Assembly of Plant Ferredoxin-NADP' Oxidoreductase in Escherichia ...

Vol. 267, No. 22, Issue of August 5, pp. 15537-15541,1392 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMI~TRY 0 1992 by The American Society far Biwhemistry and Molecular Biology, Inc.

Assembly of Plant Ferredoxin-NADP' Oxidoreductasein Escherichia coli Requires GroE ~olecularChaperones* (Received for publication, February 24, 1992)

Nestor CarrilloS(S7, Eduardo A. CeccarelliSQ, Adriana R. KrappSg, Silvana BoggioS, M. Vialeg11 Raul G. Ferreyrall**, and Alejandro From t h $Departamento de Ciencias ~ i o ~ g and i c l~l ~ e ~ r ~ a m ede n t~o ~ r o ~Facu~~ad i o ~degClencias ~ B~oquimicasy Farmac~uticU ~ ,n i v e r s i d ~Nacional de Rosario, Suipacha 531,2000 Rosario, Argentina

We have recently reported the expression in Esche- holoenzyme (plastid uptake, processing, folding, and FAD richia coli of an enzymatically competent ferredoxin- assembly) is lacking, however. Expression of recombinant, biologically active proteins in NADP+ oxidoreductase from cloned peagenes encodr constitutes ~ ~ ~ a precious tool for the study of ing either the mature enzyme orits precursor protein E s c ~ coli (Ceccarelli, E. A,, Vide, A. M., Krapp, A. R., and basic biological problems.Although many transgenic polypepCarrillo,N. (1991)J. Biol. Chern. 266,14283-14287). tides are not able to correctly fold in this bacterial host and form insoluble aggregates (7, S), a number of expressed apoProcessing to the mature form by bacterial protease($) proteins have been reported to assemble with their prosthetic and FAD assembly occurred in the bacterial cytosol. Expression of ferredoxin-NADP+ reductase in chape-groups to generate functionally active holoproteins (9-13). ronin-deficient ( g r o e ) mutants of E. coli resulted in Recent work has revealed that protein folding in uiuo may not s A series of proteins, classified as partial reductase assemblyat permissive growth tem- be a s p o n ~ n e o u process. peratures (Le. 30 "C), andin total breakdown of holo- molecular chaperones, have been found to possess the ability as aggregated in- to prevent unfolded or misfoldedpolypeptides from aggregatenzyme assembly, and accumulation clusionbodiesatnon-permissivetemperatures (i.e. ing and precipitating (14, 15). We have recently expressed a fully active FNR in E. coli 42 "C). Coexpressioninthesemutantsofacloned groESL operon from the phototrophic bacterium Chro- (16) from a pea cDNA clone (3). Regardless of whether the matium uinoeurn resulted in partial or total recoveries transit peptide sequence was includedor not in theconstrucof ferredoxin-NADP+ reductase assembly. The overall tions, the expressed p o l ~ e p t i d e swere processed in the bacresults indicate that bacterial chaperonins are required terial host, resulting in proteins that were 1-2 amino acids for the productive foldingtassembly of eucaryotic fer- longer than the mature enzyme found in chloroplasts. This expression system offers the possibility to study the mecharedoxin-NADP+ reductase expressed E. incoli. nisms of enzyme foldingand FAD assembly. In the present work we report the characteristics of FNR expression and assembly in different E. coli mutants. We found that assembly of an enzymatically competent holoenzyme in the cytosol of this host requires the functional products of the groE genes, i.e. the bacterial equivalents to chloroplast chaperonins (14, 15).

Ferredoxin-NADP~ reductase ~FNR)' is a avop protein tightly bound to the stroma surface of chloroplast thylakoid membranes (1,2). Itsstructural and catalytic properties have been thoroughly studied, including the isolation and sequencing ofcDNA clones from a number of species (3-5). The apoenzyme is synthesized in cytoplasmic ribosomes as a preMATERIALS ANDMETHODS cursor (=40 kDa), whose NH2-terminal transit peptide ( 4 P h m i d s , B a c ~ e rStrains, ~a~ anxi Growth Conxiit~ons-Details conkDa) is cleaved upon plastid import (6). A molecular mecha- cerning the construction of recombinant plasmids pCV102 and nism accounting for the conversion of precursor into mature pCV105 are given elsewhere (16). Briefly, pCV102 contains the se-

quence of pre-FNR ( 3 ) ,starting from the third codon, fused in frame to a sequence that, encodes the first 6 amino acid residues of pInvestigaciones Cientificas y Tbcnicas (CONICET, Argentina), Fun- galactosidase, whereas pCV105 contains the sequence of mature FNR daciGn Antorchas (Argentina), the ThirdWorld Academy of Sciences (3) beginning at codon -2, fused in frame to a sequence that encodes (Trieste, Italy), the International Foundation for Science (Stockholm, the 16 initial amino acid residues of @galactosidase (16). In both Sweden), and Grant CRPfARG88 from the I n ~ r n a t i o n aCentre l for cases expression of the recombinant p o l ~ e p t i d e swas placed under Genetic Engineering and Biotechnolo~ (Trieste, Italy). The costs of control of the lac promoter in plasmid pUC9 (17). Plasmid pCRFl publication of this article were defrayed in part by the payment of contains a 4-kilobase pairs Sal1 chromosomal fragment bearing the page charges. This article must therefore he hereby marked "aduer- groESL operon from the phototrophic bacterium Chrornatium tisernent" in accordance with 18U.S.C. Section 1734 solelyto indicate uinosum2 cloned in compatible sites of vector pACYC184 (18). The following E. coli K-12 strains were used in this work: JM109 this fact. (recAlendA1thihsdRl7 (lacproAB)/F'traD36 lacP (1acZ)MlS 3 Members of CONICET. ll To whom correspondence should be addressed Dept. de Ciencias proAB) (19); ARK1 ((lac)U169 ZonlOO araDl39 strA hflA15O::Tn10), BiolGgicas, Facultad de Ciencias Bioquimicas y FarmacLuticas, Sui- a Y1089 strain cured from plasmid pMC9by ethidium bromide treatment (16); CG1945 (W3110 galE chr::TnlO); CG1921 (W3110 paeha 531,2000 Rosario, Argentina. galE- groES30 chr::TnlO); and CG1943(W3110 galE groEL140 ** Fellow of CONICET. The abbreviations used are: FNR, ferredoxin-NADP+ oxidore- chr::TnlO) (20). E. coli CG strains were generous gifts of Dr. Costa ductase (EC 1.18.1.2.); IPTG, isopropyl 0-D-thiogalactopyranoside; Georgopoulos (University of Utah Medical Center, Salt Lake City, PAGE, polyacrylamide gel electrophoresis; Rbu-Pt-carboxylase, ri- UT). bulose 1,5-bisphosphatecarboxylase/oxygenase; SDS, sodium dodecyl sulfate. R. G. Ferreyra and A. M. Viale, manuscript in preparation.

* This work was supported by grants from Consejo Nacional de

15537

15538

Chaperonin-mediated Assembly

of Ferredoxin-NADP+ Reductase

E. coli JM109 and ARKl cells were grown a t 37 "C in LB medium (19) supplemented with the corresponding antibiotics, until the absorbance of the culture reached 0.2 a t 550 nm. Following induction of the lac promoter with 0.5 mM IPTG, growth wasallowed to proceed for 4 h at the same temperature before collecting the cells. E. coli cells bearing mutations in the groE genes were grown a t 30 "C until a n absorbance of 0.6-0.8 a t 550 nm was reached, supplemented with 0.5 mM IPTG, and incubated for an additional 12-h period a t either 30 or 42 "C. Cell lysates were obtained as described (16). Subcellular Localization of Transgenic Ferredoxin-NADP Reductase in E. coli Cells-Expression of FNR in E. coli JM109 bearing pCV102 or pCV105 was carried out asdescribed above. Bacteria were collected by centrifugation and freed from their cell walls using the lysozyme-EDTA treatment (21).Afterremoval of the periplasmic fraction by centrifugation (5 mina t 12,000 x g ) , pelleted spheroplasts were resuspended in 25 mM Tris-HCI, pH 7.5, 1 mM EDTA, 0.1 mM phenymethylsulfonyl fluoride (buffer A) and disrupted by sonic oscillation (30 s a t maximal intensity in a MSE-PG-830 sonicator). Lysates were centrifuged for 15 min a t 12,000 X g, and the resulting pellets were washed once with the same buffer. The supernatants obtained from these treatmentswere centrifuged for 30 min a t 45,000 X g and the cleared solution regarded as cytoplasmic extract. Malate dehydrogenase and (3-lactamase were used as cytosolic and periplasmic markers, respectively (21). To characterize the aggregated insoluble FNR forms expressed in Lon- mutants bearing plasmids pCV102 or pCV105, the cells were induced with IPTG and lysed as described (16). After centrifugation for 5 min a t 12,000 X g, the resulting pellets were washed once with buffer A and then incubated for 10 min a t 25 "C in 25 mM Tris-HCI, p H 7.6, 10 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, supplemented with either 0.5% (w/v) Sarkosyl(22) or 0.5% (v/v) Triton X-100 (23). After centrifugation (15 mina t 12,000 X g), supernatants weresaved and the pellets subjected to the same treatment. The collected supernatants were combined, and trichloroacetic acid was added to 10% (w/v). Precipitated proteins were washed twice with cold acetone, dried, and resuspended in 10 mM Tris-HCI, pH 7.5, 2% (w/v) SDS, 0.5% (v/v) (3-mercaptoethanol, 5% (v/v) glycerol (SDSloading mix). The pellets obtainedaftertheSarkosylorTriton treatments were washed thrice with buffer A and finally resuspended in SDS-loading mix. Both pellets and supernatantswere boiled for 3 min and subjected to SDS-PAGE and immunoblotting as described previously (16). Other Procedures-Total protein concentration was determined by a dye-binding assay (24). The amount of recombinant FNR in both soluble fractions and pellets obtained after sonic disruption of induced cells was measured using a quantitative slot-blot immunoassay(16). Diaphorase (25), (3-lactamase (26), and malate dehydrogenase activities (27)were measured by published procedures. Recombinant DNA methods were those of Maniatis et al. (19). RESULTS

inclusion bodies can discriminate between these possibilities (22, 29). As shown in Fig. 1 (lanes 1-4), treatment of the insoluble material generated in the lon- strain ARKl with either Triton X-100 or Sarkosyl removed most proteins associated to thepellet, without promoting thesolubilization of the reductase. Transgenic FNR in E. coli pellets could only be dissolved in the presenceof strong chaotropic agents such as guanidine HCl(6 N) or urea(8 M). Attempts to reconstitute an active reductase from these extracts by removal of the chaotropic agent in the presence of FAD and plant cell extracts have been unsuccessful. The overall results shownin this section indicate that FNR accumulates in thecytosol, where processing occurs,and that the insoluble forms of expressed FNR are not associated to bacterial membranes. Expression of Pea Ferredoxin-NADP Reductase in Chaperonin-deficient Mutants of E. coli-The amount of FNR expressed in active form varied widely among different E. coli strains. In particular, prevention of NH2-terminal proteolysis in protease-deficient bacteria resulted in the accumulation of insoluble,inactive and unassembled reductasein the cells (Fig. 1and Ref. 16). This prompted us to investigate whether FNR folding in E. coli was a spontaneous process or it was mediated by host factors. These factors, termed molecular chaperones, are ubiquitous amongliving cells (14, 15). One class of molecular chaperones, the chaperonins, are present in the cytoplasm and organelles of all organisms (15). In E. coli, chaperonins are encoded by the groE operon and represented by the products of the groES and groEL heatshock genes (14, 15). Mutants in these genes show temperature-sensitive cell growth a t 42 "C and hampered bacteriophage assembly a t permissivegrowth temperatures (30). These proteins participate in the assembly of heterologous (procaryotic) dimeric and hexadecameric Rbu-P2-carboxylase expressed in E. coli (31). In order to evaluate the possible role of chaperonins in the production of a correctly assembledFNR inE. coli, we studied the expression patterns of this protein in groE- mutants. Following induction of FNR synthesis a t 30 "C, the enzymeassociated diaphorase activity (measured as units/milligram of total soluble protein) was 50-60% lower in the mutants as compared to the parental strain (Table I). We observed no major differences in the total amount of FNR synthesized after IPTG induction in any of the strainsused (Table I). Fig. 2 showsthe distributionof FNR between solubleand insoluble

Intracellular Accumulation of Ferredoxin-NADP Reductase in E. coli-Expression of pea FNR inE. coli JM109 asa fused polypeptide to the NH2-terminal region of @-galactosidase (either the mature polypeptide found in the chloroplast or pre-FNR which contains, in addition, the transit peptide), resulted in the recovery of analmostentirely processed, soluble, and fully active enzymeafter cell disruption (16). The .bulk of FNR was found inthebacterial cytosol, and its i* )1m -20recovery in the different cell fractions showed no significant " - .. differencesfrom those of the cytoplasmic markermalate FIG.1. Analysis of insoluble ferredoxin-NADP+ reductase dehydrogenase (data not shown). We have previously observed that substantial amounts of in lysates from a lon- E. coli strain. FNR expression, cell disrupFNR were associated to bacterial pellets aftercell disruption tion, treatmentof pellets with either Triton X-100 or Sarkosyl, SDSPAGE,andimmunoblotting were carriedoutas described under in a protease-deficient (lon-) strain, specially in the case of "Materials and Methods." A , Coomassie-stained gel. E , immunoblots pCV102-transformed cells (16). These pellets showed neither of the same samples with anti-FNR antisera. Lanes: I , total E. coli diaphorase activity norassociated FAD as monitoredby fluo- pellet; 2, first Sarkosyl supernatant;3, first TritonX-100 supernatant; rographic analysis after high performance liquid chromatog- 4 , final Sarkosyl pellet; 5, final Triton X-100 pellet. Aliquots loaded raphy(not shown). Formation of insolubleaggregates by ontothe gel corresponded to 25 pg of proteininthedetergent proteins expressed in E. coli may result from binding to inner supernatants and an equivalentvolume of material in the totalE. coli and final pellets. Lane 6, protein standards (Pharmacia LKR Bio(28) or outer membranes (29), aswell as to the formationof technologies Inc.): phosphorylase b (94 kDa), bovine serum albumin cytoplasmic inclusions of aggregated polypeptides (7, 8). The (66 kDa),ovoalbumin (42 kDa), carbonic anhydrase (30 kDa), trypsin use of detergents that disrupt membranes but cannot dissolve (21 kDa), lactoalbumin (15 kDa).

-

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-

15539

Chaperonin-mediated Assembly of Ferredoxin-NADP Reductase

TABLE I Expression levelsof ferredoxin-NADP reductase directed bypCVlO2 and pCV105 in different coli E. strains The amount of FNR was determined by quantitative slot-blot using anti-FNR antisera (16). Activity and quantity of soluble FNR was measured in cell lysates after centrifugation (15 min a t 12,000 X g ) of sonicated cells. Experimental details concerning transformation of E. coli cells with the four different plasmids, as well as induction andlysis, are given in the text. Strain

Cells transformed with pCV102 groE+ groEL-

groES-

Compatible Temperature during induction

FNR-diaphoraseactivityb plasmid present

FNR cell content"

"C

%

30 42 30 42 30 42 30 42 30 42 30 42 30 42

4.2 f3.40.7 3.6 f 0.7 2.7 f 0.3 2.5 f 0.3 NDd ND ND ND 3.2 f 0.5 3.0 f 0.4 ND ND ND ND

pACYC pACYC pCRFl pCRFl pACYC pACYC pCRFl pCRFl

Unit/mg protein

Unit/mg FNR

f 0.5 3.1 f 0.6 1.7 f 0.2 0.2 f 0.2 1.6 f 0.7 0.1 f 0.1 2.7 f 0.7 2.1 f 0.7 1.8 f 0.7 0.3 f 0.1 1.7 f 0.2

95 C 16 96 f 21 104 f 10 -80C ND ND ND ND 102 f 22 -95e ND ND ND ND

co.1 2.2 f 0.8 2.0 f 0.3

Cells transformed withpCV105 groE+

123 f 15 8.4 f 0.7 8.0 f 1.0 30 133 f 19 8.2 f 0.8 8.2 f 0.8 42 115 f 14 7.2 f 0.7 4.6 f 0.5 groEL30 0.2 f 0.2 6.1 f 0.7 -160' 42 4.8 f 0.3 ND ND 30 pACYC 0.3 f 0.2 42 pACYC ND ND 6.8 f 0.4 ND ND 30 pCRFl 4.8 f 0.4 ND 42 pCRFl ND 5.2 f 0.4 7.5 f 1.0 groES30 144 f 29 6.5 f 1.3 0.2 f 0.2 -150' 42 5.0 f 0.6 ND ND pACYC 30 0.3 f 0.2 ND pACYC 42 ND 6.5 f 0.4 ND pCRFl 30 ND 5.5 f 0.5 ND ND DCRF~ 42 FNR cell content represents the sumof FNR present insoluble and insoluble fractions after sonic disruption of the cells. One FNR activity unit is defined as the amount of enzyme that catalyzes the conversion of 1 wmol of substrate/minute at 30 "C. The average of 3-12 different experiments FNR-dependent activities of E. coli pellets were negligible (16). Units depicted in the table represent the with each strain andprovide an estimation of the amountof assembled functional flavoprotein. These specific activities are approximates, due to thesmall values obtained in the activity measurements. ND, not determined.

fractions of these cells. In the parental strain, FNR is largely present in the soluble fraction (Fig. 2B, lanes 1 and 2). On the otherhand, about half of the enzyme expressed by either groEL- or groE5" mutant cells is present as an aggregated, insoluble material. These results agree well with the lower enzyme activities observed in the mutants at 30 "C (Table I), and indicate an impairment of FNR assembly in these cells. It is worth mentioning that the specific activities (units/ milligram FNR) of the enzyme present in the soluble fractions did not show significant differences in the mutants or the parental strain (Table I). The partial assembly observed in the mutant cells was nottotally unexpected. Since GroE proteins arerequired for cell viability at all temperatures(20), the mutantforms of GroEL and GroES would be expected to retain some residual or altered functionto sustaincell survival at the permissive growth temperature of 30 "C. Similar results have been reported for the assembly of hexadecameric cyanobacterial Rbu-Pz-carboxylase inE. coli groE- mutants (31). For a further characterization of the effect of chaperonin deficiency on FNR expression and assembly, we induced expression of FNR polypeptides in groE- mutants immediately aftershifting the cultures from 30 "C to a non-permissive growth temperature of 42 "C (Table I, Fig. 2). Once more, the

enzyme was recovered in soluble cell fractions of the parental strain (Fig. 2B, lanes 3 and 4 ) in a fully active form (Table I). The amount of pre-FNR observed in pCV102-transformed parental cells at 42 "C was negligible (compare lanes 1 and 3 of Fig. 2 B ) , a result that would be expected if the products of the heat-shock genes groE and lon participate inthe assembly and processing of newly synthesized FNR. This conclusion is also supported by the results observed in groE- mutants (Table I, Fig. 2). The temperature shift had only a minor effect on the total amount of FNR in the cells when compared to those found at 30 "C (Table I). However, assembly of the newly synthesized reductase was abolished at 42 "C, as deduced from the lack of FNR diaphorase activity (Table I) and the almost quantitative recovery of FNR associated to bacterial pellets (Fig. 2B, lunes 3 and 4 ) . In the case of pCV102directed pre-FNR synthesis, most of the product was in the unprocessed form (Fig. 2B, lane 4 ) . Given the pleiotropic effects of groE mutations in E. coli (20) and the natureof the heat-shock response, some caution is required in the interpretationof the above results. In order to confirm the role of GroE proteins in FNR assembly, we studied the expression patterns of FNR in groE- mutants which contained, in addition, aheterologous groE operon from

15540

Chaperonin-mediated Assembly of Ferredoxin-NADP Reductase A 1

kDa 48

GrOEL7pACYC

30 20

*-"

24

GroEUpCRFl

B

ore€+

I-

I

GroESIpACYC -

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--i- - -*=-

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GroEVpCRFl

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FIG. 2. Expression and assembly of FNR in E. coli g r o F mutants. Transformed bacteria were grown in LB medium a t 30 "C as described in the text. Following addition of 0.5 mM IPTG, 10-ml aliquots were incubated a t 30 or 42 "C, respectively, for an additional 12-h period. Cells wereharvested, lysed, and analyzed by SDS-PAGE and immunoblotting as described under "Materials and Methods." A, typical electrophoretic patterns. The two lanes at the left represent the protein staining patterns, and thenext two lanes (right side)the immunoblotting of the previous lanes, using anti-FNR antisera. The first and third lanes represent the pellets obtained from pCV102transformed groEL- cells, and thesecond and fourth lanes the soluble fractions from pCV105-transformed groEL- cells, respectively. The final positions and molecular masses of the protein standards are indicated on the left. Positions corresponding to pre-FNR, mature FNR (35 kDa) and a smaller immunoreactive product (33 kDa) are shown on the extreme right. B, E. coli cells transformed with either pCV102 (lanes 1-4, left side) or pCV105 (lanes 1-4, right side) were grown a t 30 "C (lanes 1 and 2) or 42 "C (lanes 3 and 4 ) . After cell disruption, supernatants corresponding to 50 pgof total soluble proteins (lanes I and 3 ) and pellets representing an equivalent amount of cells (lanes 2 and 4 ) were subjected to electrophoresis and immunoblotting. Lane C, right: purified pea ferredoxin NADP+ reductase (-0.3 pg).

the photosynthetic bacterium Chromatium virwsum cloned in a compatible multicopy plasmid (pCRF1). Details of plasmid construction, sequence of the C. virwsum groESL genes, and expression in E. coli cells will be published elsewhere.' An increased amount of soluble active holoenzymewas apparent in cells transformed by pCRF1, based on activity (Table I) and immunoblotting experiments (Fig. 3), which showed patterns of soluble and particulated FNR that closely resemble those obtained with the parental strain. No effect was observed when cells were cotransformed with the supporting vector pACYC and any of the FNR expression plasmids (Table I, Fig. 3). A higher content of assembled cyanobacterial Rbu-P2-carboxylaseexpressed in E. coli has also been observed when the groE operon was overexpressed in these cells (31). It was also observed that at42 "C, the presence of pCRFl drastically reduced the formation of inclusion bodies and increasedthe amount of enzymaticallyactive FNR, particularly when introduced into groEL- strains (Table I, Fig. 3). DISCUSSION

Plant ferredoxin-NADP+reductase could be expressed in E. coli (13,16). Most of the recombinant enzyme accumulates in thebacterial cytosol ina soluble, active form, after undergoing proteolytic cleavage of the NH2-terminal extension and assembly of the prosthetic group FAD. Part of the protein remains associated with E. coli pellets after cell lysis,forming

p c v l o l pcv1os 2 3 4 1 2 3 4

5:

Z"

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U-

'z,-

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FIG.3. Effect of heterologous groESL chaperonins in the expression patterns of FNR in E. coli g r o E mutants. E. coli groEL- or groES" mutants harboring plasmids pCV102 (lanes 1-4, left side)or pCV105 (lanes 1-4, right side), respectively, were cotransformed with either plasmid pACYC184, or the recombinant vector pCRF1, which expresses the C. uinosum groESLoperon.' Conditions for IPTG-induced FNR synthesis in double transformants a t either 30 "C (lanes I and 2) or 42 "C (lanes 3 and 4 ) are described under "Materials and Methods." After cell disruption, supernatants corresponding to 50 pg of total soluble protein (lanes 1 and 3 ) or pellets corresponding to an equivalent amount of cells (lanes 2 and 4 ) were subjected to SDS-PAGE and immunobloting as described previously. Lane C, purified pea ferredoxin-NADP+ reductase (-0.3 pg).

insoluble inclusion bodies that showed no FNR-dependent activities. This insoluble fraction increases when FNR is expressed in protease-deficient E. coli mutants (Fig. 1, see also Ref. 16), suggesting that survival of the transit peptide largely prevents the flavoprotein from reaching a soluble, active conformation. Wilkinson and Harrison (8) have correlated general insolubility of a number of proteins expressed in E. coli with compositional parameters of the peptides themselves, particularly the number of turn-forming amino acids, andthe average charge/residue (negative and positive correlation, respectively). Their analysis implicitly assumed spontaneous protein assembly after translation (8). When applying the Wilkinson-Harrison formula to FNR or its precursor polypeptide, insoluble peptides were predicted with probabilities greater than 90% in both cases (normalized canonical variables of 1.38 and 1.43 for FNR and pre-FNR,respectively, see Ref. 8). However, at least the mature reductase showed a fairly good solubility in a number of E. coli strains (thiswork and Ref. 16), suggestingthat folding of the eucaryotic protein in the bacterial cell was not a spontaneous process, but most probably mediated by host factors. The products of the E. coligroE operon (14,151were shown to be required for a productive folding of the eucaryotic FNR holoenzyme in the bacterial cell. This conclusion is based on the following evidence: (i) partial inhibition of FNR folding was observed whenthe plantprotein was expressed in groEE. coli mutants, at thepermissive growth temperature of 30 "C (Fig. 2, Table I); (ii) at the non-permissive temperature of 42 "C,folding and FADassembly in groE- mutants were totally abolished, resulting in quantitative recovery of expressed FNR as insoluble material, apparently in the form of denatured apoprotein as judged by the absence of bound FAD and FNR-dependent activities (Fig. 2, Table I); (iii) these effects, both at 30 or 42 "C, could be partially suppressed when the groE- cellswere cotransformed with theFNRproducing plasmid and a compatible plasmid expressing the GroEL and GroES chaperonins from a phototrophic bacterium (Fig. 3, Table I). According to current models forGroEL-GroES action (14), it is expected that FNR expression in groES" mutants would result in increased recovery of the protein in the soluble

Chaperonin-mediated Assemblyof Ferredoxin-NADP+ Reductase fraction, although inactive, unfolded and bound to GroEL. However, results obtained with groEL- and groES- mutants were essentially the same at both permissive or non-permissive temperatures (Fig. 2), suggesting that thebinding capacity of GroEL was surpassed by excess FNR. Assuming direct physical interactions between chaperonins and FNR folding intermediates (32), the involvement of E. coli GroE proteins in holoenzyme assembly of a distant eucaryotic peptide might indicate that GroE chaperonins bind to unspecific structural motifs of unfolded proteins. This would agree with the broad range of interactions reported for the groE products (30, 32). This finding might have practical implications if the role of bacterialchaperonins could be extended to other animal and plant proteins expressed in E. coli. Aggregation of the transgenicproduct could be then partially or totally overcome by overexpression of the groE genes, as it occurs with the suppression of heat-sensitive mutants (32). Alternatively, it is possible that plant FNRis a special case among eucaryotic proteins and shares structural motifs with bacterial peptides normally interacting with GroE proteins. Although the groE genes of E. coli were first identified through mutations that block bacteriophage assembly (14, 30),further work demonstrated that thegroE gene functions are essential for E. coli growth at all temperatures, participating in the host protein folding, oligomerization, and export pathways (14, 15). Direct physical interaction between GroE chaperonins and many different bacterial proteins has been demonstrated (14, 15, 32). GroE proteins are remarkably conserved across evolution, with homologues identified in organisms as diverse as bacteria, fungi, plants, and animals (14,15,33).Chloroplast GroEL (Cpn6O) has been implicated in the assembly of plant RbuP2-carboxylase(15). It forms stable associations with several imported polypeptides, both monomeric and oligomeric, although FNR-Cpn6O interaction was not tested in those experiments (34). Based on the results presented here, we consider it very likely that folding of recently imported FNR would require the participation of plastid chaperonins. The role played by FAD in the assembly process is unclear. In vitro studies have shown that FAD spontaneously binds to apo-FNR, generating an active enzyme (35). This result suggests that in vivo folding in a chaperonin-dependent process is required prior to FAD assembly. Work is currentlyin progress to investigate the physiological mechanism of FNR assembly, including the role of plastidic Cpn60, CpnlO, and the prosthetic group FAD.

15541

Acknowledgments-We thankDr. John C.Gray (University of Cambridge,United Kingdom) for the generous gift of the original pea cDNA clone for FNR, Dr. C.Georgopoulos (University of Utah Medical Center, Salt Lake City, UT) for E. coli strains from his collection, and Dr. 0. A. Roveri (Biophysics Section, Universidad de Rosario) for the use of the Gilford Response I1 Spectrophotometerin which the activity measurements were carried out. REFERENCES 1. Vallejos, R. H., Ceccarelli, E. A., and Chan, R. L. (1984) J. Biol. Chem. 259,8048-8051 2. Carrillo, N., and Vallejos, R. H. (1987) in Current Topics in Photosynthesis: The Light Reactions (Barber, J., ed) pp. 527-560, Elsevier, Amsterdam 3. Newman, B. J., and Gray, J. C. (1988) Plant Mol. Biol. 10,511-520 4. Jansen, T.,Reilaender, H., Steppuhn, J., and Herrmann, R. G. (1988) Curr. Genet. 13,517-522 5. Michalowski, 0. B., Schmitt, J.M., and Bohnert, H-J.(1989) Plant Physiol. 89,817-822 6. Grossman, A. R., Bartlett, S. G., Schmidt, G. W., Mullet, J. E., and Chua, N-H. (1982) J. Biol. Chem. 257,1558-1563 7. Marston, F. A. 0. (1986) Biochem. J. 2 4 0 , 1-12 8. Wilkinson, D. L., and Harrison, R. G. (1991) Biotechnology 9,443-448 9. Guerra, D.J., Dziewanowska, K., Ohlrogge, J. B., and Beremand, P. D. (1988) J. Biol. Chem. 263,4386-4391 10. Coughlan, V. M., and Vickery, L. E. (1989) Proc. Natl. Acad. Sci. U. S. A. Sfi ",

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