To better establish the intracellular location of the phosphatidylserine synthase of Escherichia coli and hence better understand how it is regulated in the cell, we ...
JOURNAL OF BACTERIOLOGY, Mar. 1986, p. 805-812
Vol. 165, No. 3
0021-9193/86/030805-08$02.00/0 Copyright © 1986, American Society for Microbiology
Substrate-Induced Membrane Association of Phosphatidylserine Synthase from Escherichia coli KATHRYN LOUIE,t YANG-CHANG CHEN,t AND WILLIAM DOWHAN* Department of Biochemistry and Molecular Biology, University of Texas Medical School and University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas 77225 Received 30 September 1985/Accepted 28 November 1985
To better establish the intracellular location of the phosphatidylserine synthase of Escherichia coli and hence better understand how it is regulated in the cell, we compared the size, function, and binding properties of the enzyme made in vitro with the enzyme found in cell lysates and with the purified enzyme. The enzyme made either in vivo or in an active form in vitro was found primarily associated with the ribosomal fraction of the cell and had the same apparent molecular mass as the purified enzyme. These results were unaffected by the presence of protease inhibitors. Addition of unsupplemented E. coli membranes or membranes supplemented with phosphatidylethanolamine did not affect the subcellular distribution of the enzyme in these experiments. However, addition of membranes supplemented with either the lipid substrate, CDP-diacylglycerol, or the lipid product, phosphatidylserine, resulted in membrane association by the enzyme rather than ribosomal association. Addition of membranes supplemented with acidic lipids also brought about membrane association, but this association was primarily ionic since it was disrupted by high salt concentrations. These results strongly suggest that the ribosomal location of this enzyme is not the result of some modification event occurring after cell lysis and that the normal functioning of the enzyme involves membrane association which is primarily induced by the presence of a membrane-associated substrate.
primarily through binding to its lipid substrate during catalysis. We studied the size, function, and location of the enzyme made in vivo and in vitro. The combined results of these studies and of those previously reported support the conclusion that the enzyme is functionally associated with the
The phosphatidylserine synthase of Escherichia coli, CDP-1,2-diacyl-sn-gylcerol (CDP-diacylglycerol):L-serine O-phosphatidyltransferase (EC 2.7.8.8), catalyzes the committed step in the synthesis of the major phospholipid (phosphatidylethanolamine) of this organism. This enzyme, which has a minimum molecular weight of 54,000, was purified to homogeneity (11), and its kinetic properties were partially characterized (2). By using correction of a conditional lethal mutant which affects the expression of active enzyme as a selection technique (22), the structural gene (pss locus at min 56 on the E. coli chromosome) for the enzyme was cloned into multicopy-number plasmids (17). Cells carrying such plasmids overproduce the enzyme in large
membrane fraction of the cell and not with the ribosomes. MATERIALS AND METHODS Materials. All chemicals were reagent grade or better. Ribonucleotide triphosphates and pyruvate kinase were purchased from P-L Biochemicals, Inc. Protein A-Sepharose, amino acids, all other nucleotides, phosphoenolpyruvate, E. coli tRNA, antibiotics, cycloserine, RNase A (treated at 100°C before use), N-a-p-tosyl-L-lysine-chloromethylketone, phenylmethylsulfonyl fluoride, p-aminobenzamidine hydrochloride, phosphatidylethanolamine, cardiolipin, phosphatidylglycerol, and cyclic AMP were obtained from Sigma Chemical Co. Radiochemicals were obtained from Amersham Corp. The electrophoresis reagents were from Bio-Rad Laboratories. Triton X-100 was purchased from Rohm & Haas Co. Precoated analytical silica gel thin-layer plates were purchased from E. Merck AG, and XAR-5 X-ray film from Eastman Kodak Co. was used throughout. Bacterial growth medium was obtained from Difco Laboratories. CDP-1,2-diacylglycerol was prepared as previously described (11), and phospholipid standards were obtained from Avanti Biochemicals. Bacterial strains, plasmids, and growth conditions. Strain JA200 (F+ trpE5 recA thr leu) was previously described (3). Strain RA2021 (pss-21) was obtained from C. Raetz, University of Wisconsin, Madison, and is temperature sensitive at 43°C for growth and for phosphatidylserine synthesis (20); cell extracts of the strain also have less than 1% of wild-type levels of phosphatidylserine synthase activity after treatment at 43°C. Strain CSR603 (uvrA6 phr-l recAl) was
amounts.
Extensive studies of this enzyme have not resolved the question of its intracellular location. Studies of crude cell extracts of E. coli (13, 21) clearly showed that the enzyme is tightly associated with the ribosomal fraction of thd cell. These results are similar to those reported for other gramnegative organisms (4) but are in marked contrast to those reported for gram-positive bacteria in which the enzyme was found to be membrane assoicated (5, 6). Further studies of the enzyme in crude extracts (13) suggested that the interactions with the ribosome are ionic, through the active site of the enzyme, and an artifact of the in vitro system. Studies of the purified enzyme (2) showed that the enzyme has a low affinity for hydrophobic-hydrophilic interfaces. The affinity can be greatly enhanced by the presence of the lipid substrate. Such data suggest that the enzyme is a peripheral membrane protein that interacts with the membrane surface
* Corresponding author. t Present address: Department of Ophthalmology, Baylor College of Medicine, Houston, TX 77030. t Present address: Abbott Laboratories, North Chicago, IL
60064.
805
806
LOUIE ET AL.
J. BACTERIOL. EcoRI
Sal
PLC34-44Cls I
Sal I
pPS3155 Clia I
Sal I
Sal
Sal
Sal
pPS4O17
CIa IBegI I
\
Sal I
Sal
EcoRI
Sal I
EcoRI
EcoRI
Sal
pPS5004 Cia I
I
Bgl I
I
I
Sal I I--
-u -
-
-
-
-
-
3
-
-
-
-
-
rc
- -__ -
8
18
Kilobase Pairs FIG. 1. Restriction endonuclease maps of E. coli regions of pss-containing plasmids. _, DNA derived from E. coli; -, DNA from the cloning vector (ColEl for pLC34-44 and pBR322 for the other plasmids); ------, regions of DNA deleted from each plasmid relative to pLC34-44 (deletions were generated by partial digestion with Sail endonuclease followed by ligation of the linear DNA). Tc indicates the position of the tetracycline resistance gene, and the arrow indicates the direction of transcription of the pss gene.
obtained from W. D. Rupp, Yale University, New Haven, Conn. Plasmids pPS4017 (pss+ Tcr Ap9, pPS3155 (pss+ Tcr Apr immColEl), pPS3155-X (pss+ Tcs Apr immColEl immx [N O P]x), pLC34-44 (pss+ nadB+ immColEl), and pPS5004 (Apss Tcr Ap) were previously described (17). pBR322 (Tcr Ap9 was obtained from A. Dugaiczyk, Baylor College of Medicine Houston, Tex. Partial restriction endonuclease maps of some of these plasmids are shown in Fig. 1. Preparation of plasmid DNA was carried out by using a Triton X100-lysozyme extraction method followed by CsCl buoyant density gradient centrifugation as previously described (17). E. coli cells made competent by CaCl2 treatment were transformed with plasmid DNA (12). The desired single colonies were isolated by drug resistance and screened for plasmid markers and the presence of plasmids (1). Bacteria, including strain JA200(pPS3155-A) fully induced for phosphatidylserine synthase, were routinely grown on LB agar plates or in liquid medium supplemented with the appropriate antibiotics as previously described (17). K9 salts medium (23) used in the preparation of maxicells was M9 medium (16) supplemented with 1% Casamino Acids and 0.2% thiamine. For labeling of phosphatidylserine synthase in vivo, strain JA200(pPS3155-A) was grown at 30°C in M9 medium containing MgCl2 rather than MgSO4, 13 ,ug of Na2SO4 per ml, 0.001% thiamine, 40 pug of ampicillin per ml, and 40 ,ug each of leucine, threonine, and tryptophan per ml. Cells were induced in the early log phase of growth for high overproduction of phosphatidylserine synthase by elevating the temperature to 43°C for 20 min followed by growth at 37°C. Protein was labeled by adding 5 ,uCi of 3QS04 per ml 15 min after the end of induction. Labeling was terminated after 1 to 3 h by adding excess unlabeled Na2SO4. Cell extracts for gradient centrifugation were prepared by suspending approximately 4 g (wet weight) of cells in 10 to 15 ml of 10 mM Tris hydrochloride (pH 7.4) containing 2 mM MgCl2. The suspension was passed twice through a French pressure cell, and unbroken cells were removed by centrif-
ugation at 5,000 x g for 10 min; radiolabeled cells were disrupted with a Branson W185 sonicator. The supernatants of these extracts routinely contained 15 to 20 mg of protein per ml. Protein synthesis systems. Polypeptides synthesized in maxicells (24) were labeled with [35S]methionine with CSR603 as the host strain for the indicated plasmids. Plasmid-containing cells were grown at 37°C to an optical density at 600 nm of 0.5 in K9 salts medium supplemented with the appropriate antibiotics and then irradiated with UV light at 10 J/m2. The cells were then incubated for 12 h at 37°C; during this time the medium was supplemented with cycloserine (100 ,ug/ml at 1 h and 5 h postirradiation). The cells were then washed twice with M9 salts medium and were suspended in this medium without sulfate at a density of 4 x 108 cells per ml. After preincubation for 60 min, the cells were incubated in the presence of [35S]methionine (10 ,Ci/ml) for 60 min at 37°C. The cells were harvested by centrifugation and suspended in 200 ,ul of sodium dodecyl sulfate (SDS) electrophoresis buffer (50 mM Tris hydrochloride [pH 7.5], 2% SDS, 10 mM dithiothreitol, 10% glycerol). The suspension was heated at 100°C for 2 min, and insoluble material was removed by centrifugation. The supernatant was subjected to SDS-polyacrylamide gel electrophoresis (10), or the phosphatidylserine synthase was selectively precipitated by antibody before electrophoresis as described below. Phosphatidylserine synthase was synthesized in vitro with a plasmid DNA-directed coupled transcription-translation system. Strains JA200 and RA2021 were used for the preparation of supernatants (from centrifugation at 30,000 x g) (S-30 extracts) as described by Zubay (27); such preparations had an optical density at 260 nm of 75/ml. Synthesis of protein in vitro was carried out in a final volume of 50 ,ul containing 30 mM Tris hydrochloride (pH 7.5), 150 mM potassium acetate, 15 mM magnesium acetate, ribonucleotides (ATP, GTP, UTP, and CTP) each at 2 mM, 0.1 mM cyclic AMP, E. coli tRNA at 0.2 mg/ml, 5 mM
PHOSPHATIDYLSERINE SYNTHASE
VOL. 165, 1986
phosphoenolpyruvate, pyruvate kinase at 0.01 mg/ml, 1 mM dithiothreitol, amino acids (except methionine) at 10 ,uM each, 20 ,uCi of [35S]methionine, plasmid DNA at the indicated amount, and 30 ,ul of E. coli S-30 extract. If the enzymatic activity of phosphatidylserine synthase was to be measured rather than the incorporation of radiolabel, the labeled methionine was replaced by unlabeled methionine at a concentration of 100 t.M. The protein synthesis mixtures were incubated at 37°C either for 1 h or for the indicated time, and the reactions were stopped by cooling on ice or by the addition of RNase A. Samples for SDS-polyacrylamide gel electrophoresis were precipitated either with 10% trichloroacetic acid or with antibody as outlined below. For measurement of phosphatidylserine synthase activity, 10 ,ul of the RNase A-treated sample was incubated with or without 0.1 mM CDP-diacylglycerol for 30 min at 30°C in a final volume of 50 ,ul containing 100 mM Tris hydrochloride (pH 7.5), 0.5 mM L-[U-14C]serine, 200 mM ammonium sulfate, 0.05% Triton X-100, and 1 mM AMP. The formation of phosphatidylserine dependent on CDP-diacylglycerol was quantitated as previously described (11). Preparation of membranes. Everted membrane vesicles were prepared from strain JA200 after disruption with a French pressure cell as previously described (15). Lipids to be used for membrane supplementations were dissolved in chloroform, brought to a pH of 7.0 to 7.5 with methanolic ammonia, and dried under vacuum. The lipids were then suspended in water by sonication, mixed with membranes prepared as described above, and incubated at 37°C for 30 min before use. Immunoprecipitation and SDS-polyacrylamide gel electrophoresis. Purified rabbit pooled immunoglobulin directed against purified phosphatidylserine synthase was prepared by ammonium sulfate precipitation followed by passage through DEAE-cellulose in 10 mM potassium phosphate, pH 7.0 (7). At the end of the protein synthesis incubations described above, the samples were immunoprecipitated by using specific antibody and protein A-linked Sepharose 4B (8). The final precipitates were heated to 100°C in the presence of SDS electrophoresis buffer and then subjected to SDS-polyacrylamide gel electrophoresis (10). The gels were stained with Coomassie blue R-250 to visualize protein standards, dried under vacuum, and exposed to X-ray film. Gradient centrifuation. (i) Method 1. A 20 to 50% glycerol gradient containing 10 mM Tris hydrochloride (pH 7.4) and 2 mM MgCl2 was used to separate cytoplasmic, ribosomal, and membrane fractions. Centrifugation was carried out at an average of 200,000 x g for 4 h in tubes (1.4 by 8.9 cm) as previously described (13). (ii) Method 2. The membrane fraction was separated from the cytoplasmic fraction by sedimenting the membrane vesicles onto a 70% sucrose cushion through a 20 to 50% glycerol gradient. Centrifugation was carried out in cellulose nitrate tubes (1.1 by 6.03 cm) at an average of 200,000 x g for 1.5 h to 4°C with a Beckman SW60 rotor. The 4.6-ml glycerol gradient contained 0.1 M Tris hydrochloride (pH 7.4) and 15 mM EDTA, and the 0.5-ml sucrose cushion contained 10 mM Tris hydrochloride (pH 7.4) and 2 mM MgCI2. Under these conditions only the membrane fraction sedimented onto the sucrose cushion; ribosomes and other soluble material remained in the glycerol gradient. This was verified by optical density measurements and the distribution of phosphatidylserine synthase activity. Miscellaneous procedures. Phosphatidylserine synthase was purified and assayed as previously described (17). Protein concentration was determined by the method of
807
Lowry et al. (14). Membranes containing radiolabeled lipids were extracted with chloroform-methanol as previously described (17). Radiolabeled lipid products were separated by silica gel chromatography with chloroform-methanol-acetic acid-water (50:28:4:8) and located by autoradiography relative to known standards. For quantitation, the radioactive spots were scraped from the glass plate and counted directly in scintillation fluid. RESULTS Protein synthesis in maxicells. Maxicells of strain CSR603 which had been transformed by plasmids were prepared and labeled with [35S]methionine. The labeled proteins were displayed by SDS-polyacrylamide gel electrophoresis and detected by autoradiography either directly after SDS lysis or after antibody precipitation of the SDS lysates (Fig. 2). All the plasmid-containing strains showed the ampicillin resistance gene product at 28,000 to 31,000 daltons. Only strains containing plasmids with the pss gene exhibited a product of 54,000 daltons; these bands coincided with the band for purified phosphatidylserine synthase which was visualized on the Coomassie blue-stained gel (data not shown). The very dark band visible near 37,000 daltons (lanes 4 and 6) is probably the product of the tetracycline resistance gene (24), which is more prominent in strains containing pPS4017 or pPS5004 because these plasmids confer a fourfold-higher resistance to tetracycline than other plasmids such as pPS3155 or pBR322. The identification of the 54,000-dalton species was further established by precipitation with antibody made against purified phosphatidylserine synthase (lanes 9 and 10). These results clearly demonstrate that the DNA segments in plasmids pPS3155 and pPS4017 which correct the temperature-sensitive growth phenotypes and enzyme defects in the pss mutants RA2021 (20) and RA80 (18) do in fact code for the phosphatidylserine synthase. Strains containing pPS5004 showed no band at 54,000 daltons but showed a new band near 50,000 daltons (lane 6); this band moved slightly faster than the faint background bands visible in the neighboring lanes and was weakly, but 1
2 3 4 5 6 7 8 9 10 11
_
| | | ~~~~~~~~~~~11
FIG. 2. Labeled proteins synthesized in maxicells containing plasmids. Maxicells were labeled, and either total protein (lanes 3 to 6) or protein precipitable by antibody (lanes 7 to 11) was displayed by SDS-polyacrylamide gel electrophoresis. Lanes: 1 and 7, no plasmid; 3 and 8, pBR322; 4 and 9, pPS4017; 5 and 10, pPS3155; 6 and 11, pPS5004; 2, blank. K, Molecular weight in thousands.
808
LOUIE ET AL.
J. BACTERIOL.
4K *:~~~~~~~~~-E . -31 K
i-:W28K
FIG. 3. Proteins synthesized in a coupled transcriptiontranslation system primed by plasmid DNA. Labeled protein was synthesized by using an S-30 extract prepared from strain JA200 supplemented with plasmid pPS4017 (100 p.g/ml), and either total protein (lanes 1 to 3) or protein precipitable by antibody (lanes 4 to 6) was subjected to SDS-polyacrylamide gel electrophoresis. Lanes: 1 and 4, no DNA; 2 and 5, pBR322; 3 and 6, pPS4017. K, Molecular weight in thousands.
able to direct the. synthesis of increasing amounts of active enzyme dependent on time at a fixed DNA concentration (Fig. 4A) and on DNA concentration at a fixed time (Fig.. 4B). At several data points (Fig. 4B) the incorporation of radiolabeled serine into chloroform-soluble material by the reaction mixture was shown to be time dependent. The chloroform-soluble products formed were phosphatidylserine if hydroxylamine was included in the assay mixture to inhibit endogenous phosphatidylserine decarboxylase activity (25) and a mixture of phosphatidylserine and phosphatidylethanolamine if hydroxylamine was omitted; product identification was based on mobilities on thin-layer chromatography plates relative to known standards. Therefore, the enzyme synthesized in vitro had the same motecular weight as the purified enzyme and exhibited enzymatic activity. These results suggest that no extensive posttranslational events are necessary to synthesize a functional enzyme.
.I-
"
z
w
reproducibly, precipitable with the antibody (lane 11). pPS5004 was derived from pPS4017 (Fig. l) by removing a Sall-SalI fragment from one end of the cloned E. coli DNA; this plasmid does not complement pss mutants (17). pPS5004 apparently codes for a nonfunctional truncated protein which has lost about 4,000 daltons, presumably from its carboxyl-terminal end, but is still capable of cross-reacting with antibody; therefore, transcription of the pss gene most likely proceeds in the direction indicated on pPS4O17 in Fig. I. A less likely possibility is that deletion of the amino terminus of the protein has occurred, resulting in expression of cross-reacting material by initiation of transcription from a pBR322 promoter, and initiation of protein synthesis at an internal methionine. Protein synthesis in vitro. To determine the minimum requirements for expression of phosphatidylserine synthase, the synthesis of the enzyme was studied in a coupled transcription-translation system dependent on plasmid DNA. pPS4017 was able to direct the synthesis of a 54,000dalton protein (Fig. 3, lane 3) in addition to the precursor and mature form of P-lactamase which are visible at 31,000 and 28,000 daltons, respectively. The 54,000-dalton product comigrated with authentic phosphatidylserine synthase added as a carrier and visualized with Coomassie blue (data not shown). In addition, this product was precipitable with an antibody made against the enzyme (Fig. 3, lane 6) but not with control antiserum. Identical results were obtained with an S-30 extract prepared from the pss mutant RA2021. There was no evidence of a larger form of the enzyme being made even in the presence of a protease inhibitor (1 mM N-a-ptosyl-L-lysine-chloromethylketone). As was the case in maxicells, pPS5004 coded for a 50,000-dalton protein which was weakly precipitable with antibody (data not shown). To measure the synthesis of enzymatically active phosphatidylserine synthase in vitro, S-30 extracts were made from strain RA2021 which contained less than 1% wild-type levels of phosphatidylserine synthase enzymatic activity. These extracts supplemented with pPS4017 were
N z W
,
TIME, MIN
B. .6-
-
z
I
z
.4
N z
.2
i-e 208 2S6 i00 CDNA3 JJG/ML FIG. 4. Synthesis of enzymatically active phosphatidylserine. synthase in vitro. An S-30 extract prepared from strain RA2021 was used; protein was synthesized in the absence of radiolabel. After protein synthesis was stopped, portions were assayed for phosphatidylserine synthase activity. Enzyme units refers to the CDP-diacylglycerol-dependent incorporation of radiolabeled serine (nmollmin) into chloroform-soluble product by the enzyme present in each 50-pl portion of the protein-synthesizing mixture. (A) Protein synthesis was stopped at the times indicated by the data points, and the level of enzymatic activity was measured. Symbols: 0, protein synthesis primed with pPS4017 (100 jLg/ml); unprimed control. The results with pBR322 were the same as those for the control. (B) Protein synthesis was carried out as described above for 1 h with the pPS4017 DNA concentrations indicated by the data points. After protein synthesis was stopped, enzymatic activity was measured.
5e0
*,
PHOSPHATIDYLSERINE SYNTHASE
VoL. 165, 1986
TLCK
No Inhibitors
1 2 PS synthase-_
-
-
3
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PAB
PMSF
6
5
7
8
10 11
9
12
.-
n
809
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RON
0
-PS synthase
FIG. 5. Size and distribution of phosphatidylserine (PS) synthase from cells disrupted in the presence of various protease inhibitors. Cells labeled with 35SO4 in the absence of protease inhibitors were disrupted in the absence of protease inhibitors (lanes 1 to 3) or in the presence of the inhibitor N-a-p-tosyl-t-lysine-chloromethylketone (1 mM, lanes 4 to 6), phenylmethylsulfonyl fluoride (0.5 mM, lanes 7 to 9), or p-aminobenzamidine (5 mM, lanes 10 to 12). These crude cell extracts were then subjected to glycerol gradient centrifugation (method 1) to separate them into membrane (lanes 1, 4, 7, and 10), ribosomal (lanes 2, 5, 8, and 11), and cytoplasmic (lanes 3, 6, 9, and 12) fractions. Antibody and protein A-linked Sepharose 4B-precipitated samples were separated by SDS-polyacrylamide gel electrophoresis, and the positions of the protein products were determined by autoradiography.
Size of native enzyme. The ribosomal location of the phosphatidylserine synthase may be the consequence of postlysis proteolysis of a membrane-bound form of the enzyme. To investigate this possibility strain JA200 (pPS3155-A) was labeled with 35SO4. Under these conditions plasmid-encoded proteins should be preferentially labeled since the plasmid copy number is highly elevated and cell growth is inhibited (17). After 1 h of labeling, cells were mixed with the indicated protease inhibitor, disrupted, and separated into fractions by glycerol gradient centrifugation (Fig. 5). In all cases the size of the phosphatidylserine synthase was indistinguishable frorn the purified enzyme, and the majority (greater than 90%) of the labeled enzyme was found in the ribosomal and cytoplasmic fractions; the amount of label associated with the membrane fraction was variable and was always a small fraction of the total. Assay of the gradients for phosphatidylserine- synthase -activity showed that the activity sedimented in parallel with labeled enzyme (data not shown). Direct antibody precipitation was no more specific than total protein precipitation. Since the major labeled proteins should have been products of genes carried by the plasmid, the proteins other than that at 54,000 daltons were e-ither degradation products of phosphatidylserine synthase or products not normally found in E. coli. An alternate approach used in determining the size of the enzyme in vivo was the lysis of freshly grown cells [strain JA200 or JA200(pPS3155-X) after induction] under conditions designed to minimize postlysis proteolysis. In one case the cells were lysed by direct treatment with SDS-polyacrylamide gel electrophoresis buffer at 100°C. Alternatively, cells were treated at 0°C with 0.28 M Na&H containing 1.1% P-mercaptoethanol, precipitated with 5% trichloroacetic acid, and extracted with SDS electrophoresis 'buffer at 100C. The size of the phosphatidylserine synthase in these extracts was determined by SDS-polyacrylamide gel electrophoresis followed by electrotransfer to nit-rocellulose and detection with specific antibody (6). With both methods -of cell lysis, only one antigenic species was detected which had the same mobility as the purified protein. These results establish that no extensive proteolysis of the enzyme occurred between the time of cell lysis and the time of isolation' of the pure enzyme; therefore, the possibility that a postlysis modification of the enzyme accounted for its
ribosomal association (and lack of membrane association) seems unlikely. However, the methods used were not sensitive enough to detect small differences in molecular weight. Membrane association of phosphatidyiserine synthase. Pre-
A
MEMBRANES 1 2 3
PS synthase
B
a
MEMBRANES 1 2 3 4
PS synthase-_.-..
SUPERNATANT 5 6
4
"No
-PS synthase
SUPERNATANT 5 6 7 8
umw-PS synthase
FIG. 6. Localization of in vitro-synthesized phosphatidylserine (PS) synthase in the presence of membranes supplemented with phospholipids. Membranes isolated from strain JA200 were supplemented with phospholipids at the indicated amounts and then incubated at a concentration of 1.75 mg/ml for 30 min at 37°C with the translation products made as described in the legend to Fig. 3. After incubation the mixtures -were separated into membrane and supernatant fractions by glycerol gradient centrifugation (method 2), precipitated by trichloroacetic acid, and separated by SDSpolyacrylamide gel electrophoresis. (A) Membranes supplemented with no lipids (lanes 1 and 4), with CDP-diacylglycerol at 0.6 ,umol/mg of membrane protein (lanes 2 and 5), or with phosphatidylserine at 1.4 ,umol/mg of membrane protein (lanes 3 and 6). (B) Membranes supplemented with phosphatidylethanolamine (lanes I and 5), cardiolipin (lanes 2 and 6), phosphatidylglycerol (lanes 3 and 7), or lysophosphatidylserine (lanes 4 and 8), all at 1.4 ,umol/mg of membrane protein.
810
LOUIE ET AL.
J. BACTERIOL.
200 160
120 80
a)
40
._
a)
0
-c
200
co s h._ 0
-rC0~
160 120
80 40
0 4
8
12
16
4
20
TOP
BOTTOM
Fraction No.
8
12
16
BOTTOM
20 TOP
K_i.-K
'-racitorn
Nu.
FIG. 7. Sedimentation profiles of phosphatidylserine synthase activity after glycerol gradient centrifugation in the presence of added membranes. Cell extracts of strain JA200(pPS3155-A) at 2 mg of protein per ml were mixed with phospholipid-supplemented membranes at 1.75 mg of protein per ml, incubated at 37°C for 30 min, and subjected to glycerol gradient centrifugation (method 1). Fractions were collected and assayed for phosphatidylserine synthase activity. Membranes were supplemented with phospholipids at the indicated amounts (,umoles per milligram of membrane protein). (A) No addition; (B) CDP-diacylglycerol, 0.6; (C) phosphatidylglycerol, 1.4; (D) phosphatidylethanolamine, 1.4.
vious studies indicated that the most likely method of preventing ribosomal association and inducing membrane association by the enzyme would involve the presence of membrane-associated lipid substrate under catalytically favorable conditions (2, 13). In vitro protein synthesis with an S-30 extract from strain JA200 was carried out for 1 h and terminated with RNase A. The extracts were then mixed with E. coli membranes supplemented with phospholipids as indicated and subjected to glycerol gradient centrifugation (Fig. 6). In separate experiments with labeled phospholipids, greater than 90o of the added phospholipids sedimented with the membrane fraction; since the same phospholipids remained at the top of the sucrose gradient in the absence of membranes, the lipids were assumed to be membrane associated. Addition of unsupplemented membranes caused no change in the distribution of phosphatidylserine synthase, whereas supplementation of membranes with CDPdiacylglycerol resulted in complete localization of the enzyme to the membrane fraction. Phosphatidylserine caused a similar effect but only at a higher concentration. Neither lysophosphatidylserine nor lyso-CDP-diacylglycerol (data not shown) had an effect on the distribution pattern; these phospholipids are not substrates or competitive inhibitors of the enzyme. The major phospholipid of E. coli, phosphatidylethanolamine, caused no membrane association even when supplemented at a concentration threefold higher than that of the lipid substrate. On the other hand, the minor acidic lipids of E. coli, phosphatidylglycerol and cardiolipin, did induce membrane association. This may be related to the
general affinity of the enzyme for surface-bound phosphate residues and will be discussed later. Similar experiments were carried out with cell extracts of strain JA200(pPS3155-X), but in these experiments the distribution of phosphatidylserine synthase activity was measured (Fig. 7). Similar results were observed in that membranes only or membranes supplemented with phosphatidylethanolamine induced no association, whereas both of the acidic lipids and the lipid substrate induced membrane association. Lowering the level of the supplemented lipid to 0.15 ,umol/mg of membrane protein resulted in about a 50:50 partition of enzymatic activity between the membrane and supernatant fractions. At this concentration the level of the supplemented lipid was about 50% by weight of the total lipid present in the membranes; the high level of lipid substrate required to induce significant membrane association and hence allow catalysis is actually consistent with the conclusion drawn from genetic experiments (17, 18) that the level of enzyme present in cells is in large excess over what is needed for minimum cell function. Under the conditions of the experiment the enzyme was also catalytically active. After sedimentation in the presence of radiolabeled serine, the membrane fraction was analyzed for radiolabeled phospholipid; it contained a mixture of phosphatidylserine and phosphatidylethanolamine (the product of the next enzyme in the pathway) representing about 30% conversion of the CDP-diacylglycerol to product. Previous work showed that the enzyme interacts ionically with ribosomes as evidenced by disruption of this associa-
PHOSPHATIDYLSERINE SYNTHASE
VOL. 165, 1986
7 200t j
36%
58%
B.
en i
400
°
300-
200-
-
6%
83%
100
0
4
8
12
BOTTOM
16
20
TOP
Fraction No. FIG. 8. Sedimentation profiles of phosphatidylserine synthase activity after glycerol gradient centrifugation at high ionic strength. Experimental conditions were those described in the legend to Fig. 7 except that the gradients also contained 0.8 M NaCl. The added membranes were supplemented with either CDP-diacylglycerol (A) or phosphatidylglycerol (B) at 0.6 ,umol/mg of membrane protein. The distribution between the two major peaks of the total activity applied to each gradient is indicated.
tion by high-ionic-strength buffers (13). On the other hand, interaction with lipid substrate is favored by high ionic strength (2). A similar differential effect occurred when glycerol gradients were run in the presence of 0.8 M NaCl which completely prevented membrane association induced by phosphatidylglycerol but had a much less dramatic effect on membrane association in the presence of lipid subtrate (Fig. 8). Therefore, the substrate-induced association of the enzyme with membranes exhibits a specificity beyond simply an ionic interaction.
DISCUSSION The phospholipid composition of the E. coli cell envelope is nearly invariant under a wide variety of growth conditions (19). The only means by which this composition can be varied significantly is by introducing specific mutations in genes coding for enzymes directly responsible for phospholipid synthesis (19). Overproduction of these enzymes brought about by using recombinant DNA techniques resulted in only minor alterations in phospholipid metabolism (20). Based on these observations, it appears that the primary mode of regulating phospholipid metabolism is through regulation of biosynthetic enzymes rather than through regulation of the levels of the enzymes. In addition, the
811
regulation of phospholipid metabolism in E. coli appears to be a highly coordinated process. For this reason our laboratory has actively investigated the enzymatic properties of the phosphatidylserine synthase. To fully understand the regulation of this enzyme, its functional location in vivo must be established. Previous work from our laboratory suggested that the ribosomal association demonstrated by this enzyme in vitro (13) is an artifact due to the high affinity of the enzyme for surface-bound phosphate and for cytidine. Since the association can be prevented by lipid substrate under catalytic conditions, the binding to ribosomes certainly cannot occur during catalysis. This ribosomal binding is a property of the enzyme since it also associates with ribosomes from Bacillus licheniformis, whereas the analogous enzyme from B. licheniformis (which is a membrane-bound enzyme) associates preferentially with membranes from both E. coli and B. licheniformis (6). Studies with the purified enzyme from E. coli showed that the enzyme has little affinity for hydrophobic domains since it does not bind to nonionic detergent micelles. On the other hand, binding to detergent micelles can be induced by the presence in the micelles of lipid substrate or product consistent with kinetic experiments which demonstrate a dependence on surface-bound substrate for catalysis (2). The results reported here further support the conclusion that the enzyme is functionally associated with the membrane and also strongly imply that no postlysis modification of the enzyme occurs which can account for its preferential association with ribosomes. All attempts to demonstrate the presence of a form of the enzyme larger than that purified from E. coli were unsuccessful. In addition, inclusion of a variety of protease inhibitors during cell lysis and subcellular fractionation did not alter the in vitro location of the enzyme. Finally, synthesis of the protein with two different in vitro systems resulted in a product indistinguishable from the purified enzyme. This product was enzymatically active and had the same binding properties as the purified enzyme and the enzyme found in crude cell lysates. Therefore, short of sequencing the structural gene and the isolated gene product, it appears that no posttranslational or postlysis modification occurs and that the physical and enzymatic properties of the purified enzyme are not due to a postlysis modification event. Although the functional association of the enzyme with the membrane is supported by our results, the physiological importance of the association of the enzyme with ribosomes in vivo is still not settled. The fact that in in vitro studies the enzyme had affinity for both 30S and 50S ribosomal subunits (21) does not strengthen the argument for a specific role for this association. Furthermore, the enzyme may associate with the membrane in the manner suggested by the kinetic experiments, i.e., by substrate-induced binding to a surface (2). Both the gene product made in vitro and the enzyme in crude lysates can be induced to associate with the membrane fraction if the membranes are supplemented with either the substrate, CDP-diacylglycerol, or the product, phosphatidylserine. The levels of these lipids required are much higher than those normally found in membranes, but the substrate effect is much stronger than the product effect. The lyso derivatives of these lipids are neither substrates nor inducers of membrane association. In addition, the acidic lipids of E. coli also induce membrane association when their levels are raised significantly above normal levels. This association is primarily ionic in nature and is not as strong as substrate-induced association at high ionic strength. The low level of mem-
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brane association always observed in the absence of supplementation may be a function of the levels of these lipids in the membrane. The effect brought about by the acidic lipids may have some physiological significance since increased membrane association by the enzyme might result in an increase in phosphatidylethanolamine synthesis which would compensate for the high levels of acidic lipids. As indicated above the phosphatidylserine synthase appears to be a peripheral membrane protein which has high affinities for surface-bound substrate and for phosphate residues. An important component of the regulation of the enzyme would then be the composition of the membrane surface, so that membrane association, and hence catalytic capability, would be enhanced by increases in the levels of CDP-diacylglycerol and acidic lipids in the membrane. Still unresolved is why E. coli has such a highly regulated phospholipid biosynthetic process since many of the strains with mutations in phospholipid biosynthesis have significant alterations in phospholipid compositions, yet are still viable under selective conditions (9, 16a, 18). ACKNOWLEDGMENTS This research was supported in part by Public Health Service grant GM20478 from the National Institute of General Medical Sciences. K.L. was supported in part by Molecular Basis of Cell Function Training grant GM07542 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Barnes, W. M. 1977. Plasmid detection and sizing in single colony lysates. Science 195:393-394. 2. Carman, G. M., and W. Dowhan. 1979. Phosphatidylserine synthase from Escherichia coli: the role of Triton X-100 in catalysis. J. Biol. Chem. 254:8391-8397. 3. Clarke, L., and J. Carbon. 1976. A colony bank containing synthetic ColEl hybrid plasmids representative of the entire E. coli genome. Cell 9:91-99. 4. Dutt, A., and W. Dowhan. 1977. Intracellular distribution of enzymes of phospholipid metabolism in several gram-negative bacteria. J. Bacteriol. 132:159-165. 5. Dutt, A., and W. Dowhan. 1981. Characterization of a membrane-associated cytidine diphosphate-diacylglycerol-dependent phosphatidylserine synthase in Bacilli. J. Bacteriol. 147:535-542. 6. Duft, A., and W. Dowhan. 1985. Purification and characterization of a membrane-associated phosphatidylserine synthase from Bacillus licheniformis. Biochemistry 24:1073-1079. 7. Fahey, J. L., and E. W. Terry. 1973. Ion exchange chromatography and gel filtration, p. 7.1-7.16. In D. M. Weir (ed.), Handbook of experimental immunology. Blackwell Scientific Publications, Ltd., Oxford. 8. Firestone, G. L., and E. C. Heath. 1981. The cyclic AMPmediated induction of alkaline phosphatase in mouse L-cells. J. Biol. Chem. 256:1396-1403. 9. Hawrot, E., and E. P. Kennedy. 1978. Phospholipid composition and membrane function in phosphatidylserine decarboxylase mutants of Escherichia coli. J. Biol. Chem. 253:8213-8220.
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