The Stability of Acyl Carrier Protein in Escherichia coZi*

Abstract Two techniques have been employed to examine the turnover of the protein portion of acyl carrier protein, the conjugated protein which is the acyl carrier for fatty acid biosynthesis in Escherichia coli. The protein was shown to be metabolically stable, in contrast to its covalently bound 4'-phosphopantetheine prosthetic group which was previously found to undergo turnover at the rate of 4% per min (Powell, G. L., Elovson, J., and Vagelos, P. R. (1969) J. Biol. Chem. 244, 5616–5624).

The protein was shown to be metabolically stable, in contrast to its covalently bound 4'phosphopantetheine prosthetic group which was previously found to undergo turnover at the rate of 4 % per min (POWELL, G. L., ELOVSON, J., AND VAGELOS, P. R. (1969) J.BIoL.
Acyl carrier protein, a small molecular weight protein (mol wt 8847) possessing a covalently bound 4'-phosphopantetheine prosthetic group, is the specific carrier of acyl groups during fatty acid biosynthesis in Escherichia coli (1). It appears to be localized in the bacterial membrane (2). Earlier work suggested that CoA is the biosynthetic precursor for the 4'-phosphopantetheine prosthetic group of ACPl (a), and subsequently the enzymes catalyzing the synthesis of holoACP from apoACP and CoA and that catalyzing the removal of the prosthetic group were purified and partially characterized (4,5). In vivo kinetic radioisotopic tracer analysis of the metabolism of the pantothenate compounds in E. coli has verified the precursorproduct relationship between CoA and ACP and revealed the loss and subsequent replacement of the prosthetic group from holoACP at the remarkable rate of 40/, of the total holoACP pool per min in a culture with a doubling time of 70 min (6). The biological significance of this observation is as yet unknown. In view of the known turnover of the prosthetic group of * This work was supported by a Faculty Research Grant (G. L. P.), funds from the Department of Chemistry and Geology (G. L. P.), and funds from the National Science Foundation (Grant GB-18998) (A. R. L. used is: ACP, this is used interchangeably with holoACP for acyl carrier protein with the 4'-phosphopantetheine group covalently attached. ApoACP (the apoprotein lacking the 4'-phosphopantetheine group) is used in apposition to the term holoACP. ACP, the existence of a small class of E. coli proteins which exhibit high turnover rates (7-lo), and the apparent importance of protein turnover in the control of protein concentrations in mammalian cells (ll), we undertook a study of the stability of the protein portion of holoACP.
Our observations indicate that ACP is a stable protein in E. coli.

AND MATERIALS
Pulse Experiment-E. coli M-99, a gift from P. R. Vagelos and previously employed by him in the study of ACP metabolism (3), is a /3-alanine auxotroph.
These cells were grown from a small inoculum at 30" in 500 ml of a medium modified from Pardee et al. (12) containing (at pH 7.0) 15 g of (NH&S04, 65 g of KHzP04, 0.5 g of MgS04*7HzO, and 50 g of glycerol per 5 liters of tap water and supplemented with 1.22 FCi (46.5 pg) of @-[1-Wlalanine per 500 ml of medium. At mid-log (A6?,, = 0.77) 0.288 pg of L- [4,5JH]leucine (1.0 mCi) was added. After 5 min 1.0 g of L-leucine (unlabeled and dissolved in 50 ml of medium) was added to the culture. After an additional 5 min the culture was chilled, harvested by centrifugation, and resuspended in 100 ml of unsupplemented medium which was finally added to 3130 ml of medium prewarmed to 36.5" and supplemented with 186 I.cg of &[l-Wlalanine (4.9 &i) and 10 g of L-leucine.
These experiments were carried out in a New Brunswick fermentor which was stirred at 850 rpm and aerated at a rate of 8 liters per min.
Samples were withdrawn at timed intervals, chilled, harvested by centrifugation, and frozen. ACP PurQication from Pulse Experimed-The frozen cells (1.5 to 1.7 g) were taken up in 20 ml of 0.02 M triethylamine . HCl (pH 7.5) and 10e2 M P-mercaptoethanol, broken in a French pressure cell (Aminco Instrument Company), and centrifuged at 12,000 X g for 30 min.
The supernatants were stored in liquid nitrogen.
ACP was isolated via a published procedure (13) on a small scale omitting the second DEAE-cellulose step. All operations were carried out at room temperature.
Only the peak tube of the last purification step was utilized to obtain the specific radioactivity (disintegrations per min of 3H per mg of protein) of purified ACP.
Dilution Experiment-E. coli Ilv 453 was kindly provided by H. E. Umbarger. This organism is a K-12 strain and has a deletion in the ilv E locus.
A lesion was introduced in the pan region using N, N'-nitronitrosoguanidine (14) and penicillin selection (15).
The new lesion was "tight," and no growth was 4461 by guest on March 25, 2020 http://www.jbc.org/ Downloaded from obtained in the absence of pantothenate. This organism was cultured at 37" in Vogel-Bonner mineral salts medium (16) contaming 0.2% glucose, 2 pM pantothenate, 10M4 M n-isoleucine, 10v4 M n-valine, and 10M4 M n-leucine in a G-25 New Brunswick incubator-shaker at 180 rpm. Under these conditions neither pantothenate nor isoleucine was growth limiting (valine and leucine seem to spare the isoleucine), and good exponential growth into full stationary phase (A620 -1.2) could be obtained. The pantothenate moieties and the isoleucine residues in the protein of these cells were fully labeled (to the same radioisotopic specific activity as the medium) by growth from a small inoculum of cells in 250 ml of medium of the above composition containing a final concentration of 2 pM [1-14C]pantothenate (5.2 mCi per mmole) and 10e4 M [4, 5-3H]isoleucine (1.5 mCi per mmole).
Fully labeled cells (250 ml) were harvested at room temperature by centrifugation at mid-log (A620 = 0.6), washed once with unlabeled medium, and resuspended in 600 ml of prewarmed medium (37") of composition similar to the above containing 2 PM [1-i4C] The pantothenate-containing compounds were then eluted with an increasing convex LiCl gradient using 70 ml of 0.7 M LiCl and 105 ml of 0.05 M LiCl both 0.01 M in Tris-HCl (pH 7.0) and 10u2 M in P-mercaptoethanol as previously described (6). The fractions containing ACP (determined by the characteristic conductivity and the l4C content of the fractions) were pooled and directly loaded onto a small (0.8 x 8.0 cm) column of A-25 DEAE-Sephadex which had been previously equilibrated with 0.02 M potassium phosphate (pH 6.2). Elution was accomplished using a linear gradient of 100 ml of 0.2 M LiCl and 100 ml of 0.6 M LiCl, both 0.02 M in potassium phosphate (pH 6.2) and low2 M @-mercaptoethanol. A-25 DEAE-Sephadex was found to be superior in this procedure to the A-50 DEAE-Sephadex used previously for ACP purification (13). The fractions containing 14C were pooled and frozen for further studies.
Two days were required to acquire the data for each pair of points.
Acrylamide Gel Electrophoresis of ACP-Fractions containing radioactively labeled ACP from a column chromatographic procedure were pooled, dialyzed overnight against 0.1 M urea and 10v2 M fi-mercaptoethanol, and lyophilized in glass vessels which had been treated with 100 ml of toluene containing 0.1 ml of 1,3,3,3-hexamethyldisilazane and 10 ml of dichloromethylsilane, rinsed with methanol, and oven dried (the silanation was necessary for quantitative recovery of the radioactive ACP). The lyophilized material was then taken up in 300 ~1 of electrophoresis buffer containing 10e2 M dithiothreitol, 100 ~1 of glycerol, and a small amount of methylene blue as a marker.
One such system, running at pH 7.5 was modified from System II (18). The separating gel was made by increasing the acrylamide to 7.0 g and including 0.06 g of N, N'-diallyltartaramide in place of the methylenebisacrylamide.
The stock solution was made up to 10.0 ml, and 4 ml of this stock solution were mixed with 1 ml of the buffer solution (48 ml of 1 N HCl, 6.85 g of trihydroxymethylaminomethane, and 0.45 ml of N, N, N'-tetramethylethylenediamine) and 3 ml of ammonium persulfate catalyst (14 mg/7.5 ml of water).
An aliquot (0.80 ml) of this mixture was cast in lo-cm lengths of 6-mm glass tubing.
The stacking gel was made with 1.85 g of N, N'-diallyltartardiamide in place of the methylenebisacrylamide.
The other solutions and procedures were unmodified.
The gels were removed from the glass electrophoresis tubes by first breaking the glass with the aid of a hammer, and the tough rubbery gels fractionated using a tuberculin syringe (no needle) and a razor blade much as described by Ward et al. (19).
Twenty to 30 fractions were collected in scintillation vials.
The gels were digested using 0.2 ml of 2% periodic'acid at 37" overnight.
When 2 ml of Soluene were added followed by 10 ml of a toluene-based scintillation mixture, a homogeneous sample suitable for liquid scintillation radioassay was obtained. An equally satisfactory and less expensive method consisted of solubilization of the digested gels by shaking in toluene-Triton X-100 scintillation solution (20) containing 4% Cab-0-Sil. The above mixtures do not display the strong chemiluminescence observed following hydrogen peroxide digestion of methylenebisacrylamide-containing gels. Moreover, the prolonged digestion in hydrogen peroxide required to digest the 30 y0 acrylamide gels employed earlier by us can lead to over 60% loss of label from pantothenate-labeled ACP. Other Assays and Procedures-The radioactivity of 14C-and 3H-containing samples was assayed in a toluene-Triton X-100 liquid scintillator solution (20) in the Packard Tri-Carb model 3320 liquid scintillation spectrometer or in Bray's solution (21) using the Nuclear-Chicago 720 series liquid scintillation system. Settings were chosen to essentially exclude 3H from the 14C channel and to limit the 3H efficiency in the r4C channel.
Calculations were performed in PL/l with the aid of the IBM 360/50 digital calculator.
We found it advisable to verify the specific radioactivity of [i4C]pantothenate in viva. We did this by estimating the CoA concentration (nanomoles per mg of cellular protein) in the crude extracts using an enzymatic assay for CoA (22). The disintegrations per min obtained in the CoA fraction isolated by DEAE-cellulose chromatography per mg of protein were then divided by the enzymatically estimated nanomoles of CoA to obtain the specific radioactivity in disintegrations per min per nmole.
These corrected activities were employed in subsequent calculations.
The purity of the [3H]isoleucine was verified by paper chromatography.
Protein concentration was estimated using the modified biuret procedure (23), the Lowry method (24) or, in the case of the very low concentrations encountered for the purified ACP, the spectrophotometric method of Waddell (25). ACP was assayed by the malonyl-CoA-CO2 exchange reaction as previously described (13

AND DISCUSSIOS
A number of studies on the turnover of bacterial cellular protein have been made and the results suggest that the average proteins are stable (average half-life of 30 days) in exponentially growing cells. This stability may decrease in nongrowing cells to a rate of degradation and resynthesis of 5% per hour (26). More recent studies have indicated that a small population of bacterial proteins (2 to 7%) may undergo more rapid turnover (half-life of 60 min or faster) in both growing and in nongrowing cells (7)(8)(9).
ACP is a relatively abundant cellular protein in E. coli (0.5oj, of the soluble protein), and if it were to undergo turnover, it might be expected to make a substantial contribution to the observed small population of proteins which apparently do turn over.
The class of proteins with an enhanced turnover rate may be associated with the bacterial membrane (27). A high rate of turnover of mammalian membrane proteins has also been reported (28). ACP is apparently localized at the bacterial membrane in intact cells (2). However, the conditions of cell rupture described in our paper (and probably in the former paper (27) as well) yield soluble extracts containing all of the [i4C]panto-the&e-containing compounds including ACP. ACP is a small, conjugated protein (mol wt = 8847) which is a specific coenzyme for lipid biosynthesis (1). Its covalently linked prosthetic group, 4'-phosphopantetheine, is removed and replaced at a rate of 4% of the total ACP pool per min, i.e. ti/z = 16.9 min (6). Small proteins are usually turned over more slowly than large ones in both bacterial (27) and mammalian systems (29).
Studies on the turnover of individual proteins in bacteria are few (30-33), and conjugated proteins in bacteria have not been studied in this way as far as we are aware.
The turnover or degradation of a protein in viva may be experimentally demonstrated by showing the loss of constituent amino acids from that protein as further growth proceeds.
This assay is most conveniently accomplished by incorporating radioactively labeled amino acids into the protein in intact cells, followed by periodic removal of samples of the cells during further growth and isolating the protein of interest in pure form.
The amount of radioactivity in the isolated protein, relative to the mass of the protein isolated, gives a measure of the rate of breakdown of that protein ("Dilution Experiment"). Alternatively, following a pulse of labeled amino acid in a culture of E. coli growing logarithmically, cells were grown in an unlabeled medium. Assuming no recycling of label which results from protein catabolism, the specific radioactivity of a protein made constitutively should fall at a rate inversely related to the growth rate.
For example, if the cell mass doubles, the specific radioactivity of a sample of pure protein would be halved.
We have carried out these two kinds of experiments with different strains of E. coli to characterize the rate of turnover of the protein of ACP in these cells. Although the experimental design and the purification procedures employed in each case were different, the conclusion from both was the same: the protein portion of ACP is metabolically stable. Pulse Experiment-The pulse-labeling experiment has the advantage of accentuating the labeling of the proteins under-going turnover since the stable proteins would be labeled more slowly and only as a function of cell growth.
The 4'-phosphopantetheine prosthetic group of ACP of E.
The protein portion of AC1 was labeled by introducing L- [4,5-aH]leucine into the medium at mid-log growth, followed after 5 min by a large amount of unlabeled L-leucine. The culture was washed and resuspended in a fermentor containing medium supplemented with unlabeled n-leucine and P-[lJ4C]alanine of the same specific activity as before.
Samples (1.5 to 1.7 g wet weight of cells) of this exponentially growing culture were withdrawn at timed intervals over an 8-hour period, and the ACP from each of these samples was purified using the five-step published procedure previously described (13).
The &[l-l%]alanine was employed only as a marker in these experiments as ACP is the only known protein in E. coli which has a prosthetic group containing p-alanine (4'phosphopantetheine).
The ACP concentration after the final purification step was determined by protein assay (25) and the 3H content (n-leucine in the protein) measured.
The values of aH disintegrations per min per mg of ACP protein as a function of time are shown in Fig. 1 1. Pulse experiment.
The stability of the ACP isolated after the initial pulse (0 hour) is estimated from the slope of the 3H disintegrations per min in ACP per mg of protein plotted on a log scale versus the time of sampling.
Line A is calculated from the growth rate of the cells (doubling time was 96 min).
Line B corresponds to a loss of 4% of the 3H per min.
is that expected when ACP concentration is a constant fraction of cell mass (3) and when there is no turnover (see "Appendix"). The broken line in Fig. 1 corresponds to the expected loss of [3H]leucine from ACP if the protein moiety has a turnover rate equal to the already measured (6) rate of prosthetic group exchange.
Thus it appears that the prosthetic group turnover or exchange does not involve a concomitant degradation of the protein portion of ACP.
In order for this conclusion to be valid the ACP isolated from the cells of the pulse-label experiment must be essentially homogeneous, although quantitative recovery is unimportant. If the isolated ACP contained a large amount of a contaminant which does not undergo turnover in E. coli (a condition met by the bulk of the protein of E. COG), then ACP turnover could be undetected.
The evidence for the purity of the ACP in this experiment can be summarized as follows. First, the method used for ACP purification is known to yield pure ACP when utilized on a larger scale than in the present work.
Second, the elution profile from the last step of purification shows a constant 3H:14C ratio in only those fractions which contain ACP.
Contaminating protein which would have only 3H and not 14C label would alter the 3H :14C ratio unless it eluted simultaneously with ACP.
Third, the isolated ACP was active (80% f 30% of theory) in an enzymatic assay, the malonyl-CoA-COz exchange reaction.
The variability and lower-than-theoretical amount of active ACP could be due to the difficulties in assaying minute amounts of material, the presence of denatured ACP, or the presence of apoACP in the purified ACP preparations.
Dilution Experiment-The dilution experiment was carried out on a double auxotroph and has the advantage of providing an initial 3H:14C ratio which could be related directly to the relative specific activities of [3H]isoleucine and [%]pantothenate in the medium (see "Appendix") and which provided an independent criterion for the purity of the ACP.
E. coli Ilv 453, auxotrophic for both pantothenate and iso- the exponentially growing cells were washed and resuspended in fresh medium supplemented with [1-Wlpantothenate of the same specific activity but containing only unlabeled L-isoleucine. Samples were taken immediately and at suitable intervals thereafter.
The extracts prepared from the sampled cells were fractionated first on DEAE-cellulose.
A typical separation is shown in Fig. 2. The second and last purification step, DEAE-Sephadex chromatography, is shown in Fig. 3. Only one W-containing peak is present; it is symmetrical and the 3H (isoleucine) to W (pantothenate) ratio is essentially constant across the peak. For a few of the samples, fractions representing 90% of the 14C radioactivity from A-25 DEAE-Sephadex chromatography were pooled and subjected to disc gel electrophoresis as described under "Methods and Materials," A typical result is shown in Fig. 4. A small 3H-containing peak was observed preceding the main peak; however, only one peak of 14C radioactivity was obtained.
The 3H:14C ratio was nearly constant across the peak and the value of this ratio was close to that obtained from A-25 DEAE-Sephadex chromatography (see legend, Fig. 4). Since a double auxotroph was employed, the specific radioactivity of the isoleucine and the pantothenate in the ACP should be the same as that in the medium ; the l4C content permits a direct calculation of ACP mass. Thus the values for 3H and 1% in the homogeneous ACP prepared in the dilution experiment at zero time could be used to calculate the number of residues of isoleucine per pantothenate residue (see "Appendix"). The value obtained at t = 0 (see Fig. 5) corresponds to 5.36 isoleucine residues per pantothenate residue.
The theoretical value based on amino acid analysis of ACP (I), sequence determinations (34), and now by synthesis (35) is 7.0. A value lower than theoretical would not be a sign of the presence of extraneous proteins but must reflect a certain amount of uncertainty in the values of the endogenous specific activity of the L-isoleucine since the endogenous specific activity of the [14C]pantothenate was calibrated using the enzymic assay for CoA (see "Methods and Materials").
The values of the logarithm of the %:14C ratio (see "Appendix") as a function of time arc plotted in Fig. 5 Similarly for pure ACP the content of tritiated amino acid (seven isoleucines per mole of ACP) can also be related to ACP mass : x 7 (3) or at t = 0,  (4) 4465 the holoACP must be rapidly interconverted, given the rapid turnover of the prosthetic group. Thus, the apoACP must also be stable or, if unstable, must make a very limited contribution to the demonstrated lack of turnover of the holoACP. It would be interesting to distinguish these possibilities under conditions in which apoACP was present in amounts comparable to holoACP. When the iron content of ferritin, the free iron carrier in blood of mammals, is reduced, it exhibits a decreased stability (36). Similarly, when bacterial proteins are damaged (by introduction of genetic lesions) their turnover is also increased (32,33).
Thus, it would be of interest to examine further the turnover of apoACP to see whether the presence of the prosthetic group has any bearing on the stability of ACP. We will examine the effects of pantothenate, nitrogen, phosphorus, and amino acid starvation on the turnover of this protein under conditions of growth and nongrowth as a model for the responses of this organism to such stresses. Similarly, current studies are concerned with the effect of inhibiting protein, DNA, and RNA synthesis on the turnover of ACP prosthetic group.
Earlier work demonstrated that the covalently bound 4'phosphopantetheine prosthetic group of rat liver fatty acid synthetase also undergoes turnover, analogous to the situation in E. coli (37). Moreover, it was shown that the rate of prosthetic group exchange is much faster than the rate of protein turnover of all of the subunits of the fatty acid synthetase (38). It would appear that the turnover of the 4'-phosphopantetheine prosthetic group of fatty acid synthetase and of bacterial ACP must have some intrinsic biological significance which as yet is not understood.
Since it is unlikely that the removal of the prosthetic group of ACP is an obligatory step in the de novo synthesis of a fatty acid molecule (37), this phenomena may represent an external control of either fatty acid or pantothenate metabolism.
Aclcnowledgments-We are indebted to P. Roy Vagelos for his interest and encouragement during this investigation. We appreciate the help of Joseph E. Sabaitis, Jr., in working out the conditions of the particular disc gel electrophoresis technique we used.
Phaik Foon Tan and John DeLoach helped with the ACP purifications, and their help is gratefully acknowledged.

APPENDIX
In exponentially growing cells, the concentration of ACP within the cells is constant (I).
Thus, one can relate the mass of ACP at any time, t, to the growth rate of the culture (conveniently estimated as the time required to double the cell mass) as follows: where A(aH) = amino acid specific radioactivity.  12.
17. If turnover occurs, i.e. if 3H is lost from ACP protein, the plot of log, (3H:r4C) versus time would no longer be linear and would

24
' have a greater slope than --EL. However, the intercept at t = 0 25. for pure ACP should remain as above.

26.
The above equation is generally applicable to any protein possessing a prosthetic group which can be radioisotopically labeled.
It is also valid for the pulse type experiment with the ii: proviso that in Equation  6 the value of the intercept t = 0 will be a function of the duration of the pulse and will not be equal 29. to the specific activities of the medium. The slope will be equal to -p if no turnover occurs. 3.