Folding in vivo of bacterial cytoplasmic proteins: Role of GroEL

Cell, Vol. 74, 909-917,

September

IO, 1993, Copyright

0 1993 by Cell Press

Folding In Vivo of Bacterial Cytoplasmic Proteins: Role of G roEL Arthur L. Horwich,*t K. Brooks Low,* Wayne A. Fenton,’ Irvin N. Hirshfield,§ and Krystyna Furtak’t *Department of Genetics *Department of Therapeutic Radiology Q-ioward Hughes Medical institute Yale University School of Medicine New Haven, Connecticut 06510 5Department of Biological Sciences St. John’s University Jamaica, New York 11439

Summary A general role for chaperonin ring structures in mediating folding of newly translated proteins has been suggested. Here we have directly examined the role of the E. coli chaperonin GroEL in the bacterial cytoplasm by production of temperature-sensitive lethal mutations in this essential gene. After shift to nonpermissive temperature, the rate of general translation in the mutant cells was reduced, but, more specifically, a defined group of cytoplasmic proteins-including citrate synthase, ketoglutarate dehydrogenase, and polynucleotide phosphorylase-were translated but failed to reach native form. Similarly, a monomeric test protein, maltose-binding protein, devoid of its signal domain, was translated but failed to fold to its native conformation. We conclude that GroEL indeed is a machine at the distal end of the pathway of transfer of genetic information, assisting a large and specific set of newly translated cytoplasmic proteins to reach their native tertiary structures.

Introduction The central dogma of molecular biology defines the major route for the transfer of genetic information from linear genomic DNA to three-dimensional proteins that effect structure and function. Until recently, it appeared that such transfer of information required only machinery for transcription, RNA processing, and protein translation. Yet, a profusion of recent studies indicate the presence in the cell of specialized protein components, molecular chaperones, that play essential roles in enabling polypeptides to reach biologically active forms in a variety of cellular compartments (Ellis, 1987; Ellis and van der Vies, 1991; Gething and Sambrook, 1992; Hart1 et al., 1992). One remarkable class of such components is the chaperonins, a group of ribosome-sized, ring-shaped oligomers originally found in bacteria, mitochondria, and chloroplasts and more recently identified in archaebacteria and the eukaryotic cytosol (Horwich and Willison, 1993). Studies of these components both in vivo in mitochondria and in vitro have suggested that they mediate polypeptide chain folding by sequential binding of molten globule-like folding interme-

diates within central cavities (Martin et al., 1991; Langer et al., 199213; Braig et al., 1993), followed by release mediated by cochaperonin and ATP hydrolysis. Such a mechanism does not appear to accelerate folding but, rather, prevents misfolding that often results in protein aggregation. The role in vivo for this mechanism of protein folding remains largely undefined. The most global role demonstrated to date is that of Hsp60 complex in the mitochondrial matrix, where every imported protein examined to date fails to reach its native form in an Hsp60-deficient yeast mutant (Cheng et al., 1989, 1990; Glicket al., 1992; Martin et al., 1992). Does the same general requirement for chaperonin-mediated folding extend also to newly translated proteins? In the eukaryotic cytosol, a role for TCPl complex has been demonstrated in vivo for folding newly made tubulin (Ursic and Culbertson, 1991; Yaffe et al., 1992; Frydman et al., 1992; Gao et al., 1993), but a more general role in protein folding, including a putative role in actin folding (Gao et al., 1992), remains to be established. In the bacterial cytoplasm, the scope of action of GroEL, the homolog of Hsp60, in folding newly translated proteins is unknown. groEL was originally identified asone of agroup of host cell genes that were found to be defective in Escherichia coli strains able to grow in the presence of otherwise lytic bacteriophage h (Georgopoulos et al., 1973; Sternberg, 1973). Inside the mutant cells, phage heads were found as aggregates, suggesting a role for GroEL in oligomeric protein assembly. More recently, overexpression of GroEL and its cochaperonin GroES has been associated with enhanced activity of several coexpressed foreign proteins (Goloubinoff et al., 1989a; Van Dyk et al., 1989; Lee and Olins, 1992; Wynn et al., 1992). The normal action of GroEL cannot, however, be gauged by such observations. Examination of conditional mutants, however, can directly assess the role of a component under conditions of its deficiency. Indeed, many of the original groEL mutants fail to grow at temperatures above 42%. At nonpermissive temperature they exhibit selective defects, including reduced proteolytic activity (Straus et al., 1988), slowed export of p-lactamase (Kusukawa et al., 1989), and reduced UV mutagenesis (Donnelly and Walker, 1992). A global effect on protein biogenesis, resembling the effect of Hsp60 deficiency, has not, however, been observed. Considering that groEL is essential for cell viability at all temperatures (Fayet et al., 1989), it seemed possible that more severe mutants could be generated that might demonstrate more general defects. In particular, it seemed possible that mutants could be generated that would be inviable at temperatures lower than the temperature of 42%, which already lies near the upper end of the heat shock range. To study the general role of GroEL in protein biogenesis in the bacterial cytoplasm, we have therefore produced severe mutants of groEL, selected solely for temperature-sensitive lethality at 37% and have examined protein translation and folding in one of the mutants isolated.

Cell 910

Figure 1. Construction and Analysis Regulated groE Operon

of E. coli Strain

with

Lac-

The endogenous groE promoter was replaced by transformation with linear DNA fragments. Linear DNAs containing promoter-flanking sequences, a CAM resistance marker, and indicated promoter segments were transformed into the RecE-sbcBsbcC strain KL442, and CAMresistant transformants were scored.

Results Construction of groEL Mutants Temperature-sensitive lethal (ts) mutations in E. coli groEL were produced in two steps. In a first step, the groEL promoter in the E. coli chromosome was replaced with a regulatable lac operon promoter, rendering cells dependent on lac inducer for growth. Then, in a second step, the lac inducer-dependent cells were transformed with a chemically mutagenized low copy plasmid bearing the grof operon, and transformants were selected in the absence of inducer for temperature-sensitive growth. The grof promoter was replaced by transforming a recB-sbcBsbcC E. coli strain with linear DNA molecules containing the groE region of the E. coli chromosome modified in the promoter portion (see Figure 1 and Experimental Procedures). When a linear DNA bearing a chloramphenicol (CAM) resistance marker and a lac promoter-operator segment was transformed (Figure l), CAM-resistant transformants could be isolated only in the presence of isopropyl p-o-thiogalactopyranoside (IPTG) and not in its absence. When these transformants were transferred from IPTG-containing plates to liquid L broth (LB) medium, in the presence of IPTG the cells proceeded into logarithmic growth, but in its absence growth proceeded for 3-4 hr, then slowed markedly. Defective growth was associated with a greatly reduced level of GroEL 14-mer (- 5% wild-type), detected by immunoblot analysis of native gels separating soluble extracts. Supporting the idea that it is precisely groE function that is deficient, a plasmid bearing only the gfoEoperon could rescue growth of arec’ transductant in the absence of IPTG, whereas plasmids containing various flanking segments could not rescue. To produce temperature-sensitive groE mutants, the lac inducer-dependent LG6 cells were transformed with a hydroxylamine-mutagenized low-copy plasmid, pSC1 OlgroE, bearing the groE operon and a tetracycline resistance marker (Figure 2A). Cells were plated at 23V on

LB media devoid of IPTG, containing a low concentration of tetracycline (10 bg/ml). Transformants were replica plated to 37%, and temperature-sensitive clones were identified. Six such clones were recovered from 4000 transformants. One clone, ts2, was examined in detail. Its plasmid was recovered, and the groE region was excised and recloned in a nonmutagenized pSC101 backbone. After transformation into LG6 cells, the temperaturesensitive phenotype was recapitulated. Sequence analysis of the grof region of the reconstructed plasmid, pts2R, revealed a single alteration, a G-A change of base 1851, altering grovel codon 461 from glutamic acid to lysine. This substitution apparently had an effect on the biogenesis of GroEL in ts2R cells, because, in contrast with wild-type cells, a pool of unassembled subunits was detected by immunoblot analysis of native gel-fractionated extract (data not shown). Confirming that deficiency of GroEL is responsible for temperature-sensitive growth of the reconstructed ts2R strain, we observed that introduction of a second plasmid bearing a wild-type groE operon could rescue the growth defect (see Experimental Procedures). Growth of ts2R was next examined in liquid LB medium, comparing it with LG6 cells that had been transformed with nonmutagenized pSC101 groE (Figure 26). This latter strain, which grew identically to the original MG1655 parental strain, was used as a wild-type strain in all subsequent studies. It differs from ts2R by only a single base pair, at groEL codon 461 within the pSClOlgroE plasmid. At 23% ts2R grew at a rate nearly the same as wild type (data not shown). When cells were grown first in liquid LB for 2 hr at 23% (one doubling), then shifted to 37% ts2R grew at a rate similar to wild type for the first hour after shift, then lagged behind, failing to achieve exponential growth (Figure 28). It typically reached a final optical density (OD) of 0.7-0.8. The growth-retarded cells exhibited afilamentous morphology reminiscent of thegroEmutants selected previously by resistance to h phage (Georgopoulos and Eisen, 1974). Defective 1 Phage Biogenesis To examine the lethal phenotype conferred by the E461 K substitution in ts2R cells, we first examined h phage biogenesis. Wild-type and ts2R cells were grown in liquid LB containing maltose (0.2%) at 23% for 2 hr and were shifted to 37°C for 1 hr; then equal amounts of cells were infected with bacteriophage ICI857 at a concentration of lo6 pfulml. After 40 min of further incubation at 37OC, the cultures were lysed by addition of chloroform, and the phage titer was then determined on a C600 tester strain. In the case of the wild-type lysate, a titer of 10’ per ml was obtained, indicating a 2 log burst during the 40 min period of infection. By contrast, the titer from ts2R cells remained lo6 per ml, reflecting a failure of the input phage to replicate significantly. Thus, in ts2R cells, where the groEL mutation was selected specifically for conferring a temperature-sensitive lethal growth defect, there is recapitulation of defective h biogenesis, directly selected in the original groEL mutants.

GroEL Function 911

In Vivo

were present, by immunoblot analysis of total cell extracts with anti-human OTC antiserum. This serum specifically recognizes human OTC subunits and does not crossreact with ArgF or Argl subunits. When equivalent amounts of phOTC/wt and phOTClts2R cells were solubilized at 1 or 2 hr after temperature shift, similar amounts of the 36 kd OTC subunits were observed (Figure 38). This indicated that, while OTC subunits are translated in ts2R cells at 37%, they fail to reach a biologically active conformation.

Figure 2. Production tants

and Growth of Temperature-Sensitive

GroE Mu-

(A) The strain LG6 containing the lac-regulated groEoperon was transformed with the low copy plasmid shown, after it had been subjected to mutagenesis with hydroxylamine (see Experimental Procedures). The transformed cells were plated at 23% in the absence of IPTG on media containing tetracycline (10 &ml), then replica plated to identify temperature-sensitive mutants. A wild-type control was obtained by transforming nonmutagenized DNA. (B) Defective growth of ts2R strain in liquid LB medium after shift to 37%. Cells were transferred from solid media to LB devoid of drug to a starting OD of 0.01 and incubated for 2 hr. The OD approximately doubled during this time. Cells were then shifted to 37%, and the OD was measured at various times.

Defective Biogenesis of Two Cytoplasmic Test Proteins Ornithine Transcarbamylase To examine directly the effect of GroEL deficiency on protein biogenesis, two cytoplasmic test proteins were studied. We first expressed from a plasmid, phOTC, the mature subunit of the homotrimeric human mitochondrial enzyme ornithine transcarbamylase (OTC). With its NH*-terminal mitochondrial targeting signal omitted, the mature subunit portion expressed in E. coli would be expected to reach active form in the bacterial cytoplasm. Consistent with this, in soluble extracts of phOTC/wild-type (phOTC/wt) cells growing at 23% a level of OTC enzymatic activity was detected that was substantially greater than that observed in nontransformed wild-type cells, where a baseline activity is produced from the endogenous OTC genes of E. coli, argF and argl (Figure 3A). When the level of OTC activity was measured in extracts of phOTCXs2R cells growing at 23’C, it was comparable to that of phOTC/wt, indicating normal biogenesis of the OTC enzyme in mutant cells at permissive temperature. However, after shift of mutant cells to 37% for either 1 or 2 hr, no activity above the baseline could be detected. In contrast, the level of activity in phOTC/wt cells was increased beyond that obSeNf?d at 23X, most likely reflecting more efficient transcriptional expression during more rapid cell growth at 37%. To explore the deficiency of OTC enzyme activity in mutant cells at 37X, we first assessed whether OTC subunits

wt ~ TX 2 TS

ts2R TX .2

__TENTA PS

PTS

TENTA P

S

P

C OTC

’

CP

(OTC),

Figure 3. A Cytoplasmic Version of Human OTC Is Translated Nonnative in ts2R Cells at 37OC

but

(A) OTC enzymatic activity. The strains indicated were either grown in liquid LB for 5 hr at 23OC (to an ODsso of 0.5), or were first grown at 23’YJ for 2 hr and then incubated at 37’C for 2 hr. Cells (0.3 ODeso units) were extracted with a Triton X-100 buffer (see Experimental Procedures), and the lysates were assayed for OTC activity. Activity is expressed as wg of citrulline produced per 10 min. (B) lmmunoblot analysis of wild-type and ts2R cells incubated for 2 hr at 37%. Cells (0.3 ODBxr units; duplicate amounts of cells from [A]) were collected and either directly solubilized in SDS sample buffer(T) or first extracted with the Triton X-100 buffers indicated (see Experimental Procedures) and separated into soluble (S) and insoluble (P) fractions before electrophoresis and immunodetection. (C) Substrate affinity chromatography of [3sS]methionine-radiolabeled extracts of wild-type and ts2R cells with PALO. Wild-type and ts2R cells were grown at 23OC for 2 hr and shifted to 37%, and 2 hr later equivalent ODsso amounts of cells were [%]methionine radiolabeled for 6 min. Soluble Triton X-100 extracts were prepared, and one-third of each was directly immunoprecipitated with anti-OTC antiserum, while the remaining portions were applied to PALO resin and the carbamyl phosphate (CP) eluates were immunoprecipitated and electrophoresed.

Cdl 912

To examine further the fate of OTC subunits, cells were extracted using Triton X-100, and the extracts were fractionated by centrifugation at 14,000 x g for 10 min into soluble and insoluble fractions. W h e n extracted with either a low concentration of Triton X-100 (0.2%) in a low ionic strength buffer (30 mM HEPES [pH 7.4)) or with a high concentration of Triton X-100 (1%) in 0.15 M NaCl (TENTA), substantial levels of OTC subunit were detected in soluble extracts of wild-type cells incubated at 37OC, amounting to nearly 5 0 % with TENTA extraction (Figure 38). By contrast, in the mutant cells, OTC subunits were detected only in the insoluble fraction, suggesting that in mutant cells at 37°C the newly made subunits become aggregated (Figure 36). This behavior was observed despite the fact that the level of expression of OTC was not greater than 0.1% of total soluble protein, as judged from purification steps carried out with wild-type cells (L. Kuo and A. L. H., unpublished). Thus, OTC in these experiments represented a nonabundant cytoplasmic protein that probably aggregated after synthesis in the absence of GroEL function. The foregoing studies were carried out under steady-state conditions, necessitated because expression from the phOTC plasmid proved not to be inducible. To assess directly whether newly made subunits could reach native form, cells were pulse radiolabeled for 6 min at nonpermissive temperature with [?S]methionine, Triton X-100 extracts were prepared, and thesolublefraction was incubated with a substrate analog, NG-(phosphonacetyl)-L-ornithine (PALO), which specifically binds native assembled OTC trimer. On extraction, we observed roughly equivalent amounts of immunopreciptable radiolabeled OTC subunits in the SOIUble extracts, supporting that translation of OTC subunits at 37OC in the mutant cells is largely unaffected (Figure 3C). Equal amounts of extract were applied to PALO resin, and the resin was washed with buffer and then eluted with the OTC substrate carbamyl phosphate. The carbamyl phosphate eluate in the case of wild type contained OTC subunits readily detected by immunoprecipitation, reflecting presence of assembled trimers in the wild-type extract (Figure 3C). In contrast, in the case of ts2R, OTC subunits were not detected, indicating that the newly translated subunits in the mutant lysate failed to bind to the substrate analog. W e conclude that newly translated subunits in ts2R cells, while soluble at short times after synthesis, fail to reach the native trimeric form. Failure to reach the native state appears to lead to aggregation, observed in the steady-state experiments. Maltose-Binding Protein To examine whether biogenesis of a monomeric cytoplasmic protein might also be affected in ts2R cells, we induced expression of the maltose-binding protein (MBP) devoid of the NHz-terminal peptide that normally directs its export to the periplasmic space (cytoplasmic MBP or cMBP). Signal-deleted cMBP has previously been observed to localize to the bacterial cytoplasm, where it reaches native conformation as judged by ability to bind to amylose, a polymeric form of the normal ligand, maltose. W e could thus test whether cMBP requires GroEL function to fold to its native conformation by inducing

cMBP in ts2R cells at various times after shift to 37OC, preparing cell extracts, and assaying whether soluble cMBP could bind to amylose. cMBP was induced from a lac operon promoter in a commercially available plasmid by addition of IPTG to the culture medium. The use here of IPTG could potentially have presented a problem for maintaining the GroEL-deficient phenotype, since both wild-type and ts2R strains retain a chromosomal copy of groE adjoined by a lac promoter. In particular, #activation of this otherwise functionally deficient chromosomal copy by IPTG could potentially have produced a quantity of wildtype GroEL subunits that could rescue ts2R cells. However, we observed that short times of IPTG induction did not alter the defective growth at 37OC of ts2R cells. Thus, if any wild-type GroEL was induced over short times of a few minutes, it was apparently insufficient to alter the phenotype. pMALcQ/wt and pMALc-2/ts2R cells were shifted to 37OC for 1 or 2 hr (see Figure 28) and induced with IPTG for 5 min. Triton X-100 extracts were prepared, and identical portions of the soluble fraction were incubated with amylose resin. After washing, bound proteins were recovered from the resin by direct solubilization in SDS sample buffer. lmmunoblot analysis with anti-MBP antiserum revealed relatively similar amounts of induced cMBP in soluble and insoluble fractions of both wild-type and mutant cells after induction at either 1 hr (data not shown) or 2 hr after shift (Figure 4). W h e n the relative amount of soluble cMBP bound by the amylose resin was assessed, efficient binding was observed from extracts of both wild-type and mutant cells induced at 1 hr (data not shown). By contrast, at 2 hr after shift there was a striking difference between wild-type and mutant cells (Figure 4). Whereas there was efficient binding from wild-type extracts, virtually no cMBP was detectable from the mutant extract in several independent experiments. W e conclude that cMBP is normally translated in ts2R cells, but at the later time after temperature shift, a point at which the growth defect is fully manifest, the newly translated monomeric protein fails to reach its native conformation. Insoluble Behavior of Many Newly Made Proteins in ts2R Cells Having observed failure of two newly translated cytoplasmic test proteins to reach active conformation, we examined ts2R cells for a general defect involving endogenous proteins. To do this, we examined the pattern of newly translated Triton X-lOO-soluble proteins by twodimensional (20) gel analysis. W ild-type and ts2R cells were radiolabeled with [35S]methionine at 2 hr after shift to 37OC. Because ts2R cells do not grow in minimal medium, the radiolabeling studies had to be carried out in LB and were facilitated by using 100 pCi/ml [%S]methionine and a 7 min labeling period. Triton X-lOO-soluble (TENTA) fractions were prepared and fractionated by isoelectric focusing to equilibrium followed by SDS-polyacrylamide gel electrophoresis. W e observed first that, for equivalent amounts of cell extract applied to the gels, the wild-type extract contained approximately 3-fold greater [35S]methionine. This reflects a general defect of protein

GroEL Function 913

In Vivo

s

ts2R cells, the spot patterns of wild-type and ts2R cells were observed to be strikingly different (Figure 5). Most notably, a substantial number of species observable in the soluble wild-type extract were either completely absent in the mutant extract or were far reduced relative to other proteins. Overall, of 35 major [35S]methionine-labeled proteins, 16 were affected. Given the observed behavior of human OTC, it seemed possible that at least a portion of the soluble proteins absent from the mutant extract might have been translated but have become aggregated. They might thus have localized to the Triton X-lOO-insoluble fraction and would have failed to appear in the soluble fraction applied to the 2D gel. We considered that direct urea solubilization of radiolabeled cells might disrupt such aggregates, allowing the affected species to be detectable in 2D gel analysis. This indeed proved to be the case: of 16 species that had been identified to be absent or strongly reduced in the Triton X-lOO-soluble fraction of mutant cells, 9 were detected following direct urea extraction at levels nearly identical to wild-type (identified in legend to Figure 5). Among these were three species assigned in the E. coli gene-protein data base, ketoglutarate dehydrogenase (spot 3), polynucleotide phosphorylase (spot 4) and citrate synthase (spot 11). While these species were now observed with

ts2R

wt s

S-Amy P

S-Amy P

Figure 4. A Cytoplasmic Version of Monomeric but Unable to Bind to the Substrate Amylose

MBP Is Translated

Wild-type and ts2R cells were grown at 23% for 2 hr, then shifted to 37% and incubated for an additional 2 hr. IPTG was added to equivalent ODsu, amounts of cells, and the cells were harvested after 6 min. Soluble Triton X-100 lysates were prepared; half of each lysate was directly solubilized, while the other half was incubated with 50 ul of amylose affinity resin. After 10 min, the resin was washed with 1 ml of buffer (see Experimental Procedures), then directly solubilized. All of the eluted material (SeAmy) was examined, while 50% of the amount of input soluble extract(S) and an equivalent amount of insoluble fraction (P) were examined by immunoblotting with anti-MBP antiserum.

translation occurring in ts2R ceils after shift to 37%, most noticeable by 2 hr after shift, correlating with the slowed growth of the culture by this time. After exposure times were adjusted to correct for the reduced incorporation in

ts2R

wt OH'

Ii*

OH-

Ii*

\

Figure 5. Two-Dimensional

Gel Analysis of [%S]Methionine-Radiolabeled

Proteins Translated

in Wild-Type

and ts2R Cells at 37%

Cells were grown in LB for 2 hr at 23OC and shifted to 37%. and after 2 hr equal OD Bsoamounts were radiolabeled by addition of [%S]methionine, 100 t&i/ml, to the medium. After incubation for 7 min, cells were collected, and soluble Triton X-100 extracts were prepared. Urea and buffer were added, and equilibrium isoelectric focusing (8000 V. hr) was carried out, followed by SDS-polyacrylamide gel electrophoresis in an 11.5% polyacrylamide gel (see Experimental Procedures). The gels were fluorographed, and spot patterns were compared between wild-type and ts2R. The spot patterns were also compared with the E. coli gene-protein data base (VanBogelen et al., 1992) to identify some of the major spots. Of species that were found in wild-type pattern but not in tsPR, nine could be matched with proteins in the data base with known coordinates, in five cases identifying assigned proteins: spot 1, E133.0, carbamyl phosphate synthetase large subunit; spot 2, G117, initiation factor 2a; spot 3, G97.0, ketoglutarate dehydrogenase; spot 4, C78.0, polynucleotide phosphorylase; spot 5, 860.7/E; spot 7, H54.6; spot 9, C48.7; spot IO, G48.6; spot 11, H47.4, citrate synthase. Four species were found in ts2R but not in wild type, and two could be matched to assigned proteins: spot a, F88.0, methionine synthase (tetrahydropteroyllriglutamate methyltransferase); spot b, G43.0, cystathionine lyase. When the same analysis was carried out with total urea extracts of wild-type and ts2R cells (data not shown), we observed many of the spots that were absent in the soluble Triton X-100 extract to be detected at levels similar to wild-type, including spots 3, 4, 7, 8, 9, 11, 14, 15, and 16.

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urea extraction, other species, including carbamyl phosphate synthetase large subunit (spot 1) and initiation factor 2a (spot 2) failed to be observed with direct urea extraction. They might have either failed to be solubilized, been proteolytically degraded, or failed to be translated. Notably, all of the assigned species are proteins localizing to the bacterial cytoplasm. In addition to many species absent from soluble extracts of ts2R cells at 37%, four “new” spots were detected in ts2R lysates that were not present in wild-type extracts. Two of them could be identified in the gene-protein data base: spot a is assigned as themetEgene product, methionine synthase, and spot b as the product of metC, cystathionine lyase. Remarkably, these twoenzymescarryout the last two steps in the pathway of methionine biosynthesis in prokaryotes. Neither is a known heat shock component. Selective induction of these enzymes could represent a physiological response to deficiency of intracellular methionine, but amino acid analysis of cell extracts showed equal methionine levels in mutant and wild type. MetE was further examined. It was virtually undetectable in ts2R cells at 23%, but after shift to 37%, it was immediately and massively induced, becoming the single most abundant protein in the cell, judging by Coomassie staining of one-dimensional SDS gels (data not shown) and confirmed by NH,-terminal sequence analysis. MetE was similarly induced when LG6cells were placed in the absence of IPTG. Induction of enzymes in the pathway of methionine leads inescapably to consideration that this represents a mechanism of regulation of translational initiation, perhaps a means by which a component involved in protein folding can communicate back to the translational machinery. Discussion Defective Protein Translation and Folding in GroEL-Deficient E. coli W e have described here a lethal mutation in the essential E. coli protein, GroEL, that leads to failure of many newly translated cytoplasmic proteins to reach their native forms. A broad effect on protein biogenesis has not previously been reported for the longstanding class of groE!- mutants that were isolated by resistance to I phage infection. It seems likely that these latter mutants have a milder phenotype, considering that they are able to grow in minimal medium and that their cutoff temperature for growth is 42%. By contrast, the strain described here appears more severely affected, as it could not grow on minimal medium at any temperature and exhibited a cutoff temperature in LB of 35%. Consistent with the expression of a more severe effect on the same cellular function, the phenotype observed in the ts2R strain appears to encompass the phenotypic properties of the previously isolated mutants, including defective phage biogenesis and filamentous morphology. Importantly, the severe phenotype observed here was selected without any bias as to the function of GroEL in the cell-selection was carried out solely for conditional loss of the essential function of GroEL required for growth.

Deficiency of GroEL in ts2R cells affected protein translation. This was repeatably observed in both short-time and pulse radiolabeling studies as a general 3- to 4-fold reduction in intensity of the radiolabeled species relative to wild-type. This effect could reflect a primary requirement for GroEL function in the translation process, e.g., involving its interaction with nascent polypeptide chains. Arguing against this, however, are recent observations that DnaJ and DnaK, but not GroEL, are found in association with nascent chains on ribosomes isolated from E. coli (G. Gaitanaris, personal communication). Reduced translation in ts2R cells seems more likely to be a secondary effect. Factors required for initiation or elongation could become limiting in concentration. In particular, we observed that initiation factor 2a, which normally binds in a complex with fmet-tRNAf to the 30s ribosomal subunit, was absent from the soluble fraction of radiolabeled ts2R extracts (see Figure 5). The more immediate effect of GroEL deficiency appears to involve inability of newly translated cytoplasmic proteins to reach their native states. Studies of two programed test proteins, one a monomer, the other a trimer, showed that at nonpermissive temperature they were efficiently translated but failed to reach active forms. More generally, examination of newly translated endogenous proteins revealed many to be insoluble upon Triton X-100 extraction, suggesting that they had aggregated in the cell. While many of the major newly translated species were affected in this way, we did not observe the wholesale aggregation of all newly translated proteins, accompanied by production of inclusion bodies, that has recently been reported in E. coli rendered generally deficient of heat shock proteins by deletion of the transcription factor 032 (Gragerov et al., 1992). In contrast with the effects observed on newly translated proteins, an effect was not observed on preexistent proteins. For example, when mutant cells expressing OTC were shifted to 3 7 % in the presence of CAM, the preexistent enzymatic activity did not turn over any faster than in wild-type cells. Likewise, activity of the endogenous enzyme citrate synthase was unaffected. A Subset of Cytoplasmic Proteins Are Affected by GroEL Deficiency While many newly translated endogenous proteins seem subject to aggregation, others may remain largely soluble despite nonnative conformation, as in the case of the test protein cMBP. The 2D gel analysis would not have identified such species, and thus may underestimate the number of misfolded proteins. Still other proteins may be proteolytically degraded if misfolded and could be part of the class of species in 2D gel analysis that failed to be detected following direct urea extraction of mutant cells. While many cytoplasmic proteins are affected, it seems clear that not all are involved. For example, we failed to observe any effect on the specific activity or solubility of the homotetrameric enzyme 6-galactosidase, induced at nonpermissive temperature. The lack of effect in vivo corresponds well with our failure to observe interaction of 6-galactosidase with GroEL in vitro (K. Braig, unpublished data). Indeed, the requirement for GroEL function in vivo

GroEL Function 915

In Vivo

may correlate with ability to observe interaction in vitro, supported by observations of human OTC and citrate synthase (Zheng et al., 1993; Buchner et al., 1991; Zhi et al., 1992). More generally, 2D gel analysis of GroEL-deficient cells shows that approximately 30% of species are affected; this agrees with observations in vitro indicating that approximately 50% of soluble protein species of E. coli are bound by GroEL upon dilution from chaotrope (Viitanen et al., 1992; A. L. H., unpublished data). It thus seems likely that there is a specific set of cytoplasmic proteins that depend on GroEL function to reach native form. Other polypeptides may be able to fold without assistance from GroEL, either utilizing assistance from other components or perhaps folding spontaneously. The class of exported proteins seemed also not to require GroEL function stringently. Proteins including alkaline phosphatase, OmpA, and OmpC were not affected in the 2D gel analysis, although we cannot exclude that the rate of their export was slowed, as previously reported for B-lactamase. While we have identified a set of cytoplasmic proteins affected by GroEL deficiency, we cannot exclude the possibility that this set of proteins comprises only those most dependent on GroEL function. Whether even more severe conditional lethal mutants than ts2R could be isolated to address a potentially wider range of function remains to be seen. In the setting of GroEL deficiency, the lack of induction of DnaK and other heat shock components was surprising. To date, however, a heat shock response has been observed only in mutants affecting DnaK, DnaJ, and GrpE (Straus et al., 1990). Perhaps the conformations resulting from deficient function of GroEL are not recognized by components like DnaK or DnaJ that normally recognize intermediates that have less native structure (Flynn et al., 1991; Landry et al., 1992; Langer et al., 1992). This would leave these components available to dictate normal levels of the heat shock transcription factor o32. Alternatively, perhaps 032 itself fails to be properly folded. Our study supports a role in vivo for GroEL in polypeptide chain folding but does not specifically address a potential role in oligomeric protein assembly. In particular, we saw effects on a monomeric protein, a signal-deleted form of maltose binding protein, that failed to reach a native conformation and was unable to bind a substrate. Whether effects we observed both on human OTC and on endogenous proteins known to be oligomeric were the result exclusively of misfolding of component subunits or were also contributed by an effect at the step of oligomeric assembly could not be distinguished. We remark, however, that recent studies with OTC in vitro have shown that monomers can be detected following release from GroEL under conditionsof high dilution (Zheng et al., 1993). These subunits remain soluble, in contrast with subunits that misfold, e.g., after dilution from chaotrope in the absence of GroEL. Both these observations and studies carried out with dimeric RUBISCO (Goloubinoff et al., 1989b) fail to demonstrate a role of GroEL in vitro in protein assembly. This is consistent with other recent observations in vitro that polypeptides bound to GroEL localize within a central cavity (Langer et al., 1992a; Braig et al., 1993)-in general, there would be

insufficient interior space to accommodate more than one polypeptide. Experimental Procedures Strains KL442 (F- meE70

trpA605 rpsLl71

gyrA19 rec621 sbc615

sbcC250

Xc/n&) is a lac+pro+derivative, cured of a cryptic PI prophage, of strain KL635 (Schellhorn and Low, 1991), obtained by a cross with Hfr KL226 (Low, 1973). Transformation of KL442 with the PI&AM linear DNA molecule (Figure 1), designated groEL200, produced a strain, LGI (also denoted AHI) with the CAM marker and lac segment integrated in place of the grof promoter, demonstrated by DNA blot analysis. A ret’ transductant, LG6 (also denoted KL446), was obtained by transduction of MGl655 (Bachmann, 1987) by Pl,,, grown on LGI, selecting for CAMR. Strains designated as wild-type (also denoted AH2) and ts2R (also denoted AH3) were obtained by transformation of LG6 with, respectively, plasmids pSClOlgroE and pSClOlgroE carrying a G-A mutation at base 1851 (Hemmingsen et al., 1988), denoted groEL201. DNA Manipulations and Transformation and Pl Transduction groE promoter alterations were carried out through a series of cloning steps in pBR322-based plasmids carrying the groE region of the E. coli chromosome. This region was initially subcloned from Kohara phage 1649 (Kohara et al., 1987). First, for ease of making alterations, an EcoRl site was engineered into the groE operon downstream from the RNA start site, at base 90 in the sequence of Hemmingsen et al. (1988). Next, a synthetic DNA adapter was inserted between a naturally occurring Bell site that crosses base 1 and the engineered EcoRl site. Then a CAM resistance marker, carried on a 1600 bp filled in Hincll-Accl fragment, was inserted into a Smal site in the upstream end of the adaptor, and synthetic promoter segments were afterward inserted directionally between Xbal and Xhol restriction sites carried in the downstream end of the adaptor. Linear DNA fragments for transformation were derived by digestion of the derived pBR322-based plasmids. For insertion of the wild-type grof operon into pSClO1 (Cohen et al., 1973), the groE-bearing plasmid pOF39 (Fayet et al., 1988) was first modified by insertion of an EcoRl linker into a unique Smal site downstream from the groEL stop codon. Hydroxylamine mutagenesis of the derived plasmid, pSClOlgroE, was carried out as described by Busby et al. (1982). The period of mutagenesis was varied so as to yield - 10-200~ the number of tetracycline-resistant clones produced by the same amount of nonmutagenized DNA. Typically, 15 min or 22 min of incubation at 75OC was required. The plasmid phOTC. directing expression of cytoplasmic human OTC, was derived by insertion of an EcoRl fragment bearing a tat promoter into a unique EcoRl site carried in a plasmid, pSPROTC (Horwich et al., 1986). This positions the regulatory element to drive expression of the mature portion of OTC, initiating translation at an ATG adjoined to codon 5 of the mature subunit. The plasmid directing expression of a cytoplasmic-localized MBP, pMAL-c2, was obtained from New England Biolabs. Preparation of competent bacteria and DNA transformation were carried out using methods described by Hanahan (1985). All cloning steps were carried out using the strain DH5, except for manipulations involving Bell, where DNA was prepared in the dam- strain GM2163 (Woodcock et al., 1989). Pl transduction was carried out according to Low et al. (1971), with selection for resistance to CAM, 12 uglml. For rescue of ts2R cells with a wild-type gfoE operon, compatibility was maintained with the already present pSCl01-based plasmid by utilizing a pBR322 plasmid bearing the wild-type operon. Transformation was carried out at 23%, and cells were directly plated at 37% on media containing both ampicillin and tetracycline. Cell Growth, Radiolabeling, and Extraction Wild-type and mutant strains were maintained at 23% during the period of most of the experiments, on LB plates containing IO pglml tetracycline and in some cases also 12 kg/ml CAM, although this latter resistance, conferred by an integrated marker, proved to be nonreverting. Where pBR322-based plasmids encoding test proteins were

Cell 916

carried in the wild-type and mutant strains, ampicillin (75 @g/ml) was also included in the solid media. Strains were streaked every 2 to 3 days. Colonies were observed to be in all cases uniform in size, with the ts2R mutant clones somewhat smaller than wild-type. Every few days the mutant was also streaked to 37OC to assure that the temperature-sensitive phenotype was present. Onlyaminimal rateof reversion, in the form of a few colonies in the most dense part of the streak, was ever observed. For studies in liquid culture, cells were inoculated from plates growing al 20°C into liquid LB devoid of antibiotics, to produce an OD,,, of 0.010. Cells were then grown for at least 2 hr at 23’C. Both wild-type and ts2Rculturesapproximatelydoubledduringa2 hrperiod. Cultures for all experiments were followed by OD,,, measurement both while at 23% and after temperature shift. Growth curves both before and after temperature shift were virtually identical from day to day. Radiolabeling was carried out 2 hr after shift to 37%. [35S]methionine (100 &i/ml, 1000 Cilmmol; Amersham) was added lo 2 ml of ts2R culture and to an equivalent OD amount of wild-type culture, typically 0.5 ml. We verified that, despite acquisition of filamentous morphology, given optical density amounts of the mutant cultures contained the same amount of total cell protein as equivalent OD amounts of wild-type cultures. For induction of cMBP, IPTG was added to equal ODssO amounts of wild-type and ls2R cells incubated at 37”C, to a final concentration of 1 mM, and the cells were harvested after 6 min. No MBP was detectable by immunoblot analysis of uninduced cells. For most experiments, cells were harvested by immediate centrifugation at 4OC, requiring - 1 min. Cell pellets were typically resuspended in 20 VI of an ice-cold solution of 25% sucrose-50 m M Trls (pH 7.4), and then 5 VI of lysozyme, 10 mglml, was added. After 3 min, spheroplasts were extracted with typically 80 ul of a solution (TENTA) containing 1% Triton X-l 00 (v/v), 5 m M EDTA, 150 m M NaCI, and 20 m M Tris (pH 7.5). In one case a solution (Tx.2) of 0.2% Triton X-100 (v/v), 30 m M HEPES (pH 7.4) was employed. For analysis of MBP, the foregoing steps were all carried out at 37% to inhibit possible spontaneous refolding of the monomeric protein during extraction. For 2D gel analysis, either TENTA extracts were directly processed for gel analysis, or they were first centrifuged at 14,000 x g for 10 min and then the supernatant was processed. In both cases the extracts were treated first with DNAase I and RNAase A, each at 50 pg/ ml final concentration, for 20 min at 4%, and then 100 mg urea was added to the mixture. An additional 200 ~1 of isoelectric focusing sample buffer was then added, containing 8 M urea, 2% Nonidet P-40, 0.7 M mercaptoethanol, and ampholines (Pharmacia LKB) at 1% (pH 5-7) and 0.25% (pH 3.5-10). Protein Analysis lmmunoblot analysis was carried out according to Harlow and Lane (1988). Rabbit anti-human OTC antiserum was kindly supplied by F. Kalousek. Rabbit anti-MBPantiserum wasobtainedfrom New England Biolabs. OTC activity was determined according to Kalousek et al. (1978). For both wild-type and ls2R cells, OTC assay results were the same whether total extracts or Triton X-lOO-soluble fractions were analyzed. Affinity chromatography on PALO was carried out as described by Hoogenraad et al. (1980); OTC subunits in the applied and eluded fractions were identified by immunoprecipitation with anti-OTC antiserum. For affinity chromatography of cell extracts on amylose resin, 100 ~1 out of 200 PI of soluble TENTA extract was incubated with 50 MI of amylose resin (New England Biolabs) that had been prewashed in a buffer containing 10 m M KPO,. 0.5 M NaCI, and 1 m M dithiothreitol. After 10 min of incubation at 37%, the resin was collected by brief centrifugation in a microfuge, washed with 1 ml of the buffer at 37%, then resuspended directly in 50 @I of SDS sample buffer and boiled for 2 min. The mixture was centrifuged, and the solubilized supernatant was analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting. Two-dimensional gel analysis was carried out as described by O’Farrell (1975). using focusing to equilibrium, 8000 V’hr, in a 1.36% (pH 5-7) and 0.33% (pH 3.5-10) carrier ampholyte mixture, followed by second-dimension SDS-polyacrylamide gel electrophoresis in 11.6% polyacrylamide. The gels were then fluorographed (Horwich et al., 1980).

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