Proc. NatL Acad. Sci. USAVol. 79, pp. 2255-2259, April 1982Biochemistry

Post-translational modification and processing of Escherichia coliprolipoprotein in vitro

(signal peptidase/protein secretion/lipoprotein/globomycin)

MASAO TOKUNAGA, HIROKO TOKUNAGA, AND HENRY C. WUDepartment of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814

Communicated by M. J. Osborn, January 15, 1982

ABSTRACT Escherichia coli strain MM18 cells containingmalE-lacZ hybrid protein was reported to accumulate prolipopro-tein when they were induced with maltose [Ito, K., Bassford, P.J. & Beckwith, J. (1981) Cell 24, 707-717]. We have shown that theprolipoprotein accumulated in maltose-induced MM18 cells is notmodified, lacking covalently linked glyceride. When the cell en-velope of MM18 containing unmodified prolipoprotein was incu-bated in the presence ofdetergent with [2-3H]glycerol-labeled cellenvelope of strain JE5505 lacking murein lipoprotein, incorpo-ration of [2-3H]glycerol radioactivity into both prolipoprotein andprocessed mature lipoprotein was observed. Likewise, when [3H]-palmitate-labeled JE5505 cell envelope was incubated with theMM18 cell envelope containing unmodified prolipoprotein in thepresence of detergent, [3H]palmitate radioactivity was incorpo-rated into prolipoprotein by ester linkage and into mature lipo-protein by both ester and amide linkages. These results indicatethat our in vitro system contains activities of prolipoprotein mod-ification and processing enzymes, including glyceryltransferase,O-acyltransferase, signal peptidase, and N-acyltransferase. Thesignal peptidase activity in our in vitro system was completely in-hibited by globomycin. At pH 5.0, glyceryltransferase was inac-tive. Signal peptidase was active at pH 5.0, provided that proli-poprotein had been modified by glyceryltransferase (and O-acyl-transferase) during a prior incubation at pH 9.1. These resultsstrongly suggest that the modification of prolipoprotein by glycer-yltransferase (and O-acyltransferase) precedes, and may in factbe a prerequisite for, the processing of prolipoprotein by signalpeptidase.

It has become increasingly clear in recent years that many outermembrane and periplasmic proteins in Escherichia coli are firstsynthesized as precursor proteins containing signal sequencesat the NH2 termini (1-4). The number and specificity of signalpeptidases that process these precursor proteins remains oneof the major unresolved questions in the mechanism of exportof outer membrane and periplasmic proteins (3).

Braun's lipoprotein contains a novel lipoamino acid at theNH2 terminus of the mature protein (5). Recent work on themechanism of action of a cyclic peptide antibiotic globomycinhas revealed two interesting and unexpected findings: (i) pro-lipoprotein can be modified to contain covalently linked gly-ceride when the proteolytic processing by the signal peptidaseis inhibited by globomycin (6), and (ii) additional lipoproteinscontaining glyceride are found in both the cytoplasmic and outermembranes ofthe E. coli cell envelope, and processing of thesenew prolipoproteins is also inhibited by globomycin (7). Wehave recently shown that penicillinase from Bacillus licheni-formis synthesized in E. coli is also a lipoprotein, containingglyceride-cysteine, presumably at its NH2 terminus (8). E. coli

met___ _Gly-Cys Lys

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PhosphatldylglycerolGlyceryl transferase

glyceryl'met Gly-Cys Lys

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PhosphipidAcyl Transferase

GlyceryLemet Gly-Cys Lys

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GlycerideNH2-Cs.

IPhospholipidAcyl Transferase

GlycerideAcyI-NH-Cys

IPeptidoglycan

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Lipid-freePropoprotein

Glyceryl-Prohipoprotein

Glyceride-Proipoprotein

Glyceride-Lipoprotein

Free-formLipoprotein

.Lys Bound-form

NH Lipoprotein

C=om-DAP-Peptidoglycan

FIG. 1. Postulated pathway for the biosynthesis of murein lipo-protein in Escherichia coli. m-DAP, meso-diaminopimelic acid.

mutants containing lipid-deficient lipoprotein can be readilyobtained by globomycin selection; all of them have so far beenshown to contain uncleaved and unmodified prolipoprotein,due to mutations in the structural gene for murein lipoprotein(9). E. coli mutants containing cleaved but unmodified lipid-deficient lipoprotein are yet to be isolated. On the basis oftheseobservations, we have recently postulated that a common mod-ification and processing enzyme system exists in E. coli that isresponsible for the biogenesis of lipoproteins in E. coli, and theproteolytic processing of prolipoproteins requires prior modi-fication of prolipoproteins by the transfers of glyceryl moietyand fatty acids from phosphatidylglycerol and phospholipids,respectively (Fig. 1) (10).

Abbreviations: LP, lipoprotein; PLP, prolipoprotein.

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Ito et al. (11) have recently shown that, as a result of the de-fective export of the hybrid protein encoded by a malE-lacZfused gene, many precursor proteins of outer membrane andperiplasmic proteins, including prolipoprotein, accumulate incells grown in media containing maltose. In this paper, we showthat the prolipoprotein accumulated in cells containing thismalE-lacZ hybrid protein contains unmodified cysteine. Usingthis prolipoprotein as the substrate, we have succeeded in thein vitro modification and processing of prolipoprotein to formthe mature form of Braun's lipoprotein. Our results stronglysuggest that the processing of prolipoprotein by the globomy-cin-sensitive signal peptidase requires prior modification ofpro-lipoprotein to form glyceride-containing prolipoprotein.

MATERIALS AND METHODSBacterial Growth. MM18 (a gift of J. Beckwith, Harvard

Medical School) was grown in M63 minimal medium containingthiamin at 1 gg/ml and 0.5% glycerol (11). For labeling with[2-3H]glycerol, 0.5% sodium succinate was used as the carbonsource. JE5505 was grown in proteose peptone beef extractbroth medium. Cells were grown at 30'C with shaking.

Labeling Conditions. All labeling experiments were carriedout at 30'C. For the labeling ofphospholipids, MM18 cells werefirst induced for the synthesis of the malE-lacZ hybrid proteinby growing the cells in media containing maltose (final concen-tration 0.2%) for 2 hr, followed by the addition of either [2-3H]glycerol (10 Ci/mmol) at 20 ,Ci/ml or [3H]palmitate (11.8Ci/mmol) at 40 ACi/ml for 30 min (1 Ci = 3.7 X 1010 becque-rels). Trichloroacetic acid (final concentration 5%) was addedto the culture medium to terminate the labeling.

For in vitro enzymatic studies, MM18 cells were grown for2 hr in the presence of maltose and subsequently labeled for 2min with [ S]cysteine (871.5 Ci/mmol) at 0.2 ,uCi/ml. JE5505cells (ODr00 0.4-0.5) were labeled with [2-3H]glycerol at 20,uCi/ml or [3H]palmitate at 20 ,Ci/ml for 15 min.Membrane Preparation and in Vitro Enzyme Assays.

[35S]Cysteine-labeled MM18 cells and [2-3H]glycerol-labeledJE5505 cells were suspended in 50mM Tris HCl buffer (pH 8.0)with or without 0.2 M KC1, respectively, and sonicated for 2 minin an ice bath. The homogenates were centrifuged at 220,000x g for 2 hr at 4°C and the crude membrane fractions were sus-pended in 50 mM Tris HCl buffer (pH 8.0).Enzyme reactions were carried out at 37°C in mixtures (250

1,u final volume) containing [35S]cysteine-labeled MM18 mem-brane (0.70 mg of protein), [2-3H]glycerol-labeled JE5505membrane (0.29 mg of protein), 100 mM Tris HCl buffer (pH8.0), 10 mM ATP, 0.2 mM GTP, 1 mM CTP, 0.1 mM mag-nesium acetate, 0.2% 2-mercaptoethanol, 25 ug of chloram-phenicol, and the nonionic detergent Nikkol, which was presentat a final concentration of 0.25%. Before the addition of deter-gent, membrane fractions were frozen and thawed twice. In-cubation was terminated by the addition of 0.1 vol of 10%NaDodSO4 followed by boiling for 2 min. After the removal ofpeptidoglycan by centrifugation (Eppendorf 5412, 15 min), im-munoprecipitation with anti-lipoprotein antiserum was carriedout as described (12). Immunoprecipitates were washed twicewith washing solution (13), once with acetone, and twice withchloroform/methanol, 2:1, (vol/vol).

Other Techniques. NaDodSOjurea gel electrophoresis(14), electrophoresis of amino acids on cellulose plates (15) andfluorography (16) were carried out as described. NaDodSO4 gelelectrophoresis was carried out according to Inouye et al. (17)except that 12.5% acrylamide and 0.27% bisacrylamide wereused.

Chemicals. [2-3H]Glycerol (10 Ci/mmol), [9,10-3H(N)]-palmitate (11.8 Ci/mmol) and L-[35S]cysteine (871.5 Ci/mmol)

were purchased from New England Nuclear. Globomycin wasa gift from Mamoru Arai (Sankyo, Tokyo). Nikkol (Octa-ethyleneglycol mono-n-dodecyl ether) was obtained from Nikko Chem-icals, Tokyo). Chemicals used were of the best grade commer-cially available.

RESULTSProlipoprotein Accumulated in the Maltose-Induced MM18

Cells Is not Modified with Glycerol. When MM18 cells wereinduced with maltose for the synthesis ofmalE-lacZ hybrid pro-tein, precursor forms of periplasmic proteins (maltose-bindingprotein and alkaline phosphatase) and outer membrane proteins(ompF, ompA, and lipoprotein) accumulated (11). Prolipopro-tein has been shown to accumulate in E. coli cells treated withglobomycin (18, 6), benzyloxycarbonylalanine chloromethyl ke-tone (19), or membrane perturbants such as toluene (20). Thelipoprotein accumulated in the cells treated with globomycincontains covalently linked glyceride (6).To determine whether prolipoprotein that accumulates in

MM18 cells after induction with maltose is modified by the ad-dition of glycerol and fatty acyl moieties, MM18 cells were in-duced with maltose for 2 hr and then labeled with either [2-3H]glycerol or [3H]palmitate for 30 min or with [3S]cysteinefor 2 min, respectively. The labeling was terminated by the ad-dition of trichloroacetic acid (final concentration 5%). The tri-chloroacetic acid precipitates were solubilized with 1% Na-DodSO4 and immunoprecipitated with anti-lipoprotein anti-serum. Fig. 2 shows the fluorogram of the slab gel afterNaDodSO4 gel electrophoresis of the immunoprecipitates. Asshown in lanes A and B, uninduced cells contained only maturelipoprotein that was labeled with both [3S]cysteine and [2-3H]glycerol. After 2-hr maltose induction, both prolipoproteinand lipoprotein were labeled with [35S]cysteine (lane G). Underthe same conditions, [2-3H]glycerol and [3H]palmitate were in-corporated into the band corresponding to mature lipoproteinbut not into the prolipoprotein (lanes C, D, and F). In the gel

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A B C D) E F

- (PLPL

- PLP

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FIG. 2. Accumulation of unmodified unprocessed prolipoproteinin maltose-induced MM18 cells. Strain MM18 cells were labeled with[2-3H]glycerol, [3H]palmitate, and [35S]cysteine. Lipoprotein immu-noprecipitates were analyzed by NaDodSO/polyacrylamide slab gelelectrophoresis (acrylamide/bisacrylamide, 12.5%:0.27%). The fluo-rogram of the slab gel is shown above. LanesA and B, uninduced cells;lanes C through G, maltose-induced cells; lanes A, C, and F, [2-3H]glycerol-labeled; lane D, [3Hlpalmitate-labeled; lanes B, E, and G,[35S]cysteine-labeled. Samples in lanes A through E were not reducedwith 2-mercaptoethanol; samples in lanes F and G were reduced with2-mercaptoethanol. Phosphorylase B (92,500), bovine serum albumin(66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybeantrypsin inhibitor (21,500), and cytochrome c (12,400) were used as themolecular weight standards. PLP, prolipoprotein; LP, lipoprotein.

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system used in the present study, the mobility ofprolipoproteinin MM18 cells was almost the same (but slightly faster) than theprolipoprotein accumulated in globomycin-treated cells (datanot shown). When immunoprecipitates were solubilized in thebuffer without 2-mercaptoethanol and analyzed by NaDodSO4gel electrophoresis, an additional band was observed that waslabeled with [3S]cysteine but not with [2-3H]glycerol or

[3H]palmitate (lane E). The apparent molecular weight of thisband was about 20,000, and the mobility of this band was thesame as that of the unmodified prolipoprotein dimer found inthe mipA mutant described previously,(21). These results in-dicate that the prolipoprotein accumulated in maltose-inducedMM18 cell contains unmodified cysteine and therefore can forma dimer in the absence of a reducing agent.

In Vitro Modification and Processing of Prolipoprotein toForm Mature Form of Lipoprotein. Our previous in vivo ex-

periments indicate that phosphatidylglycerol is the donor oftheglycerol moiety of lipoprotein and that the fatty acyl moietiesof phospholipids are the donors of the fatty acid residues of li-

poprotein (22, 23). Accordingly, we incubated a [2-3H]glycerol-or [3H]palmitate-labeled cell envelope fraction of strain JE5505[which lacks Braun's lipoprotein (24) but contains precursors forthe lipid moieties of lipoprotein] with an [3S]cysteine-labeledMM18 cell envelope fraction (which accumulates unmodifiedprolipoprotein) in order to detect the in vitro modification andprocessing of prolipoprotein to mature form.

The reaction was terminated after a 5-hr incubation by theaddition of NaDodSO4 (final concentration 1%) and boiling for2 min. Immunoprecipitates were analyzed by NaDodSOJurea gel electrophoresis. As shown in Fig. 3A, there was no

incorporation of [2-3H]glycerol into prolipoprotein or lipopro-tein in the absence ofdetergent. In addition, prolipoprotein wasnot processed to lipoprotein.

However, [2-3H]glycerol was incorporated into both proli-poprotein and lipoprotein in the presence of0.25% Nikkol (Fig.3B). A similar result was obtained when Nikkol was replacedby 0.2% sodium cholate/0. 1% sodium deoxycholate (data notshown).

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FIG. 3. Incorporation of [2-3H]glycerol and [3H]palmitate radioac-tivities into [35S]cysteine-labeled prolipoprotein in vitro. Cell enve-

lopes from [35S]cysteine-labeled maltose-induced MM18 cells and [2-3H]glycerol- or [3H]palmitate-labeled JE5505 cells were prepared andthe in vitro modification of prolipoprotein was carried out as describedin the text. (A) Incubation without Nikkol; (B-D) incubation in thepresence of 0.25% Nikkol; (A-C) [2-3H]glycerol-labeled JE5505 mem-brane; (D) [3H]palmitate-labeled JE5505 membrane; (C) globomycin(final concentration 10 gg/ml) was included in the incubation. Count-ing efficiencies were 42% for 3H and 75% for 36S. c, Cytochrome cmarker.

These results strongly indicate that both glyceryltransferaseand signal peptidase activities are active in the presence of de-tergent. The difference in the [2-3H]glycerol to [3S]cysteineratio between prolipoprotein and lipoprotein is-most likely dueto the presence of unmodified [ S]cysteine-labeled prolipo-protein in the prolipoprotein peak, because the mobilities ofmodified and unmodified prolipoprotein differ only by one slice(1 mm) in this gel system. When the immunoprecipitate of [2-3H]glycerol- and [ S]cysteine-labeled reaction product was ox-idized with performic acid and then hydrolyzed in constant-boiling HC1 at 1050C for 20 hr, 3H- and 'S doubly labeled gly-ceryl-cysteine sulfone was identified by both paper chromatog-raphy (1-butanoVpyridine/acetic acid/water, 15:10:3:12 byvolume, Hf 0.21) and by cellulose plate electrophoresis in for-mic acid/acetic acid buffer (pH 1.9) (15) (data not shown).The processing -of prolipoprotein to lipoprotein was com-

pletely inhibited by globomycin (10 jAg/ml) in our in vitro assay(Fig. 3C). However, a comparison of the [2-3H]glycerol to[3S]cysteine ratios in Fig. 3 B and C indicates that globomycindoes not affect the activity of the glyceryltransferase.The mutant prolipoprotein from E. coli strain E610 (mlpA),

which contains a single amino acid substitution within the signalsequence (Gly-14 -- Asp-14) (25) was tested as the substrate forglyceryltransferase in vitro. As shown in Table 1, mlpA proli-poprotein was a very poor substrate for glyceryltransferase ascompared with the wild-type prolipoprotein present in themaltose-induced MM18 cells. This result is in full agreementwith the in vivo observation that a minimal amount (4%) ofmrpAprolipoprotein is modified and processed to form mature lipo-protein (26). No incorporation of [2-3H]glycerol was seen whenmembranes from uninduced MM18 cells were used. These re-sults and the data in Fig. 3 further support the conclusion thatthe glyceryltransferase activity demonstrated in this assay isrelevant to the modification of prolipoprotein in vivo.

[3H]Palmitate was also incorporated into both prolipoproteinand lipoprotein (Fig. 3D), indicating that the enzymes involvedin the transfer of fatty acids to prolipoprotein or mature lipo-protein are also active in our in vitro system. To determine thenature of the linkage of [3H]palmitate to prolipoprotein and li-poprotein in the in vitro modified products, the [3H]palmitate-labeled immunoprecipitate was hydrolyzed with 0.1 M NaOHat 37°C for 2 hr. As shown in Table 2, 59% of [3H]palmitateradioactivity in lipoprotein was resistant to mild alkali hydroly-sis. Under the same condition, 99% of [3H]palmitate radioac-tivity in phospholipids was released. This result suggests thatthe in vitro modified lipoprotein contains both amide-linkedand ester-linked palmitate. In contrast, these data indicate thatin vitro modified prolipoprotein contains only ester-linked pal-mitate, as expected.

Table 1. Acceptor activities of prolipoproteins forglyceryltransferase

[2-3H]GlycerolAcceptor incorporation, dpm

MM18 induced cell envelope 5470MM18 uninduced cell envelope 102E610 (mlpA) cell envelope 8

MM18 cells with or without maltose induction and E610 (mlpA) cellswere grown in M63 minimum medium supplemented with 0.5% glyc-erol and 0.4% glucose, respectively. Cells were labeled with [35S]cysteine(0.2 ,uCi/ml) for 2 min at an ODr,0, of 0.700. [36S]Cysteine-labeledmembranes were incubated with [2-3H]glycerol-labeled JE5505 mem-brane. Globomycin (10 jg/ml) and 0.2% sodium cholate/0.1% sodiumdeoxycholate were used instead of 0.25% Nikkol. [2-3H]Glycerol ra-dioactivities incorporated into prolipoprotein were normalized per1000 dpm of [35S]cysteine in prolipoprotein.

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Table 2. Release of [3H]palmitate radioactivity from in vitromodified prolipoprotein and lipoprotein by mild alkali treatment*

[3H]PamIn vitro [3H]Pam, [35S]Cys, [3H]Pam/ releasedproduct Alkali dpm dpm [35S]Cys (a - b)/a %tPLP - 15,576 846 18.4 (a) 0.79 100

+ 1,301 331 3.9 (b) .7 10

LP - 45,903 782 58.7 (a) 2+ 10,630 268 39.7 (b) 0.3 41

Pam, palmitate.* [35S]Cysteine-labeled MM18 cell envelope and [3H]palmitate-labeledJE5505 cell envelope were incubated as described in the text. Halfof the immunoprecipitate was treated with 200 A.l of 0.1 M NaOHat 370C for 2 hr. The control and alkali-treated immunoprecipitateswere analyzed by NaDodSO4/urea gel electrophoresis.

t Normalized percentage based on 0.79 as 100%.

Proteolytic Processing ofProlipoprotein by Signal PeptidaseRequires Prior Attachment of the Glyceride Moiety to Proli-poprotein. In preliminary experiments to define the pH optimaof the glyceryltransferase and signal peptidase, we noticed thatthe glyceryltransferase is inactive at pH 5.0, whereas both en-zymatic activities occur at pH 9.1. As shown in Fig. 4A, therewas virtually no incorporation of [2-3H]glycerol into prolipo-protein at pH 5.0. Furthermore, the profiles of [3S]cysteine-labeled prolipoprotein and lipoprotein were unaltered by thepresence of globomycin during incubation at pH 5.0 (Fig. 4 Aand B), further indicating that processing had not taken place.(The small shoulder of [ S]cysteine radioactivity at the lipo-protein position represents mature lipoprotein labeled in vivo.)As shown in Fig. 4C, however, at pH 9.1 both glyceryltrans-ferase and signal peptidase are active, because [2-3H]glycerolradioactivity was found in both prolipoprotein and lipoprotein.Incorporation of [3H]palmitate into both prolipoprotein and li-poprotein also took place at this pH (data not shown).The lack ofprocessing ofprolipoprotein in vitro atpH 5.0 may

be due to an inactive signal peptidase at the acidic pH. Alter-natively, the processing of prolipoprotein by signal peptidasemay require prior modification of the cysteine residue by glyc-eryltransferase, which is inactive at pH 5.0, as shown directlyby the results in Fig. 4A. If the latter possibility is true, thenmodification ofprolipoprotein by glyceryltransferase must pre-cede processing by signal peptidase. To test this hypothesis, thefollowing experiment (described in Fig. 4 D and E) was carriedout. The reaction mixture was first incubated at pH 9.1 for 1.5hr. Next, the pH was adjusted to 5.0'by the addition of citratebuffer (pH 5.0) and 1 M HC1, and the incubation was continuedfor an additional 1.5 hr. Fig. 4D shows that glyceride-containingprolipoprotein, which remained at the end of the incubation atpH 9.1 (Fig. 4C),was completely processed at pH 5.0 to themature form of lipoprotein. Fig. 4E shows that this processingcould be completely inhibited by globomycin. Whereas [2-3H]glycerol-labeled prolipoprotein in Fig. 4C was almost com-pletely processed to mature lipoprotein upon further incubationat pH 5.0, the increase in [5S] cysteine labeled lipoproteinduring the second incubation was relatively small (Fig. 4D).This is probably due to the difference in the specific radioac-tivities in [2-3H]glycerol- and [35S]cysteine-labeled prolipopro-teins.The data clearly show that signal peptidase for prolipoprotein

is active at pH 5.0, but it is active only towards modified pro-lipoprotein. This property ofthe prolipoprotein signal peptidasein vitro strongly suggests that the modification ofprolipoproteinby glyceryltransferase precedes the processing of modified pro-lipoprotein by the signal peptidase.

FIG. 4. Effect of pH on glyceryltransferase and signal peptidasein vitro. [MS]Cysteine-labeled MM18 cell envelope and [2-3Hlglycerol-labeled JE5505 cell envelope were incubated at pH 5;0 or pH 9.1 eithersingly or in succession. (A and B) Incubation at pH 5.0 (50mM sodiumcitrate buffer) for 1.5 hr; (C) incubation atpH 9.1 (50mMsodium boratebuffer) for 1.5 hr; (D and E) incubation at pH 9.1 for 1.5 hr, followedby an additional 1.5-hr incubation at pH 5.0. Incubations in B and Ewere carried out in the presence of globomycin (10 ,ug/ml). c, Cyto-chrome c.

DISCUSSION

The data presented here demonstrate that the prolipoproteinaccumulated in maltose-induced MM18 cells contains unmodi-fied cysteine. This is probably due to the accumulation of themalE-lacZ hybrid protein, which prevents the proper targetingof the unmodified prolipoprotein to the subcellular site wherethe modification and processing of prolipoprotein normallytakes place. In addition, we have utilized the unmodified anduncleaved prolipoprotein that accumulates in maltose-inducedMM18 cells as a substrate for demonstrating the modificationand processing of prolipoprotein in vitro. At least four of theenzyme activities involved in the biosynthesis of mature lipo-protein could be demonstrated in our cell-free system: glycer-yltransferase, O-acyltransferase(s), signal peptidase, and N-acyltransferase. This in vitro system can be used profitably bothfor the biochemical characterization ofthese individual enzymesand their substrate and for the identification of potential mu-tants in this pathway.

Both in vivo and in vitro results reported here clearly indicatethat proteolytic processing of prolipoprotein can occur post-translationally. Because of the small size of prolipoprotein, thisconclusion is not surprising. Whether the insertion of nascentprolipoprotein chain into the cytoplasmic membrane occurs

cotranslationally or post-translationally in vivo remains unknown.We have previously suggested that proteolytic processing of

prolipoprotein is preceded by the addition of glyceryl and fattyacyl moieties to prolipoprotein, and that the modified glyceride-cysteine may even constitute a recognition site for a uniqueprolipoprotein signal peptidase that is inhibited by globomycin(10). This model would account for the existence of multiplestructurally unrelated lipoproteins in E. coli synthesizing B.licheniformis penicillinase. Globomycin may be a structuralanalog for lipid-containing prolipoprotein due to both its hy-drophobicity and its structural similarity with-the signal peptidein prolipoprotein. Alternatively, globomycin may bind lipid-containing prolipoproteins noncovalently and interfere withtheir processing by signal peptidases.The experiments described in this paper provide strong sup-

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port for the conclusion that processing of prolipoprotein by sig-nal peptidase requires prior modification by glyceryltransferaseand 0-acyltransferase. This observation, plus the apparentspecificity of globomycin, is consistent with the possibility thatprolipoprotein signal peptidase is unique and distinct fromother signal peptidase(s). Many precursor proteins contain theamino acid sequence Ala-X at the cleavage site (3), thus sug-gesting a certain degree of specificity for signal peptidases. Thesignal peptidase for phage M13 procoat protein has been ex-tensively purified and characterized (27). It is still conceivablethat a single signal peptidase [e.g., Wickner's leader peptidase(27)] recognizes and cleaves the modified prolipoprotein as wellas the precursor forms of other exported proteins. In contrastto the signal peptidase described in this paper, which is activeat pH 5.0 towards modified prolipoprotein, Wickner's enzymeappears to be inactive at pH 5.6 (28). The final resolution of thequestion as to the number, specificity, and topological locali-zation of this important group of membrane-bound enzymesawaits further biochemical and genetic studies.The extent of modification and processing of prolipoprotein

in our in vitro system is very low, even after prolonged incu-bation. This may be a reflection ofthe inadequacy ofour in vitrosystem. It is likely, however, that the bulk of the unmodifiedprolipoprotein in MM18 cell envelope exists in a conformationor topological orientation unfavorable for the modification andprocessing reactions. This possibility is supported by an in vivopulse-chase experiment, which indicated that only a small frac-tion of pulse-labeled prolipoprotein can be chased into maturelipoprotein (data not shown).

Although we have routinely used [2-3H]glycerol-labeledJE5505 membranes and [asS]cysteine-labeled MM18 mem-brane as the donor of glyceryl residue and as a source of pro-lipoprotein acceptor, respectively, both the glyceryltransferaseand the signal peptidase activities can be demonstrated in vitroby using the prolipoprotein accumulated in MM18 membranepreparation as the acceptor and exogenous [2-3H]glycerol-la-beled phospholipids as the donor. In both the single membraneand the mixed membrane incubations, detergent is required forthe modification and processing activities. All four enzyme ac-tivities are recovered in the 100,000 X g supernatant fractionunder our in vitro assay conditions (unpublished data). There-fore, the requirement of the detergent may be to bring the do-nor and acceptor molecules in physical proximity to one anotherin a manner similar to the in vivo situation, where these reac-tions take place in the hydrophobic interior of a lipid bilayer,rather than to promote the fusion of the donor and acceptormembranes. In this respect, our results differ from the recentreports by Wickner and his colleagues in which they have dem-onstrated the insertion and processing of M13 procoat proteininto cytoplasmic membrane or reconstituted liposomes in theabsence of detergent (29, 30).

The modification and processing of prolipoprotein in vivoproceeds so rapidly that intermediates in the pathway outlinedin Fig. 1 cannot be detected even by pulse labeling for veryshort times. Our in vitro system, while low in its over-all effi-ciency, is capable of four consecutive modification and pro-cessing reactions. Moreover, the cleavage of prolipoprotein bysignal peptidase appears to be the rate-limiting step. These ob-servations suggest the possibility that these enzymes may be

present as a complex in vivo and that they function in a con-certed manner to modify and process structurally unrelatedprolipoproteins.

We are most grateful to Dr. Jonathan Beckwith of Harvard MedicalSchool for the gift of strain MM18, to Dr. Mamoru Arai of Sankyo (To-kyo) for the gift of globomycin, and to Dr. Taiji Nakae of Tokai Uni-versity School of Medicine (Isehara, Japan) for the gift of Nikkol. Wethank Drs. Paul Rick, Robert Wellner, and Joe Giam for critical readingof the manuscript. This work was supported by U. S. Public Health Ser-vice Grant GM-28811 and by Grant 81-663 from the American HeartAssociation.

1. DiRienzo, J. M., Nakamura, K. & Inouye, M. (1978) Annu. Rev.Biochem. 47, 481-532.

2. Wickner, W. (1979) Annu. Rev. Biochem. 48, 23-45.3. Osborn, M. J. & Wu, H. C. P. (1980) Annu. Rev. Microbiol 34,

369-422.4. Emr, S. D., Hall, M. N. & Silhavy, T. J. (1980)J. Cell Biol 86,

701-711.5. Braun, V. (1975) Biochim. Biophys. Acta 415, 335-377.6. Hussain, M., Ichihara, S. & Mizushima, S. (1980)J. Biol. Chem.

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