute p-buffer containing various amounts of MgClz (2). The content of the dialysis bags was then centrifuged at 37,000g for 1 hour in the cold. Both sediment and supernatant fluid were assayed for protein and radioactivity. Sucrose gradient centrifugation. Samples (0.15 ml) of labeled membranes or membrane reaggregates were layered over 4.2 ml of linear sucrose gradients (28-70% sucrose). The gradients were centrifuged at 39,000 rpm in an SW 39 head of a Spinco model L-2 ultracentrifuge for 2 hours at 4”. Fractions (0.095 ml) were collected by puncturing the bottom of the centrifuge tubes, and were assayed for protein and radioactivity. Polyacrylamide gel electrophoresis. Membranes solubilized in 0.02 M SDS were analyzed in a gel system composed of 7.5yo Cyanogum 41 (a mixture of 95% acrylamide and 5% N,N-methylenebisacrylamide, supplied by E-C Apparatus Co. Philadelphia, Pennsylvania) in TEB buffer (Tris, 60.5 gm; EDTA, 6 gm; boric acid, 4.6 gm; and water to 1000 ml; final pH without adjustment, 8.8-8.9) diluted I:4 with water in 6 X IOO-mm glass tubes. Polymerization was induced by addition of 0.05 ml N,N,N’,N’-tetramethylethylenediamine (TMED) and 10 mg of ammonium persulfate to 20 ml of the Cyanogum solution. Three identical samples(lOO-150 pl, each containing about 100 pg protein) of SDS-solubilized membranes were mixed with 50 pl of a 607, sucrose solution and deposited on top of three of the gels. The tubes were filled up carefully with TEB buffer diluted 1:20 with water. The same dilute buffer was used to fill the reservoirs of the electrophoresis apparatus. Electrophoresis was carried out at room temperature for 1 hour at a constant current of 5 mA per tube; the lower electrode was used as anode. One of the gels was stained with 0.5yo Amido Black 10 B in 7yo acetic acid for 30 minutes. The second gel was cut into small sections which were transferred to scintillation vials and assayed for radioactivity. The third gel was cut into small sections, proteins were eluted from the gel sections by boiling in 1 ml of 0.1 N NaOH for 30 minutes, and protein was determined on the eluates. Radioactivity measurements. Radioactivity was determined in a Packard Tri-Carb scintillation counter with 10 ml of a scintillation mixture composed of 800 ml of dioxane, 150 ml toluene, 50 gm naphthalene, 10 gm PPO, and 150 mg POPOP (Packard Instrument Co. Inc., Illinois). Analytical ultracentrifuge analysis. Disaggre-
AND
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s oziz 4 “0 Ol-
l TRITON X-100
CONCENTRATION
FIG.
branes
1. Solubilization detergents.
OF DETERGENT
of
M.
(mg/ml)
Zaidlawii
mem-
by
gated membranes, membrane protein, or membrane lipid solubilized in 0.02 M SDS were centrifuged at 59,780 rpm in a Spinco model E analytical ultracentrifuge at 20’. Sedimentation peaks were observed by use of schlieren optics. Sedimentation coefficients were calculated without correcting for medium viscosity or density. Preparations of specimens for electron microscopy. A drop of the material to be studied was placed on collodion-coated, carbon-reinforced grids and mixed on the grid with a drop of a 1.7% solution of potassium phosphotungstate (PTA), pH 7. The material was mixed with the stain in a Pasteur pipette for about 30 seconds, and the excess fluid was removed from the grid with a fragment of filter paper. The grids were dried briefly in air and examined at 100 kV in an RCA EMU 3G electron microscope. RESULTS
Xolubili.zation of the membranes by detergents. Membranes of M. laidlawii and M. gallisepticum could be solubilized by several
ionic and non-ionic detergents. The ionic detergents SDS and CTAB were more effective in membrane solubilization than the non-ionic detergent Triton X-100, but Triton A-20 was totally ineffective (Fig. 1). The solubilization of the membranes by SDS caused at least a partial separation of membrane lipid from protein. Polyacrylamide gel electrophoresis of the solubilized membrane material showed a highly variable ratio of labeled lipid to protein in the different gel sections (Fig. 2). Removal of
REASSEMBLY PROTEIN
(Pd
LABELLED M.LAIDLAWII
MYCOPLASMA
LIPID
(cpm) 4815 200 62 36 22
500 346 32 6 0
OF
. - - __
10 0 0 0 0
M. GALLISEPTICUM 386 402 360 242 19 160 30 12 0 0
FIG. 2. Polyacrylamide Mycoplasma membranes S~~MATERIALSANDMETHODS mental procedure.
39 50 500 160 Ll 11&O 1700 1450 160 21 0
gel erectropnoresrs solubilized by fordetailsofexperi-
or SDS.
MEMBRANES
49
the SDS before electrophoresis by Sephadex G-25 did not change the electrophoretic patterns. Ultracentrifugal analysis of membrane cornponents. Figure 3A shows the sedimentation patterns of the hydrophobic protein fraction and of the isolated lipid of M. laidlawii membranes. The uncorrected sedimentation coefficient of the single protein peak was 2.8, and the sedimentation coefficients of the lipid peaks were 2 and 6.3. Figure 3B shows the sedimentation patterns of mixtures of membrane protein and lipid in various proportions. A single symmetrical schlieren peak was exhibited by both mixtures. The mixture containing equal amounts of lipid and protein had a sedimentation coefficient of 6.5, and that containing a protein to lipid ratio similar to that of original membranes had a sedimentation coefficient of 3.5. Removal of detergents and reaggregation of membrane material. Prolonged dialysis in the cold of SDS-solubilized membranes against dilute P-buffer containing 1 X 10-Z M magnesium caused the reaggregation of
FIG. 3. Sedimentation patterns of the hydrophobic protein and lipid of M. Zaidlawii membranes dissolved in 0.02 M SDS. (A) Upper schlieren peak = hydrophobic membrane protein. Lower schlieren peak = membrane lipid.(B) Upper schlieren peak = a mixture of 2 mg protein and 2 mg lipid. Lower schlieren peak = a mixture of 4 mg protein and 2 mg lipid.
50
ROTTEM,
FIG. 4. Mycoplasma duced
by the osmotic
STEIN,
AND
RAZIN
laidlawii membranes negat,ively stained shock are most pronounced. X42,CW.
membrane material to membrane-like structures (2). Negatively stained preparations and thin sections of isolated M. laidlawii and M. gallisepticum membranes showed them to be relatively free of cytoplasmic contaminants, such as ribosomes. Figure 4 shows membranes of M. laidlawii. The large holes produced in the membranes by osmotic shock are most prominent. Negative staining of the reaggregated membrane material of both Mycoplasma strains showed it to consist of spherical vesicles of very different diameters (Fig. 5). Ultrathin sections showed the vesicles to be limited by a triple-layered membrane of !bout the same thickness (approximately 90A) as the original Mycoplasma membranes. The use of radioactive SDS facilitated the study of detergent removal and its cor-
with
PTA.
The
holes
pro-
relation with the membrane reformation phenomenon. Figure 6 shows that over 99% of the detergent is removed by dialysis against dilute buffer alone, but more detergent is left in the dialysis bag when magnesium is added to the dialysis buffer. A higher concentration of SDS used for membrane solubilization caused a higher percentage of the detergent to stay in the dialysis bag, which interfered with the reaggregation of the membrane material. Sephadex G-25 columns were found most effective in removing SDS from the,, solubilized membrane material-over 99.99 % was removedbut magnesium was still essential for reaggregation. Dialysis was apparently ineffective in removing Triton X-100 and CTAB. Triton X-100 could be efficiently removed by filtering the solubilized membrane material
REASSEMBLY
OF MYCOPI,ASMA
FIG. 5. IReaggregated M. Zaidluu;ii membrane and dialysis against dilute buffer containing X23,250.
through a Sephndex G-200 column. The detergent was eluted in fractions 22-24 as determined by its precipitation with Na2C03, while membrane material emerged in fractions 15 and 16, but again magnesium was required for reaggregation. Most of the CTAB could be removed by filtration through a Sephadex G-25 column, but unlike the SDS-solubilized membranes, the CTAB-solubilized membrane material that emerged from the column reaggregated spontaneously without magnesium. Requirement C$ cations for reaggregation. Divalent cat,ions such as Ca2+, MI?+, Zn2+, and Cu2+ were as effective as Mg2+ in reaggregating the membrane material. Once it was formed t’he reaggregated membrane material was not solubilized by prolonged dialysis against dilute buffer without
MEMBRANES
mat.erial after solltbilization in 0.02 M SDS 0.02 M hIg2+. Kegattive st,aining with PTA.
divalent cations, even when EDTA (up to 5 X 10P2 31) was added. The reformed membranes were also not solubilized by incubation in 0.1 M EDTA for 30 minutes at 37”. Repeated washings of the reaggregates with dilute buffer resulted in a loss of membrane material, but the lipid protein ratio in the residual material remained con&ant. Hence the loss was probably due to t,he vesicles breaking up into smaller nonsedimentable vesicles during washing. Addibion of ;\Ig2+ to the washing fluid reduced the loss of membrane material. Although sodium ions were able to reaggregate the SDS-solubilized membrane material, much higher concentrations than that of ,11g2+lvere required (Table I). Unlike t,he reaggregates formed in the presence of divalent cations, the reaggregates produced
52
ROTTEM,
STEIN,
AND
RAZIN TABLE REAGGREGATION ~UEMBRANES BY
NaCl
TIME
FIG. 6. The effect of SDS by dialysis membranes.
OF DIALYSIS
of magnesium of solubilized
(hrs)
on the removal Mycoplasma
with sodium solubilized when resuspended in deionized water. The magnesium concentration required for reaggregation of membrane material depended on the pH of the dialysis buffer. Figure 7 shows that by lowering the pH of the buffer to 3.5, about 60% of the original membrane protein was incorporated into the reaggregate at a magnesium concentration as low as 2 X 1O-3 M. Lipid-to-protein ratio in membrane reaggregates. The initial study of Razin et al. (a), revealed a higher ratio of lipid to protein in the reformed M. laidlawii membranes than in the original membranes. This finding has been further investigated. The lipid-toprotein ratio in the reaggregate was found to depend on the magnesium concentration in the dialysis buffer. The reaggregates formed in the presence of low Mg2+, especially those of M. gallisepticum, showed a high lipid-to-protein ratio, since very little membrane protein was incorporated into these reaggregates. By increasing the magnesium concentration more protein was found in t’he precipitable membrane reaggregates, and the ratio of lipid-to-protein decreased (Table II). All reaggregates appeared to consist of vesicles in preparations stained with PTA. The higher lipid content of reaggregates of both M. laidlawii and M. gallisepticum membranes formed at lovv Mg2+ concentration was reflected in their lower density as determined by centrifugation on sucrose gradients (Figs 8 and 9). At a concentra-
in dialysis bufier,‘(ac)
OF SODIUM
CHLORIDE”
Protein reaggregate
0 0.25 0.50 1.00 2.00 4.00 a The SDS.
I
SDS-SOLUBILIZED Mycoplasma laidlawii OF
in (mg)
Lipid reaggregate
0.02 0.41 1.80 2.02 2.48 2.54 membranes
were
314 2400 11,730 13,000 15,560 15,720 solubilized
5.103
pH OF
THE
in (cpm)
DIALYSIS
in
H Mg
0.02
M
2+
BUFFER
FIG. 7. Effect of pH on the concentration of magnesium required for reaggregation of M. Zaidlawii membranes solubilized in 0.02 M SDS.
tion of 2 X lop2 M Mg2+, the density of M. laidlawii reaggregates was very close to that of the original membranes (1.168 gm/cm3 compared with 1.172 of the original membranes), but the density of M. gallisepticum reaggregates formed at this Mg2+ concentration was still markedly lower than of the original membranes (1.162 gm/cm3 compared with 1.198 of the original membranes). When the Mycoplasma membranes were disaggregated by a mixture of SDS + DOC and dialyzed against dilute buffer containing 2 X 10e2 M Mg2+, a white precipitate appeared in the dialysis bag. The precipi-
TABLE EFFECT
OF
hfAGNESIUM
ON
OF
M. laidlawii Magnesium in dialysy puffer hI
2.5 5.0 1.0 2.0 4.0
0 X x x x
lo-” 10-z 10-Z 10-Z
x
10-Z
Q The
Protein reaggregatc
No
in (mg)
X0
1.70
1.92
membranes
were
solubilized
L~fyCOphmU
SOLUBILIZED
-
I
cpm/w
Lipid in reaggregate(cpm)
aggregate 0 0.64 1.38
II
&AGGREGATION
Protein
protein
aggregate 120 15,600 24,420 24,500 24,700
in
reaggregate
So
24,300 17,700 15,000
13,300
(mg)
~%MBRANES~
Lipid reaggregate
aggregate 0.068 0.27 0.67 0.78 0.81
Ko
in ccpm)
aggregate 4000 5730 8900 9100 9000
cpm/mg protein
59 ) 000 21,200 13,400 11,700 11,100
in 0.02 M SDS
MEMBRANES
79
G 501 3 -= w
25
‘d
0
[L a
75
157;
50
; 10 E 5
1;1 25
0 REAGGREGATE
0 5
FORMED
w c a (L a
n
0 REAGGREGATE
2 0 a A
FORMED
75
15 y
w z
50
2 10 F
; m
25
5:
=
0
7s
0
4T 5.163 M tAgz+
0
REPIGGREGtJE FORMED
FRACTION
zA
!I d
15
NUMBER
FIG. 9. Density FRACTION
NUMBER
FIG. 8. Density gradient analysis of M. laid. Zawii membranes and reaggregates. Linear sucrose gradients (28-70% sucrose) were used. The top of the gradient is on the right.
tate contained most of the original membrane protein, but very little of the lipid. When the detergents were removed before dialysis by Sephadex G-25 gel filkation, the reaggregate formed upon dialysis had t’he usual lipid protein ratio for a i\Ig2+ concentration of 2 X lo+ M (Table III), and showed a vesicular membraneous structure in the electron microscope.
lisepticunl sucrose The top
gradient analysis of M. gelmembranes and reaggregat,es. Linear gradients (28-70% sucrose) were used. of the gradient is on the right. DISCUSSION
The separat’ion of a large part of membrane lipid from protein by electrophoresis of SDS-solubilized membranes on polyacrylamide gels, and the formation of reaggregated membrane material with a varying lipid-to-protein ratio, support, the conclusion of Engelman et al. (4) that the SDS-solubilized membrane material of M. Zaidlazcii does not consist of homogeneouslipoprotein subunit’s. The single symmetrical schlieren
54
ROTTEM, TABLE
COMPOSITION
OF
Mycoplasma SOLUBILIZED
Detergent
SDS DOC SDS SDS
+ DOG + DOC”
STEIN,
III
REAGGREGATES
FORMED laidlawuii MEMBRANES BY SDS AND DOC”
FROM
Protein in reaggregate (md
Lipid in reaggregate (cw)
cpm/ms protein
1.18 0.65 0.89 1.14
34,600 26,460 2500 34,100
29,000 41,000 2820 29 ) 020
a The concentration of detergents SDS and 0.025 M DOC. * The detergents were removed G-25.
was by
0.01
M
Sephadex
peak exhibit’ed by the SDS-solubilized material does not provide evidence for the existence of such subunits, because lipid-free membrane proteins solubilized in SDS also gave a single peak of a similar S value (5), while the sedimentation pattern of membrane lipids solubilized in SDS gave two peaks with S values close to that of the protein (Fig. 3). When membrane protein and lipid were mixed together in SDS, a single schlieren peak appeared. It seems that the analytical ultracentrifuge is unable to separate membrane protein from lipid when they are solubilized in SDS, and this has given rise to the fallacy that the single peak represents lipoprotein subunits. Although the membrane lipid is separated from the protein in the SDS-disaggregated membranes, these membrane components are able upon removal of the detergent to reassemble spontaneously to membranelike structures. The reassembly of membrane material solubilized by a mixture of SDS + DOC is a striking example of the ability of membrane protein to reassociate with membrane lipid after the removal of the detergent. This detergent mixture has been found to be very effective in the separation of membrane lipid from hydrophobic membrane proteins (5). Dialysis, unlike Sephadex G-25, apparently does not remove all the SDS + DOC, and the residual detergents interfere with the reassociation of lipid with protein. The hydrophobic membrane protein precipitates, but the
AND
RAZIN
lipid remains in the supernatant fluid and forms mixed micelles with the residual detergents. Formation of membrane-like structures by reassembly of membrane components on removal of the detergent has been described for a great variety of membranes (6, 13). A detailed morphological description of the structures obtained by reaggregation of M. laidlawii membrane material has been presented by Terry et al. (3). Our limited electron-microscopic studies confirm the other reports by showing that vesicles bounded by a triple-layered membrane form the predominant structure in the reaggregated membrane material. Cations are essential for the reassembly of Mycoplama membrane components. The divalent cations are far more effective in the reassembly process than the monovalent cations. It seems that the monovalent and divalent cations act by neutralizing negatively charged groups on membrane lipids and proteins, which interfere with membrane reassembly by electrostatic repulsion. The divalent but not the monovalent cations further contribute to the stability of the reaggregated membranes by forming salt bridges between negatively charged groups on neighboring molecules. This difference between the action of monovalent and divalent cations may explain the much higher stability of the reaggregates formed in the presence of divalent cations. Similar differential effects of mono- and divalent cations on the stability of membranes of the halophilic bacteria have been recorded (14, 15). The primary function of the divalent cations could not have been to neutralize the negative charges of the SDS molecules bound to the membrane material, because Mg2+ was still necessary for reaggregation when the SDS was completely removed from the disaggregated membrane material by a Sephadex G-25 column. Moreover, Mgz+ was also required for reaggregation of M. laidlawii membranes solubilized in the non-ionic detergent Triton X-100. However, membranes solubilized by the cationic detergent CTAB reaggregated spontaneously upon filtration through Sephadex
G-25 in the absence of Mg2+. It is possible that the Sephadex G-25 did not remove all the CTAB. The small amounts of detergent that were left could not interfere with the reassembly of membrane components, but were sufficient to neutralize the negatively charged groups in t,his mat’erial and could thus replace t,he need for magnesium ions. The reaggregates formed at different RIg2+ concentrations had a varying lipid-to-protein ratio and consequently different densities. The density of ~11.laidlawii reaggregates formed at 0.02 JI Mg*+ was very close to that of the original membranes (see also Ref. 3), but the A/. gallisepticum reaggregates consistently showed a significantly lower density than that of the original membranes. Preliminary chemical analysis of M. gallisepticum membranes showed them to consist of about 79% protein and 19% lipid, as against 59% protein and 36% of lipid in M. laidlazoii membranes. It is very probable that the high protein content of M. gallisepficum membranes results from the presence of the bleb, a unique structure of this flIycopZasnaa. This structure closely associat)ed with the cell membrane does not comain nucleic acids (16) and possibly may consist of protein. Reformed AI. gallisepticum membranes contain 110 blebs, and t’herefore their density is close t’o that, of reformed 31. laidlawii membranes. It may be concluded that the use of SDS for membrane disaggregation has so far failed to demonstrate lipoprotein subunits in Mycoplasma membranes. However, this should not be taken as evidence against the subunit theory, because SDS by virtue of its ability to separate lipid from protein might disrupt the lipid protein association in subunits. The spontaneous reassembly of membrane protein and lipid to a membraneous structure indicates that these components contain sufficient structure-determining in formation to interact spontaneously under suitable conditions and produce membraneous structures in the absence of templates of preexisting membranes (3). The mechanism of reassembly is not yet clear. The higher lipid-to-protein ratio in reaggregates obtained at low 1\Ig3+ concentrat’ions
may be explained according to the Danielli and Davson model (17) by the initial formation of a bimolecular leaflet of lipid up011 which membrane prot#ein can be bound t)o a varying degree, possibly depending 011 t’he neutralizat,ion of electrostatic repulsive forces and formation of salt bridges by divalent cations. However, the resuhs of Terry et al. (3) do not seem to agree with such a mechanism. They claim that upon removal of the detergent in the absence of n!rg*+, membrane protein is supposed to reassociate with the lipid to form lipoprotein particles, which on addition of ;\Ig*+ aggregate to a membraneous struct,ure. The different lipid-to-protein ratio in the reaggregates obtained in our study does not agree with this result. In addition, we were able to separate membrane protein from lipid by polyacrylamide gel electrophoresis after the complete removal of SDS by Sephadex G-25, which indicates that this material does not consist of lipoprotein subunits. REFERENCES 1. RMXN, S., dnn. S.Y. dead. Sci. 143,115 (1967). 2. RAZIN, S., MOROWWZ, II. J., .IND TERRII, T. Al., Proc. Xail. Acad. Sci. IFS. 54, 219 (1965). 3. TERRY, T. 1\L., ENGELX~N, II. >I., .\ND MoaoWITZ, H. J., Biochim. Biophys. Acta 135, 391 (1967). 4. ENGELIVUN, II. AI., TERRY, T. XI., IND MwtoWITZ, H. J., Biochim. Biophys. Acfa 135, 381 (1967). 5. RODWEZLL, A. W., R.IZIN, S., ROTVX, S., .\ND ARG.DUN, M., Arch. Biochem. Biophys. 122, 621 (1967). 6. GREEN, I). E., ALLMINN, 1). W., BACHMINN, a., B.~uM, H., KOPACZYIC, K., KORIH.\N, E. F., LIPTOX, S., M.xLI~:xx~N, 1). II., MCCONNI;LL, 1). G., PERDV~~, J. F., RWSKI,:, J. S., .\ND TZAGOLOFF, A., Arch. Biochem. Biophys. 119, 312 (1967). 7. ROTTEM, P., .INI) R.\zIN, S;., J. Bacterial. 94, 359 (1967). 8. R.IZIN, S., J. Gen. Microbial. 33, 471 (1963). 9. RO~RISH, S. A., IND M.\RH., A. G., J. Bacterial. 83, 158 (1962). 10. POLLXI~, J. II., R~ZIN, S., .\ND CLEVERDON, R. C., J. Bacterial. 90, 617 (1965). 11. LOWRY, 0. H., ROS~BROUGH, N. J., F.\RR, A. L., AND RAND.~LL, R. J., J. Bid. Churl. 193, 275 (1951).
56
ROTTEM,
STEIN,
12. FOLCH, J., LEES, M., AND SLOAN-STANLEY, G. H., J. Biol. Chem. 226, 497 (1957). 13. ERNSTER, L., SIEKEVITZ, P., AND PALADE, G. E. J. Cell BioZ. 16, 541 (1962). 14. BROWN, A. D., Biochim. Biophys. Acta 76, 425 (1963).
AND
RAZIN
15. ONISHI, H., AND KUSHNER, D. J., J. Bacterial. 91, 646 (1966). 16. MANILOFF, J., MOROWITZ, H. J., AND BARRNETT, R. J., J. Bacterial. 90, 193 (1965). 17. DANIELLI, J. F., AND DAVSON, H., J. Cell Comp. Physiol. 6, 495 (1935).
OF
BIOCHEMISTRY
Reassembly
AND
BIOPHYSICS
46-56
126,
of Mycoplasma
(1968)
Membranes
Disaggregated
by
Detergents’ S. ROTTENI, Department
of
OLGA
STEIN,
AND
S. RAZIN
Clinical Microbiology, and Department of Experimental Medicine and The Hebrew University-Hadassah Medical School, Jerusalem, Israel Received
August
Cancer
Research,
28, 1967
Plasma membranes of Mycoplasma laidlawii and Mycoplasma gallisepticum were solubilized by several ionic and non-ionic detergents. Solubilization of the membranes by sodium dodecyl sulfate (SDS) separated membrane lipid from protein as demonstrated by polyacrylamide gel electrophoresis of the solubilized material. The solubilized membrane material reaggregated spontaneously on removal of the detergent by dialysis or by Sephadex G-25, and formed vesicles limited by a triplelayered membrane of about the same thickness as the original Mycoplasma membrane. A divalent cation (e.g., Mg2+) was essential for membrane reassembly. The ratio of lipid to protein in membrane reaggregates varied considerably according to the Mg*+ concentration. At a low Mg2+ concentration reaggregates contained a higher percentage of lipid. The present results seem to bear out the suggestion of Engelman et al. [Biochim. Biophys. Acta 136, 381 (1967)] that the SDS-solubilized membrane material does not. consist of homogeneous lipoprotein subunits but of separate SDS-lipid and SDS-protein complexes. The reassembly of solubilized membrane lipid and protein on remov.11 of the detergent indicates that these components contain sufficient structure-determining information to interact spontaneollsly in the presence of Mg2+ and produce membraneous structures.
centrifugally homogeneous lipoprotein subunits (2). If so, then in the det’ergent-solubilized material, membrane lipid should be bound to membrane protein. Alt,hough electrophoresis on cellulose acetate strips, and Sephadex G-200 filtration failed to separate the lipid from protein in the solubilized membrane material, the lipid peak did not coincide with the protein peak on centrifugation in a sucrose density gradient. Hence at least part of the lipid had separated from the protein (5). Engelman et al. (4) recently succeeded in separating the lipid from the protein in SDS-solubilized Mycoplasma laidlawii membranes by prolonged centrifugation in density gradients. Accordingly, the disaggregated membrane material does not consist of lipoprotein subunits. This report provides additional evidence for the separation of the lipid from protein
Mycoplasma are bounded by a single lipoprotein membrane, which can be readily isolated by osmotic lysis of the organisms (1). The mode of organization of protein and lipid in this membrane has been the subject of several recent reports (l-5). Disaggregation of Mycoplasma membranes by sodium dodecyl sulfate (SDS) resulted in a clear solution exhibiting a single symmetrical schlieren peak in the analytical ultracentrifuge. It was thought that this peak, which had an uncorrected sedimentation coefficient of about 3S, represented 1 This work was supported by grant FG-Is-174 from the United States Department of Agriculture under Public Law 480. 2 Taken in part from a dissertation to be submitted by the senior author in partial fulfillment of the requirements for the Ph.D. degree from the Hebrew University, Jerusalem, Israel. 46
REASSEMBLY
OF
illYCOPLASMA
in SDS - disaggregated Mycoplaslna membranes. Removal of the detergent from the disaggregated membrane material by dialysis against dilute buffer containing magnesium has resulted in the reassembly of the solubilized membrane components to a membraneous structure (2, 3). This spontaneous reassembly of disaggregated membrane material has been taken in support of the subunit hypot’hesis of membrane struct’ure (2, 6). The present investigation of the fat t,ors governing the self-assembly of membrane mat’erial was undertaken to elucidate the mechanism of this interesting phenomenon. K2TERIALS
AND
METHODS
Organisms and growth conditions. Mycoplasma Zaidlawii (oral strain) was isolated from the human oral cavity and was found to be related to Hycoplasma laidlawii strain A (7). Mycoplasma gallisepticutn strain A5969 was obtained from 11. E. Tourtellotte (Department of Animal Diseases, University of Connecticut, Storrs). The organisms were grown in 3- to g-liter volumes of a modified Edward medium (8) containing 2yo PPLO serum fraction (Difco). To label the membrane lipids, 1 PC of uniformly labeled sodium acetate-14C and 1 PC of oleic acid-l-‘% (The Radiochemical Centre, Amersham, England) were added to each liter of the growth medium. The organisms were harvested after 24-48 hours of incubation at. 37” by centrifugation at 13,000g for 10 minutes. The sedimented cells were washed twice in 0.25 hl NaCl. Isolation of cell membranes. Cell membranes were isolated by osmotic lysis of the organisms. For osmotic lysis of $2. Zaidlawii, the washed sedimented cells were resuspended in 250 ml of deionized water and incubated at 37” for 15 minutes. The more osmotically resistant Jf. galZisepticllm cells were lysed by a modification of the method devised by Robrish and Marr (9). The cells were resuspended in 5 ml of a 2 M glycerol solution and incubated at 37” for 10 minutes. The suspension was t,hen rapidly injected into 250 ml of deionized water and incubated at 37” for 15 The treated Mycoplasw~a suspensions minutes. were centrifuged at 8OOOg for 5 minutes to remove clumps of unbroken cells. The supernatant fluid was then cent,rifuged for 30 minutes at 34,OOOg to collect the membranes. The membranes were washed ten times alternatively with deionized m-ater and 0.05 M NaCl in 0.01 M phosphate buffer, pH 7..5. The washed membranes were re-
MEMBRANES
47
suspended in p-buffer (NaCl, 0.15 M; Tris, 0.05 31; 2-mercaptoethanol, 0.01 hf; in deionized water adjusted to pH 7.4 with HCl; Ref. lo), diluted 1:20 in deionized water (will be referred to as “dilute p-buffer”), and kept at -20” until used. The amount of protein in membrane suspensions was determined according to Lowry et 01. (11). Only traces of nucleic acids could be found in the membranes. Thin sections and negative staining of the membrane preparations with phosphotungstic acid displayed membraneous structures and showed little contamination with nonmembraneous material. Assessment of membrane solubilization by detergents. Various amounts of the tested detergents were added to 4 ml of membrane suspension in dilute p-buffer. The degree of membrane solubilization was estimated after incubation at 37” for 1,s minutes by measuring the decrease of the optical density of the membrane suspension at 500 m*. Removal of detergents by gel $ltration. Sephadex G-25 (Pharmacia AB, Uppsala, Sweden) was used to remove sodium dodecyl sulfate (SDS) or cetyltrimethylammonium bromide (CTAB) from membrane material, and Sephadex G-200 was used to remove Triton X-100. The columns (1.8 X 34 cm) were equilibrated with dilute p-buffer containing 5 X lo-” M EDTA. For the removal of SDS, onehalf ml of membrane suspension (containing 1.5-3 mg protein) solubilized in 0.02 M SDS containing 1 ~tc of 3%-SDS (The Radiochemical Centre, Amersham, England) was placed on the column. Elution was performed with dilute pbuffer containing 5 X 1OW M EDTA. Two-ml fractions were collected and assayed for protein and radioactivity. For the removal of Triton X-100, 0.2 ml of membranes (containing 2-3 mg protein) solubilized by Triton X-100 were placed on the column. Elution and fraction collection were performed as above. The fractions were assayed for protein and for Triton X-100. The presence of Triton X-100 in the fractions was determined by adding 0.5 ml of 2Oc/ Na&Os in I N NaOH to 1 ml of each fraction. Turbidity indicated the presence of Triton X-100. Isolation of membrane protein and lipid. Hydrophobic membrane proteins were separated from membrane lipids by solubilization of the membranes with a mixture of SDS and sodium deoxy cholate (DOC) and precipitation with ammonium sulfate (;i). Membrane lipids were extracted with chloroform-methanol (2: 1, v/v) and washed according to Folch et al. (12). The lipid solution in chloroform-methanol was brought to a volume of 0.5 ml by evaporation under nitrogen. Dilute p-buffer cont,aining 0.02 Y SDS (1.3 ml) was added
48
ROTTEM,
STEIN,
to the lipid solution. The resulting turbid emulsion cleared when the solvents were removed completely by a stream of nitrogen. Reaggregation of solubilized membranes. Labeled membranes solubilized by detergents were dialyzed for 34 days at 4” against 500-1000 volumes of dilute p-buffer containing various amounts of MgClz (2). The content of the dialysis bags was then centrifuged at 37,000g for 1 hour in the cold. Both sediment and supernatant fluid were assayed for protein and radioactivity. Sucrose gradient centrifugation. Samples (0.15 ml) of labeled membranes or membrane reaggregates were layered over 4.2 ml of linear sucrose gradients (28-70% sucrose). The gradients were centrifuged at 39,000 rpm in an SW 39 head of a Spinco model L-2 ultracentrifuge for 2 hours at 4”. Fractions (0.095 ml) were collected by puncturing the bottom of the centrifuge tubes, and were assayed for protein and radioactivity. Polyacrylamide gel electrophoresis. Membranes solubilized in 0.02 M SDS were analyzed in a gel system composed of 7.5yo Cyanogum 41 (a mixture of 95% acrylamide and 5% N,N-methylenebisacrylamide, supplied by E-C Apparatus Co. Philadelphia, Pennsylvania) in TEB buffer (Tris, 60.5 gm; EDTA, 6 gm; boric acid, 4.6 gm; and water to 1000 ml; final pH without adjustment, 8.8-8.9) diluted I:4 with water in 6 X IOO-mm glass tubes. Polymerization was induced by addition of 0.05 ml N,N,N’,N’-tetramethylethylenediamine (TMED) and 10 mg of ammonium persulfate to 20 ml of the Cyanogum solution. Three identical samples(lOO-150 pl, each containing about 100 pg protein) of SDS-solubilized membranes were mixed with 50 pl of a 607, sucrose solution and deposited on top of three of the gels. The tubes were filled up carefully with TEB buffer diluted 1:20 with water. The same dilute buffer was used to fill the reservoirs of the electrophoresis apparatus. Electrophoresis was carried out at room temperature for 1 hour at a constant current of 5 mA per tube; the lower electrode was used as anode. One of the gels was stained with 0.5yo Amido Black 10 B in 7yo acetic acid for 30 minutes. The second gel was cut into small sections which were transferred to scintillation vials and assayed for radioactivity. The third gel was cut into small sections, proteins were eluted from the gel sections by boiling in 1 ml of 0.1 N NaOH for 30 minutes, and protein was determined on the eluates. Radioactivity measurements. Radioactivity was determined in a Packard Tri-Carb scintillation counter with 10 ml of a scintillation mixture composed of 800 ml of dioxane, 150 ml toluene, 50 gm naphthalene, 10 gm PPO, and 150 mg POPOP (Packard Instrument Co. Inc., Illinois). Analytical ultracentrifuge analysis. Disaggre-
AND
RAZIN
s oziz 4 “0 Ol-
l TRITON X-100
CONCENTRATION
FIG.
branes
1. Solubilization detergents.
OF DETERGENT
of
M.
(mg/ml)
Zaidlawii
mem-
by
gated membranes, membrane protein, or membrane lipid solubilized in 0.02 M SDS were centrifuged at 59,780 rpm in a Spinco model E analytical ultracentrifuge at 20’. Sedimentation peaks were observed by use of schlieren optics. Sedimentation coefficients were calculated without correcting for medium viscosity or density. Preparations of specimens for electron microscopy. A drop of the material to be studied was placed on collodion-coated, carbon-reinforced grids and mixed on the grid with a drop of a 1.7% solution of potassium phosphotungstate (PTA), pH 7. The material was mixed with the stain in a Pasteur pipette for about 30 seconds, and the excess fluid was removed from the grid with a fragment of filter paper. The grids were dried briefly in air and examined at 100 kV in an RCA EMU 3G electron microscope. RESULTS
Xolubili.zation of the membranes by detergents. Membranes of M. laidlawii and M. gallisepticum could be solubilized by several
ionic and non-ionic detergents. The ionic detergents SDS and CTAB were more effective in membrane solubilization than the non-ionic detergent Triton X-100, but Triton A-20 was totally ineffective (Fig. 1). The solubilization of the membranes by SDS caused at least a partial separation of membrane lipid from protein. Polyacrylamide gel electrophoresis of the solubilized membrane material showed a highly variable ratio of labeled lipid to protein in the different gel sections (Fig. 2). Removal of
REASSEMBLY PROTEIN
(Pd
LABELLED M.LAIDLAWII
MYCOPLASMA
LIPID
(cpm) 4815 200 62 36 22
500 346 32 6 0
OF
. - - __
10 0 0 0 0
M. GALLISEPTICUM 386 402 360 242 19 160 30 12 0 0
FIG. 2. Polyacrylamide Mycoplasma membranes S~~MATERIALSANDMETHODS mental procedure.
39 50 500 160 Ll 11&O 1700 1450 160 21 0
gel erectropnoresrs solubilized by fordetailsofexperi-
or SDS.
MEMBRANES
49
the SDS before electrophoresis by Sephadex G-25 did not change the electrophoretic patterns. Ultracentrifugal analysis of membrane cornponents. Figure 3A shows the sedimentation patterns of the hydrophobic protein fraction and of the isolated lipid of M. laidlawii membranes. The uncorrected sedimentation coefficient of the single protein peak was 2.8, and the sedimentation coefficients of the lipid peaks were 2 and 6.3. Figure 3B shows the sedimentation patterns of mixtures of membrane protein and lipid in various proportions. A single symmetrical schlieren peak was exhibited by both mixtures. The mixture containing equal amounts of lipid and protein had a sedimentation coefficient of 6.5, and that containing a protein to lipid ratio similar to that of original membranes had a sedimentation coefficient of 3.5. Removal of detergents and reaggregation of membrane material. Prolonged dialysis in the cold of SDS-solubilized membranes against dilute P-buffer containing 1 X 10-Z M magnesium caused the reaggregation of
FIG. 3. Sedimentation patterns of the hydrophobic protein and lipid of M. Zaidlawii membranes dissolved in 0.02 M SDS. (A) Upper schlieren peak = hydrophobic membrane protein. Lower schlieren peak = membrane lipid.(B) Upper schlieren peak = a mixture of 2 mg protein and 2 mg lipid. Lower schlieren peak = a mixture of 4 mg protein and 2 mg lipid.
50
ROTTEM,
FIG. 4. Mycoplasma duced
by the osmotic
STEIN,
AND
RAZIN
laidlawii membranes negat,ively stained shock are most pronounced. X42,CW.
membrane material to membrane-like structures (2). Negatively stained preparations and thin sections of isolated M. laidlawii and M. gallisepticum membranes showed them to be relatively free of cytoplasmic contaminants, such as ribosomes. Figure 4 shows membranes of M. laidlawii. The large holes produced in the membranes by osmotic shock are most prominent. Negative staining of the reaggregated membrane material of both Mycoplasma strains showed it to consist of spherical vesicles of very different diameters (Fig. 5). Ultrathin sections showed the vesicles to be limited by a triple-layered membrane of !bout the same thickness (approximately 90A) as the original Mycoplasma membranes. The use of radioactive SDS facilitated the study of detergent removal and its cor-
with
PTA.
The
holes
pro-
relation with the membrane reformation phenomenon. Figure 6 shows that over 99% of the detergent is removed by dialysis against dilute buffer alone, but more detergent is left in the dialysis bag when magnesium is added to the dialysis buffer. A higher concentration of SDS used for membrane solubilization caused a higher percentage of the detergent to stay in the dialysis bag, which interfered with the reaggregation of the membrane material. Sephadex G-25 columns were found most effective in removing SDS from the,, solubilized membrane material-over 99.99 % was removedbut magnesium was still essential for reaggregation. Dialysis was apparently ineffective in removing Triton X-100 and CTAB. Triton X-100 could be efficiently removed by filtering the solubilized membrane material
REASSEMBLY
OF MYCOPI,ASMA
FIG. 5. IReaggregated M. Zaidluu;ii membrane and dialysis against dilute buffer containing X23,250.
through a Sephndex G-200 column. The detergent was eluted in fractions 22-24 as determined by its precipitation with Na2C03, while membrane material emerged in fractions 15 and 16, but again magnesium was required for reaggregation. Most of the CTAB could be removed by filtration through a Sephadex G-25 column, but unlike the SDS-solubilized membranes, the CTAB-solubilized membrane material that emerged from the column reaggregated spontaneously without magnesium. Requirement C$ cations for reaggregation. Divalent cat,ions such as Ca2+, MI?+, Zn2+, and Cu2+ were as effective as Mg2+ in reaggregating the membrane material. Once it was formed t’he reaggregated membrane material was not solubilized by prolonged dialysis against dilute buffer without
MEMBRANES
mat.erial after solltbilization in 0.02 M SDS 0.02 M hIg2+. Kegattive st,aining with PTA.
divalent cations, even when EDTA (up to 5 X 10P2 31) was added. The reformed membranes were also not solubilized by incubation in 0.1 M EDTA for 30 minutes at 37”. Repeated washings of the reaggregates with dilute buffer resulted in a loss of membrane material, but the lipid protein ratio in the residual material remained con&ant. Hence the loss was probably due to t,he vesicles breaking up into smaller nonsedimentable vesicles during washing. Addibion of ;\Ig2+ to the washing fluid reduced the loss of membrane material. Although sodium ions were able to reaggregate the SDS-solubilized membrane material, much higher concentrations than that of ,11g2+lvere required (Table I). Unlike t,he reaggregates formed in the presence of divalent cations, the reaggregates produced
52
ROTTEM,
STEIN,
AND
RAZIN TABLE REAGGREGATION ~UEMBRANES BY
NaCl
TIME
FIG. 6. The effect of SDS by dialysis membranes.
OF DIALYSIS
of magnesium of solubilized
(hrs)
on the removal Mycoplasma
with sodium solubilized when resuspended in deionized water. The magnesium concentration required for reaggregation of membrane material depended on the pH of the dialysis buffer. Figure 7 shows that by lowering the pH of the buffer to 3.5, about 60% of the original membrane protein was incorporated into the reaggregate at a magnesium concentration as low as 2 X 1O-3 M. Lipid-to-protein ratio in membrane reaggregates. The initial study of Razin et al. (a), revealed a higher ratio of lipid to protein in the reformed M. laidlawii membranes than in the original membranes. This finding has been further investigated. The lipid-toprotein ratio in the reaggregate was found to depend on the magnesium concentration in the dialysis buffer. The reaggregates formed in the presence of low Mg2+, especially those of M. gallisepticum, showed a high lipid-to-protein ratio, since very little membrane protein was incorporated into these reaggregates. By increasing the magnesium concentration more protein was found in t’he precipitable membrane reaggregates, and the ratio of lipid-to-protein decreased (Table II). All reaggregates appeared to consist of vesicles in preparations stained with PTA. The higher lipid content of reaggregates of both M. laidlawii and M. gallisepticum membranes formed at lovv Mg2+ concentration was reflected in their lower density as determined by centrifugation on sucrose gradients (Figs 8 and 9). At a concentra-
in dialysis bufier,‘(ac)
OF SODIUM
CHLORIDE”
Protein reaggregate
0 0.25 0.50 1.00 2.00 4.00 a The SDS.
I
SDS-SOLUBILIZED Mycoplasma laidlawii OF
in (mg)
Lipid reaggregate
0.02 0.41 1.80 2.02 2.48 2.54 membranes
were
314 2400 11,730 13,000 15,560 15,720 solubilized
5.103
pH OF
THE
in (cpm)
DIALYSIS
in
H Mg
0.02
M
2+
BUFFER
FIG. 7. Effect of pH on the concentration of magnesium required for reaggregation of M. Zaidlawii membranes solubilized in 0.02 M SDS.
tion of 2 X lop2 M Mg2+, the density of M. laidlawii reaggregates was very close to that of the original membranes (1.168 gm/cm3 compared with 1.172 of the original membranes), but the density of M. gallisepticum reaggregates formed at this Mg2+ concentration was still markedly lower than of the original membranes (1.162 gm/cm3 compared with 1.198 of the original membranes). When the Mycoplasma membranes were disaggregated by a mixture of SDS + DOC and dialyzed against dilute buffer containing 2 X 10e2 M Mg2+, a white precipitate appeared in the dialysis bag. The precipi-
TABLE EFFECT
OF
hfAGNESIUM
ON
OF
M. laidlawii Magnesium in dialysy puffer hI
2.5 5.0 1.0 2.0 4.0
0 X x x x
lo-” 10-z 10-Z 10-Z
x
10-Z
Q The
Protein reaggregatc
No
in (mg)
X0
1.70
1.92
membranes
were
solubilized
L~fyCOphmU
SOLUBILIZED
-
I
cpm/w
Lipid in reaggregate(cpm)
aggregate 0 0.64 1.38
II
&AGGREGATION
Protein
protein
aggregate 120 15,600 24,420 24,500 24,700
in
reaggregate
So
24,300 17,700 15,000
13,300
(mg)
~%MBRANES~
Lipid reaggregate
aggregate 0.068 0.27 0.67 0.78 0.81
Ko
in ccpm)
aggregate 4000 5730 8900 9100 9000
cpm/mg protein
59 ) 000 21,200 13,400 11,700 11,100
in 0.02 M SDS
MEMBRANES
79
G 501 3 -= w
25
‘d
0
[L a
75
157;
50
; 10 E 5
1;1 25
0 REAGGREGATE
0 5
FORMED
w c a (L a
n
0 REAGGREGATE
2 0 a A
FORMED
75
15 y
w z
50
2 10 F
; m
25
5:
=
0
7s
0
4T 5.163 M tAgz+
0
REPIGGREGtJE FORMED
FRACTION
zA
!I d
15
NUMBER
FIG. 9. Density FRACTION
NUMBER
FIG. 8. Density gradient analysis of M. laid. Zawii membranes and reaggregates. Linear sucrose gradients (28-70% sucrose) were used. The top of the gradient is on the right.
tate contained most of the original membrane protein, but very little of the lipid. When the detergents were removed before dialysis by Sephadex G-25 gel filkation, the reaggregate formed upon dialysis had t’he usual lipid protein ratio for a i\Ig2+ concentration of 2 X lo+ M (Table III), and showed a vesicular membraneous structure in the electron microscope.
lisepticunl sucrose The top
gradient analysis of M. gelmembranes and reaggregat,es. Linear gradients (28-70% sucrose) were used. of the gradient is on the right. DISCUSSION
The separat’ion of a large part of membrane lipid from protein by electrophoresis of SDS-solubilized membranes on polyacrylamide gels, and the formation of reaggregated membrane material with a varying lipid-to-protein ratio, support, the conclusion of Engelman et al. (4) that the SDS-solubilized membrane material of M. Zaidlazcii does not consist of homogeneouslipoprotein subunit’s. The single symmetrical schlieren
54
ROTTEM, TABLE
COMPOSITION
OF
Mycoplasma SOLUBILIZED
Detergent
SDS DOC SDS SDS
+ DOG + DOC”
STEIN,
III
REAGGREGATES
FORMED laidlawuii MEMBRANES BY SDS AND DOC”
FROM
Protein in reaggregate (md
Lipid in reaggregate (cw)
cpm/ms protein
1.18 0.65 0.89 1.14
34,600 26,460 2500 34,100
29,000 41,000 2820 29 ) 020
a The concentration of detergents SDS and 0.025 M DOC. * The detergents were removed G-25.
was by
0.01
M
Sephadex
peak exhibit’ed by the SDS-solubilized material does not provide evidence for the existence of such subunits, because lipid-free membrane proteins solubilized in SDS also gave a single peak of a similar S value (5), while the sedimentation pattern of membrane lipids solubilized in SDS gave two peaks with S values close to that of the protein (Fig. 3). When membrane protein and lipid were mixed together in SDS, a single schlieren peak appeared. It seems that the analytical ultracentrifuge is unable to separate membrane protein from lipid when they are solubilized in SDS, and this has given rise to the fallacy that the single peak represents lipoprotein subunits. Although the membrane lipid is separated from the protein in the SDS-disaggregated membranes, these membrane components are able upon removal of the detergent to reassemble spontaneously to membranelike structures. The reassembly of membrane material solubilized by a mixture of SDS + DOC is a striking example of the ability of membrane protein to reassociate with membrane lipid after the removal of the detergent. This detergent mixture has been found to be very effective in the separation of membrane lipid from hydrophobic membrane proteins (5). Dialysis, unlike Sephadex G-25, apparently does not remove all the SDS + DOC, and the residual detergents interfere with the reassociation of lipid with protein. The hydrophobic membrane protein precipitates, but the
AND
RAZIN
lipid remains in the supernatant fluid and forms mixed micelles with the residual detergents. Formation of membrane-like structures by reassembly of membrane components on removal of the detergent has been described for a great variety of membranes (6, 13). A detailed morphological description of the structures obtained by reaggregation of M. laidlawii membrane material has been presented by Terry et al. (3). Our limited electron-microscopic studies confirm the other reports by showing that vesicles bounded by a triple-layered membrane form the predominant structure in the reaggregated membrane material. Cations are essential for the reassembly of Mycoplama membrane components. The divalent cations are far more effective in the reassembly process than the monovalent cations. It seems that the monovalent and divalent cations act by neutralizing negatively charged groups on membrane lipids and proteins, which interfere with membrane reassembly by electrostatic repulsion. The divalent but not the monovalent cations further contribute to the stability of the reaggregated membranes by forming salt bridges between negatively charged groups on neighboring molecules. This difference between the action of monovalent and divalent cations may explain the much higher stability of the reaggregates formed in the presence of divalent cations. Similar differential effects of mono- and divalent cations on the stability of membranes of the halophilic bacteria have been recorded (14, 15). The primary function of the divalent cations could not have been to neutralize the negative charges of the SDS molecules bound to the membrane material, because Mg2+ was still necessary for reaggregation when the SDS was completely removed from the disaggregated membrane material by a Sephadex G-25 column. Moreover, Mgz+ was also required for reaggregation of M. laidlawii membranes solubilized in the non-ionic detergent Triton X-100. However, membranes solubilized by the cationic detergent CTAB reaggregated spontaneously upon filtration through Sephadex
G-25 in the absence of Mg2+. It is possible that the Sephadex G-25 did not remove all the CTAB. The small amounts of detergent that were left could not interfere with the reassembly of membrane components, but were sufficient to neutralize the negatively charged groups in t,his mat’erial and could thus replace t,he need for magnesium ions. The reaggregates formed at different RIg2+ concentrations had a varying lipid-to-protein ratio and consequently different densities. The density of ~11.laidlawii reaggregates formed at 0.02 JI Mg*+ was very close to that of the original membranes (see also Ref. 3), but the A/. gallisepticum reaggregates consistently showed a significantly lower density than that of the original membranes. Preliminary chemical analysis of M. gallisepticum membranes showed them to consist of about 79% protein and 19% lipid, as against 59% protein and 36% of lipid in M. laidlazoii membranes. It is very probable that the high protein content of M. gallisepficum membranes results from the presence of the bleb, a unique structure of this flIycopZasnaa. This structure closely associat)ed with the cell membrane does not comain nucleic acids (16) and possibly may consist of protein. Reformed AI. gallisepticum membranes contain 110 blebs, and t’herefore their density is close t’o that, of reformed 31. laidlawii membranes. It may be concluded that the use of SDS for membrane disaggregation has so far failed to demonstrate lipoprotein subunits in Mycoplasma membranes. However, this should not be taken as evidence against the subunit theory, because SDS by virtue of its ability to separate lipid from protein might disrupt the lipid protein association in subunits. The spontaneous reassembly of membrane protein and lipid to a membraneous structure indicates that these components contain sufficient structure-determining in formation to interact spontaneously under suitable conditions and produce membraneous structures in the absence of templates of preexisting membranes (3). The mechanism of reassembly is not yet clear. The higher lipid-to-protein ratio in reaggregates obtained at low 1\Ig3+ concentrat’ions
may be explained according to the Danielli and Davson model (17) by the initial formation of a bimolecular leaflet of lipid up011 which membrane prot#ein can be bound t)o a varying degree, possibly depending 011 t’he neutralizat,ion of electrostatic repulsive forces and formation of salt bridges by divalent cations. However, the resuhs of Terry et al. (3) do not seem to agree with such a mechanism. They claim that upon removal of the detergent in the absence of n!rg*+, membrane protein is supposed to reassociate with the lipid to form lipoprotein particles, which on addition of ;\Ig*+ aggregate to a membraneous struct,ure. The different lipid-to-protein ratio in the reaggregates obtained in our study does not agree with this result. In addition, we were able to separate membrane protein from lipid by polyacrylamide gel electrophoresis after the complete removal of SDS by Sephadex G-25, which indicates that this material does not consist of lipoprotein subunits. REFERENCES 1. RMXN, S., dnn. S.Y. dead. Sci. 143,115 (1967). 2. RAZIN, S., MOROWWZ, II. J., .IND TERRII, T. Al., Proc. Xail. Acad. Sci. IFS. 54, 219 (1965). 3. TERRY, T. 1\L., ENGELX~N, II. >I., .\ND MoaoWITZ, H. J., Biochim. Biophys. Acta 135, 391 (1967). 4. ENGELIVUN, II. AI., TERRY, T. XI., IND MwtoWITZ, H. J., Biochim. Biophys. Acfa 135, 381 (1967). 5. RODWEZLL, A. W., R.IZIN, S., ROTVX, S., .\ND ARG.DUN, M., Arch. Biochem. Biophys. 122, 621 (1967). 6. GREEN, I). E., ALLMINN, 1). W., BACHMINN, a., B.~uM, H., KOPACZYIC, K., KORIH.\N, E. F., LIPTOX, S., M.xLI~:xx~N, 1). II., MCCONNI;LL, 1). G., PERDV~~, J. F., RWSKI,:, J. S., .\ND TZAGOLOFF, A., Arch. Biochem. Biophys. 119, 312 (1967). 7. ROTTEM, P., .INI) R.\zIN, S;., J. Bacterial. 94, 359 (1967). 8. R.IZIN, S., J. Gen. Microbial. 33, 471 (1963). 9. RO~RISH, S. A., IND M.\RH., A. G., J. Bacterial. 83, 158 (1962). 10. POLLXI~, J. II., R~ZIN, S., .\ND CLEVERDON, R. C., J. Bacterial. 90, 617 (1965). 11. LOWRY, 0. H., ROS~BROUGH, N. J., F.\RR, A. L., AND RAND.~LL, R. J., J. Bid. Churl. 193, 275 (1951).
56
ROTTEM,
STEIN,
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