From the Département de Biochimie
Médicale, Centre Médical Universitaire, 1 rue
Michel-Servet, CH-1211 Genève 4, Switzerland and
Laboratoire de Microbiologie et Génétique
Moléculaire, CNRS-UMR 5100, 118 route de Narbonne,
F-Toulouse Cedex 31062, France
Received for publication, September 15, 2000, and in revised form, December 1, 2000
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ABSTRACT |
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Bacteriophage T4-encoded Gp31 is a functional
ortholog of the Escherichia coli GroES cochaperonin
protein. Both of these proteins form transient, productive complexes
with the GroEL chaperonin, required for protein folding and other
related functions in the cell. However, Gp31 is specifically required,
in conjunction with GroEL, for the correct folding of Gp23, the major
capsid protein of T4. To better understand the interaction between
GroEL and its cochaperonin cognates, we determined whether the
so-called "pseudo-T-even bacteriophages" are dependent on host
GroEL function and whether they also encode their own cochaperonin.
Here, we report the isolation of an allele-specific mutation of
bacteriophage RB49, called The Escherichia coli GroEL and GroES proteins form a
complex, known as the GroE chaperone machine, that is crucial to the functions related to protein folding (reviewed in Refs. 1-4). The
GroEL and GroES proteins are often referred to as chaperonin and
cochaperonin, respectively (5). Both GroES and GroEL have been shown to
be essential for E. coli viability under all conditions tested (6). Extensive structural analysis has revealed that both
proteins are organized into rings with a 7-fold rotational axis (7, 8).
GroEL is arranged in two head-to-head stacked rings of seven subunits
in whose central cavities various substrate proteins can bind,
primarily through hydrophobic interactions (Ref. 9; reviewed in Refs. 2
and 4). GroES is a heptameric ring of 10.5-kDa subunits from which
mobile loops extend and interact with GroEL (10, 11).
A typical GroEL substrate is first captured in either of the GroEL
central cavities, and the subsequent binding of ATP and GroES to the
same ring (referred to as the cis ring) results in the
formation of a dome-like structure over the substrate, which is now
released into the GroEL cis cavity. The subsequent binding of ATP and substrate (and potentially GroES as well) in the
trans GroEL ring results in the release of GroES from the
cis ring (4, 12). If the substrate in the cis
ring is properly folded, or in a form that no longer binds GroEL, it is
released into the medium, and this GroEL ring will become the new
trans ring in a new GroEL folding cycle. Houry et
al. (13) have shown, by using coimmunoprecipitation experiments,
that ~10% of E. coli's polypeptides bind transiently to
GroEL. Some of these proteins are encoded by essential genes, in
agreement with the genetic observation that the groES and
groEL genes are both essential for E. coli growth
(6).
The bacteriophage T4, which is dependent on the host GroEL function for
folding of its major capsid protein, Gp23, encodes its own GroES
ortholog, termed Gp31. There is very little sequence similarity between
GroES and Gp31 (14, 15), although there is a limited conservation in
certain regions, most notably the mobile loop that becomes immobilized
upon binding to GroEL (10, 16, 17). The functional importance of the
mobile loop was first shown by sequencing mutations in groES
that blocked bacteriophage Comparative studies of GroES and Gp31 have shown that while Gp31 can
substitute for GroES, the reverse is not true; i.e. Gp31 can
suppress the temperature-sensitive phenotype of the E. coli groES42 mutant and can participate in the in vitro
folding of various substrates, but GroES is unable to substitute for
Gp31 in T4 bacteriophage assembly, even when overproduced (21). This may not be totally surprising, since it is likely that Gp31 evolved as
a specialized ortholog of GroES that specifically folds the Gp23
substrate. Gp23 is 56 kDa, near the maximum limit allowed by the
"Anfinsen cage" formed under the dome of the GroEL-GroES complex
(reviewed in Refs. 3 and 4). Structural studies suggest that the
GroEL-Gp31 complex may result in the formation of a larger
cavity under the dome of the Gp31 heptamer due to a longer mobile loop,
the lack of the roof loop present in GroES, and lack of a conserved
aromatic residue, which extends into the cavity of GroES (22, 23).
Recently, a large number of bacteriophages with T4-like morphology have
been characterized according to their sequence homology with the
classical T-even bacteriophages, T2, T4, and T6 (24). The vast majority
of these bacteriophages are very closely related to T-evens at the DNA
sequence level. However, a few of them have DNA sequences that differ
significantly, and these have been termed "pseudo-T-even
bacteriophages" (25). We investigated the genomes of the
pseudo-T-even bacteriophages to determine whether they possess a gene
encoding a homolog of either GroES or Gp31. Here, we report our
findings on the bacteriophage RB49 gene 31 homolog, which we
have named cocO (cochaperonin
cognate).
Strains--
The various strains, bacteriophages, and plasmids
used in this study are listed in Table
I.
Media--
Bacteria were grown in Luria-Bertani broth (LB;
10 g of tryptone, 5 g of NaCl, 5 g of yeast extract per
liter, pH 7) or on LB-agar (10 g of agar/liter of LB). Top agar (6 g of
agar/liter of LB) was used for seeding lawns with cells and/or
bacteriophage. Ampicillin and tetracycline were added to the media at
final concentrations of 100 or 15 µg/ml, respectively, when
necessary. TSG buffer (0.01 M Tris-HCl, pH 7.4-7.5, 0.15 M NaCl, 0.03% gelatin) was used for making serial
dilutions of bacteriophage and for storing concentrated bacteriophage stocks.
DNA Library Construction--
One liter of an RB49 lysate
(2 × 109 pfu/ml) was concentrated by polyethylene
glycol precipitation (34), followed by CsCl banding on a step gradient.
The concentrated bacteriophage was dialyzed for 30 min in 500 ml 3 M NaCl, 0.1 M Tris, pH 7.4, followed by two
30-min dialyses in 500 ml of 0.3 M NaCl, 0.1 M
Tris, pH 7.4. The dialyzed bacteriophage was treated with proteinase K, followed by phenol/chloroform extraction (34). The DNA was partially digested using Sau3A (New England Biolabs), and fragments of
1.5-5 kb were purified from an agarose gel. The fragments were ligated to the vector pMPMA4 (a derivative of pMPMA4 Marker Rescue--
Cells growing exponentially at a density of
A595 = 0.1 were infected with the appropriate
bacteriophage at a multiplicity of infection of 0.02 bacteriophage/bacterium. The cultures were aerated at 37 °C until
lysed, ~2 h. Chloroform was added to ensure lysis and the killing of
all remaining cells. To check for wild type recombinants, 0.1 ml of the
lysate was mixed with 0.3 ml of a saturated culture of
groEL140 cells and 3 ml of molten top agar and then poured
onto an LB-agar plate. The plates were incubated at 37 °C overnight
to allow plaque formation.
Suppression of the groES42 Temperature-sensitive
Phenotype--
E. coli groES42 cells were transformed with
either the pcocO+34-2,
p31+, or pMPMA4 parental vector plasmids.
(p31+ is a plasmid that complements the T4
31amNG71 mutant, which has an amber (nonsense) mutation in
gene 31. The plasmid was isolated from a T4 genomic library
cloned into the vector pBAD18 (33).) Transformants were selected for
ampicillin resistance at 30 °C. After overnight growth, two colonies
from each transformation were resuspended in 100 µl of LB broth each.
Three 5-fold dilutions were made of each culture. Ten µl of each
dilution and the original suspension were spotted on LB-agar-ampicillin
plates. The plates were incubated at 30 or 43 °C, and bacterial
colony-forming ability was monitored following overnight incubation.
DNA Sequencing--
Chain termination reactions were performed
using the ThermoSequenase kit (Amersham Pharmacia Biotech), and the
reactions were sequenced on a Li-Core sequencing apparatus (MWG
Biotech). The primers used for sequencing and polymerase chain reaction
amplification are as follows: cocO pBAD 5',
5'-CGCAACTCTCTACTGTTAATGTAGATGGGCTGAC-3'; cocO 5'
RI/NdeI, 5'-GGAATTCATATGACCGTTAAAACTCCTAAAGC-3';
cocO 3' XbaI,
5'-GCTC TAGAGGATATTCCCGCCGTTAAA-3'.
Cloning of the Minimal cocO Gene for Overexpression--
The
cocO 5'RI/NdeI and cocO 3'
XbaI primers were used to polymerase chain reaction-amplify
the minimal cocO gene from wild type RB49 bacteriophage. The
product was cloned into the EcoRI and XbaI sites
of the pMPM201 vector under the regulation of the PBAD
promoter as previously described (32). The resulting plasmid was named
pFScocO+. The cocO Purification of the CocO Protein--
Six 1.5-liter cultures
inoculated with DA1368 bacteria containing plasmid
pFScocO+ were grown in LB plus 100 µg/ml
ampicillin at 37 °C. Synthesis of CocO was induced at
A600 ~0.6 with 0.02% arabinose for
4 h. Cells were harvested by centrifugation and washed once in
buffer containing 50 mM Tris, pH 7.5, 1 mM
EDTA, 5 mM
Purification of the mutant CocO Refolding of Citrate Synthase--
The procedure for
chaperonin-dependent renaturation of pig heart citrate
synthase has been described previously (35). Briefly, citrate synthase
at 33 µM was denatured in 6 M guanidine
hydrochloride, 3 mM dithiothreitol, and 2 mM
EDTA for 30 min at 25 °C. Denatured citrate synthase was diluted to
0.2 µM into a renaturation mix containing 20 mM potassium phosphate, pH 7.4, 10 mM
MgCl2, 2 mM ATP, 1 mM oxaloacetic
acid and various combinations of the chaperonins at 4.2 µM (concentration given for monomers) as indicated under "Results." The refolding reactions were performed at 27 °C in a
total volume of 400 µl, and citrate synthase activity was measured after 60 min. The enzyme activity was assayed by measuring the decrease
in absorption at 232 nm due to the cleavage of acetyl CoA and the
utilization of oxaloacetic acid.
Dependence of RB49 on groEL--
Both polymerase chain reaction
amplification and DNA hybridization experiments with the pseudo-T-even
bacteriophage RB49 suggested that this bacteriophage has no gene
31 homolog with significant DNA sequence similarity to the
T4 gene (data not shown). Thus, we determined if RB49 growth is
dependent on the host GroEL and GroES functions. We found that, like
bacteriophage T4, bacteriophage RB49 is totally dependent on the
E. coli GroEL function for successful completion of its life
cycle but is not dependent on GroES function. Table
II shows that wild type RB49, which
plates with an efficiency of 1.0 on the wild type host, does not form
plaques on either the E. coli groEL44 or groEL515
mutant hosts but does propagate on the groEL140 and
groES42 hosts. Since bacteriophage T4 plaques normally on
groEL515, it appears that RB49 is more sensitive to the
in vivo biological effects of this mutation.
Isolation of RB49 Mutations in the Putative Gene 31 Homolog--
In analogy with the isolation of the bacteriophage T4
31
We obtained further evidence for a Gp31 homolog in RB49 by showing that
the T4 gene 31 product complements the Cloning and Genetic Properties of cocO--
To identify the RB49
gene where the
We transformed the entire library into the E. coli groEL44
mutant, which is permissive for RB49
Besides the ability to recombine with the Isolation of an Amber Mutation in the cocO Gene--
Following the
successful cloning of the cocO+ gene, it was
transformed into CG3014 sup+ bacteria. We tested
the resulting CG3014 (pcocO+34-2) strain for
permissiveness toward a collection of RB49 amber mutants that were
isolated on the basis of propagating on CR63 supD bacteria
but not on the CG3014 sup+ host. We found that
of all the RB49 amber mutants tested, only RB49 amE45 was
capable of growth on the CG3014 (pcocO+34-2)
bacteria. Thus, it was renamed RB49 cocOamE45. Not only did
the pcocO+34-2 plasmid enable normal growth of
RB49 cocOamE45 (Table III), but 1% of the progeny were wild
type RB49 recombinants. These results, taken together with those
described above, genetically demonstrate that the
pcocO+34-2 clone indeed carries the wild type
gene that corresponds to the RB49 The Sequence and Predicted Structure of the CocO Protein--
The
predicted sequence of the RB49 cocO gene product is shown in
Fig. 2. It encodes a 107-amino acid
residue protein with a predicted molecular mass of 11,732 Da and a
theoretical pI of 6.84. It is 34% identical and 55% similar at the
amino acid sequence level to T4 Gp31 (36). The
Alignment of the GroES, Gp31, and CocO amino acid sequences reveals a
stretch of 12 amino acid residues (residues 79-90 in CocO) conserved
between the two bacteriophage-encoded cochaperonins but absent in GroES
and the rest of its cochaperonin homologs. In the three-dimensional
structure of Gp31, this region constitutes an extra loop extending down
from the external face of the Gp31 dome (22).
Sequencing of the RB49 cocOamE45 mutation showed that it is
localized in codon 100, being a CAG to TAG transition mutation. Thus,
the corresponding CocOamE45 protein should be eight amino acids shorter
than wild type CocO when expressed in the CG3014 sup+ (nonsuppressing) bacterial host. The lethal
phenotype of this mutation clearly indicates that the last eight amino
acids of CocO are important for its correct functioning and/or assembly.
Purification of CocO Protein--
The CocO wild type protein was
overproduced and purified from bacteria carrying the
pFScocO+ plasmid (see "Experimental
Procedures" for details). Similarly, the CocO Functional Analysis of the CocO Wild Type and CocO Although the x-ray crystallographic structures of the GroEL
chaperonin and its GroES cochaperonin have been solved, both as a
complex and as individual proteins, many details of their interaction with each other and their substrates are not understood (2, 7, 8, 11).
The primary contact that GroES makes with GroEL has been localized to a
16-amino acid residue segment of GroES. This so-called mobile loop,
although relatively unstructured in free GroES, adopts a Genetic and biochemical studies have identified the bacteriophage
T4-encoded Gp31 protein (product of gene 31) as essential for the correct folding of Gp23 (product of gene 23), the
major bacteriophage capsid protein (39-42). In the absence of a
functional gene 31, the Gp23 capsid protein aggregates in
lumps on the E. coli inner membrane (39). The realization
that some groEL mutations (but none of the groES
mutations) block bacteriophage T4 morphogenesis by interfering with the
action of Gp31 led to the proposal that Gp31 may serve as a more
specialized GroES-like homolog, capable of assisting the correct
folding of Gp23 by GroEL (14, 43) despite the fact that the GroES and
Gp31 proteins share little sequence identity (~14%; Refs. 14-16).
Gp31 is indeed a cochaperonin for GroEL; it substitutes for GroES in
protein folding, binds GroEL, and modulates its ATPase activity as
GroES does and even suppresses the temperature-sensitive phenotype of
groES mutations (21). But only Gp31, and not GroES, can
assist in correctly folding Gp23 in vivo (21, 44).
What then is the feature(s) of Gp31 that distinguishes it from GroES?
Hunt et al. (22) emphasized four structural features that
distinguish Gp31 from GroES and all of the other GroES-like cochaperonins. First, since Gp31's mobile loop is substantially longer
(22 versus 16 amino acids) compared with that of GroES (10,
19), this may result in the formation of a "taller" dome structure
over GroEL. Such a larger space in the GroEL-Gp31 cavity could better
accommodate Gp23, a relatively large substrate at 56 kDa. A large
mobile loop is also a feature of the CocO cochaperonin (Fig. 2), which
is actually one amino acid longer than that of Gp31. The Hunt et al. (22) also noted the presence of a universally
conserved aromatic amino acid residue at position 71 in GroES and its
homologs. This residue juts into the central cavity of GroES and limits
the volume available within the GroEL-GroES complex for substrate
folding. This aromatic amino acid is absent from the equivalent
position in both Gp31 and the CocO cochaperonin (Fig. 2). Another
feature that distinguishes both Gp31 and CocO from GroES and the other
cochaperonins is the lack of a "roof loop" structure at the top of
the GroES dome (Fig. 2). Again, this difference could allow Gp31 and
CocO to fold a larger substrate, such as Gp23. Finally, both Gp31 and
CocO possess an extra loop at their C-terminal region that is absent in
GroES and all other GroES-like cochaperonins. In the Gp31 crystal
structure, this extra loop domain is located outside of the dome.
Although it is not clear what role it plays in Gp31 and CocO
cochaperonin function, deletion of this loop domain abolishes Gp31
specificity for Gp23 but not for its E. coli protein
substrates (45).
Despite the various structural differences between the
bacteriophage-encoded Gp31(CocO) cochaperonin class and the GroES-type cochaperonins, the molecular mechanism(s) that underlies the unique capacity of the Gp31(CocO) cochaperonin to fold the Gp23 capsid protein
remains enigmatic. The T-even bacteriophages assemble an icosahedral
head structure composed of 960 Gp23 subunits (46). If one assumes a
burst size of 200 bacteriophage particles per infected bacterium, then
~200,000 Gp23 subunits must mature during the short time (10-20 min
at 37 °C) available for virion morphogenesis. In addition to being
relatively large, the Gp23 capsid protein also has a tendency to
aggregate, as judged by the formation of lumps in the absence of Gp31
(39). Thus, Gp23 may necessitate many cycles of GroEL binding and
release for its proper maturation. It could be that the
Gp31(CocO)-GroEL folding cycle is substantially shorter than
that of GroES-GroEL, thus favoring the fast and efficient maturation of Gp23. Another possibility is that the Gp31(CocO) cochaperonin can bind to the trans ring of GroEL more
efficiently than GroES and, in doing so, accelerate the release of Gp23
from the cis ring. Finally, the size and biochemical
properties of the Gp31(CocO)-GroEL cavity could be specifically
customized for Gp23 folding. Of course, none of these possibilities are
mutually exclusive, and they could all contribute to the correct and
rapid maturation of Gp23. Whatever this difference between Gp31 (CocO) and GroES may be, it is clear that the Gp31(CocO) cochaperonin class is
capable of folding all of E. coli's essential GroEL
substrates, since either Gp31 or CocO can totally substitute for GroES
in bacterial growth.2 Thus,
Gp31 (CocO) must maintain all features necessary for the correct
folding of many polypeptide substrates. Clearly, more experiments are
necessary, such as domain swap experiments, to pinpoint the Gp31 (CocO)
features that enable preferential Gp23 folding.
One aspect of this work deserves further comment. Here, we have studied
the structure and function of the Gp31 protein of an evolutionarily
distant T4-type bacteriophage and compared its properties with those of
the T4 homolog and the E. coli ortholog. There is extremely
little sequence homology between bacteriophage T4 Gp31 and E. coli GroES, although their cochaperonin functions are very
similar. More sequence homology is apparent between GroES and RB49 CocO
sequences, particularly in the N-terminal portion of these proteins. As
Gp31 homologs are analyzed from additional distant T4-type
bacteriophages, we anticipate that their evolutionary relationship to
the GroES protein will become increasingly obvious. Such an expanded
phylogenetic analysis of Gp31 could also provide valuable information
about the motifs and domains that are critical for function and thus conserved.
22, which permits growth on the E. coli groEL44 mutant but not on the isogenic wild type host. RB49
22 was used in marker rescue experiments to identify the
corresponding wild type gene, which we have named cocO
(cochaperonin cognate). CocO has extremely
limited identity to GroES but is 34% identical and 55% similar at the
protein sequence level to T4 Gp31, sharing all of the structural
features of Gp31 that distinguish it from GroES. CocO can substitute
for Gp31 in T4 growth and also suppresses the temperature-sensitive
phenotype of the E. coli groES42 mutant. CocO's predicted
mobile loop is one residue longer than that of Gp31, with the
22
mutation resulting in a Q36R substitution in this extra residue. Both
the CocO wild type and
22 proteins have been purified and shown
in vitro to assist GroEL in the refolding of denatured
citrate synthase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
growth (
requires both GroEL and
GroES host functions for growth (10, 18)). Similarly, T4 gene
31 mutations, which allow T4 to propagate on certain
E. coli groEL mutants, also affect the Gp31 mobile loop (14,
19, 20).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Bacterial strains, bacteriophage, and plasmids
(32) in which digestion
by XbaI was used to remove the
fragment encoding
spectinomycin resistance), which had been digested with
BglII. The ligation reaction was transformed into competent
DA1368 (MC1061
endA::tetR)1
bacteria, and transformants were selected on LB-agar-ampicillin plates
at 37 °C.
22 allele was
cloned in the same manner, using RB49 cocO
22 DNA as
template, resulting in plasmid pFScocO
22.
-mercaptoethanol, and 15% (v/v) glycerol.
The pellet was stored at
20 °C and gently thawed before lysis.
Cells were resuspended in buffer A (10 mM Tris, pH 7.7, 100 mM NaCl, 1 mM EDTA, 1 mM
-mercaptoethanol) and lysed by two passes through a French press at
1,000 p.s.i. The following steps are adapted from the purification
procedure used for Gp31 (21). After removal of the debris by low speed centrifugation (10 min at 10,000 × g), followed by
ultracentrifugation for 60 min at 145,000 × g, the
supernatant was fractionated by a streptomycin sulfate (5% of 25%
(w/v)) precipitation. The CocO-containing supernatant was further
fractionated by a 35% (w/v) ammonium sulfate precipitation. Again,
CocO remained in the supernatant. CocO was dialyzed into buffer B (20 mM Tris, pH 7.7, 1 mM EDTA, 1 mM
-mercaptoethanol) and applied to a Q-Sepharose column, which binds
acidic proteins. Because of its neutral pI, CocO eluted in the
flow-through. After a 70% (w/v) ammonium sulfate precipitation, CocO
was resuspended and dialyzed into buffer C (20 mM sodium
phosphate, pH 6.8, 5 mM
-mercaptoethanol, 1 mM EDTA) and loaded onto an hydroxyapatite column to which
a 20 mM to 0.5 M sodium phosphate, pH 6.8, gradient was applied. CocO eluted at ~0.2 M sodium
phosphate. After exchanging into buffer D (100 mM Tris, pH
7.5, 1 mM EDTA, 1 mM
-mercaptoethanol), final impurities were removed by gel filtration (Hiload 16/60 Superdex
G200 on a Waters HPLC). CocO eluted at 16 min, suggesting an
approximate native molecular mass of 80,000 Da.
22 protein was executed following its
overproduction from plasmid pFScocO
22, using the exact steps described for the wild type protein.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Plating properties of various bacteriophages on various bacterial hosts
at 37 and 42 °C
6.
1 mutant (23, 31), spontaneous mutants of RB49 can be
isolated at a frequency of ~5 × 10
8
that grow on groEL44 mutant bacteria. One such isolate, RB49
22, forms wild type-size plaques on the E. coli groEL44
mutant host at 37° (Table II). It was found that RB49
22 cannot
form plaques on either wild type, groEL140, or
groES42 hosts at 37 °C; however, it does form plaques on
these same hosts at 42 °C. For comparison, we include the T4 mutant
T4 31
1 in Table II, which was originally isolated as a
spontaneous plaque former on groEL44 (31). Previous analysis
has shown that the T4 31
1 mutation results in an L35I
substitution in the middle of the mobile loop of Gp31 (14, 19, 23).
22 mutation (Table
III), allowing bacteriophage RB49
22
growth on the restricting E. coli wild type host at
37 °C. In contrast, a plasmid overexpressing both the E. coli
groEL and groES wild type genes does not complement the
22 mutation (data not shown). Earlier results had shown that overproduction of groES also does not complement the T4
31
1 defect (21).
Ability of plasmid-encoded Gp31 and CocO proteins to specifically
complement the growth of bacteriophage 31 or cocO mutants
22 mutation resides, we constructed an RB49 DNA
library in the multicopy plasmid pMPMA4. Although the construction of
the RB49 DNA library was initiated with ~1.5-5.0-kb-long DNA
fragments, the resulting library was enriched in small DNA insert
fragments, probably because of the toxicity of many of the RB49 DNA
sequences in E. coli.
22 and used this library in a
marker rescue scheme. Since it was unclear whether the wild type clone
corresponding to
22 would be underrepresented in our RB49 DNA
library, we started with an initial pool of ~105
independent groEL44 transformants obtained with plasmid DNA
purified from the original amplified library (itself consisting of
~106 independent clones). This pool was divided into 45 groups each containing ~2,000 transformants and allowed to amplify.
RB49
22 was grown on these 45 groups of transformants, and 0.1 ml of
each lysate was plated on the nonpermissive groEL140 mutant
strain at 37 °C (Table II) to detect the presence of RB49 wild type
recombinants or revertants. Whereas most of the transformant pools gave
fewer than 20 plaques per plate, five of them gave significantly higher numbers, ranging from 50 to 200. The marker rescue scheme was repeated
(five independent lysates each) for two of these pools, confirming that
the elevated number of plaque formers (putative wild type recombinants)
was not due to statistical fluctuation. One of these pools was chosen
for further screening and, by repeating the marker rescue experiment
with progressively smaller pools of transformants (250, 10, and finally
individual transformants), we progressively obtained higher frequencies
of RB49 wild type recombinants. Thus, we were able to eventually
isolate a single transformant carrying a plasmid
(pcocO+34-2) encoding the RB49 wild type allele
that corresponds to the
22 mutation and which we have named
cocO (cochaperonin cognate).
22 mutation, the following
experiments strongly suggested that the
pcocO+34-2 clone carries a bona fide
Gp31 homolog. First, the presence of pcocO+34-2
enables both the T4 31amNG71, which carries an amber
(nonsense) mutation in gene 31, and RB49
cocO
22 mutant bacteriophages to propagate on their
otherwise restrictive wild type host, CG3014 sup+, which lacks a nonsense suppressor. Second,
we could show that plasmid pcocO+34-2 suppresses
the temperature-sensitive phenotype of the E. coli groES42
mutant, as was previously shown with the cloned T4 gene 31 (Fig. 1; Ref. 21).
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Fig. 1.
Suppression of the groES42
temperature-sensitive growth phenotype by the RB49 cocO
gene. Strain DA1415 (groES42) was transformed
with either plasmid pMPMA4 (control), p31+
(expressing wild type Gp31), or pcocO+34-2
(expressing wild type CocO). See "Experimental Procedures" for
details.
22 and amE45 alleles.
22 mutation is a
transition mutation, resulting in the change of a glutamine codon (CAA)
to arginine (CGA) at amino acid position 36 (Q36R). Neither Gp31 nor
GroES have this particular residue in their mobile loop. In analogy with Gp31, the predicted mobile loop of CocO is longer than that of
GroES and contains the highly conserved glycine and the three consecutive hydrophobic residues found at positions 32-34 in CocO (Fig. 2). Similar to Gp31, CocO encodes neither the roof loop (composed
of residues 48-55 in GroES) nor the tyrosine at position 71 of GroES,
both highly conserved features among the other GroES cochaperonin
homologs (22, 23). Crystallography of T4 Gp31 has led to the suggestion
that these modifications may result in a larger Anfinsen cage in the
GroEL-Gp31 complex (22). Perhaps this allows a better accommodation of
the 56-kDa Gp23 capsid protein, which is at the upper limit of the size
permitted by the GroEL-GroES complex.
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Fig. 2.
Comparison between Gp31, CocO, and GroES
predicted amino acid sequences. The alignment between Gp31 and
GroES is taken from the article by Hunt et al. (22).
Alignment between CocO and Gp31 was performed using BLAST (36).
22 mutant protein was
overproduced and purified from bacteria carrying the
pFScocO
22 plasmid. Both proteins were at least 90% pure,
as judged by staining of the purified protein preparations following
separation by SDS-polyacrylamide gel electrophoresis (Fig.
3A). The elution profiles of
both proteins during HPLC gel filtration suggest an approximate
molecular mass of 80,000 Da, consistent with their putative heptameric
forms (data not shown).
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Fig. 3.
Purification and analysis of the
bacteriophage RB49 CocO protein. A, 15% (w/v)
SDS-polyacrylamide gel electrophoresis analysis of purified CocO wild
type protein (a), CocO 22 mutant protein (b),
and T4 Gp31 wild type protein (c). Molecular weight markers
were low range prestained SDS-polyacrylamide gel electrophoresis
standards from Bio-Rad. See "Experimental Procedures" for details
of the overproduction and purification of the various proteins.
B, chaperonin-dependent refolding of citrate
synthase. The yield of folded protein is expressed as a percentage of
the activity determined for an equal quantity of native citrate
synthase (nCS). Citrate synthase activity was measured after
1 h of refolding at 27 °C with the indicated combinations of
chaperonins. The presented data are the average of three separate
experiments, and error bars indicate the S.E.
dCS, denatured citrate synthase diluted into refolding
buffer in the absence of chaperones.
22
Proteins--
To test whether the purified CocO wild type and
CocO
22 proteins are active and capable of interacting with their
GroEL partner, we carried out in vitro folding assays with
denatured citrate synthase. Previously, it was established that the
correct in vitro folding of pig heart citrate synthase
requires both the GroEL and GroES proteins (37, 38). As shown in Fig.
3B, the CocO cochaperonin is indeed capable of assisting
wild type GroEL in the folding of denatured citrate synthase. Even more
convincing are the observations that (a) the wild type CocO
protein cannot assist the GroEL44 mutant protein in citrate synthase
folding, thus reproducing the in vivo inability of
bacteriophage RB49 to propagate on the groEL44 mutant host
(Table II), and (b) the CocO
22 mutant protein restores
the ability of the GroEL44 mutant protein to fold citrate synthase,
again in agreement with the in vivo result that the
bacteriophage RB49 cocO
22 mutant grows perfectly well on
the groEL44 mutant host (Table II). These experiments have
been performed using various chaperonin/cochaperonin ratios, with
essentially the same results (data not shown). In contrast to the
in vivo bacteriophage plating phenotype (Table II), the CocO
22 mutant protein was capable of assisting the wild type GroEL
protein in citrate synthase folding. This result is not entirely
unexpected, since bacteriophage RB49 cocO
22 can form plaques on CG3014 wild type bacteria at high temperatures, thus demonstrating significant interaction between GroEL wild type and
CocO
22 proteins, at least at high temperature (Table II). Most
likely, citrate synthase, although a relatively large substrate (monomer is 51,629 Da), is not as problematic to fold as the RB49 major
capsid protein.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hairpin
structure upon binding to GroEL (10). In the crystal structure, the
only direct contact that GroES makes with GroEL is with a conserved,
hydrophobic tripeptide located in the middle of its mobile loop (10,
11). The contributions of other elements to both the initial binding of
GroES to GroEL and to the subsequent stabilization of the GroEL-GroES
complex are unknown. Another detail that remains to be clarified is
whether GroES binding to the trans ring of GroEL can take
place while GroES is still bound to the cis ring
("footballs") and whether such binding may be required for the
efficient release of some substrates from the cis ring of GroEL.
22 mutation
(Q36R) that results in an efficient interaction with the GroEL44 mutant
chaperonin actually alters this extra residue in CocO. Previous
biochemical analyses have shown that Gp31 and GroEL44 do not
efficiently form a complex in vitro (20). Thus, CocO and
GroEL44 could suffer from a similar defect, which is overcome by the
Q36R substitution of CocO
22. The inability of the GroEL44-CocO wild
type pair to correctly interact in vivo could be reproduced
in vitro where they are unable to refold citrate synthase.
Furthermore, the GroEL44-CocO
22 pair of mutant proteins that
functions normally in vivo for bacteriophage RB49
morphogenesis also correctly folds citrate synthase in
vitro. In this respect, it is interesting that the only mutation
of bacteriophage T4 gene 31 that is capable of restoring
substantial interaction with GroEL44 is the T4 31
1
mutation, an L35I substitution in Gp31's mobile loop (23). This
substitution results in a more hydrophobic amino acid (isoleucine 35 for leucine) in the first position of the hydrophobic tripeptide of
Gp31. In contrast, the Q36R substitution in CocO's mobile loop enables
it to interact even better with GroEL44, as judged by the fact that T4
31
1 (L35I) grows on groEL44 hosts at 37 °C
but not at 42 °C, while RB49 cocO
22 (Q36R) grows on
groEL44 bacteria at both 37 and 42 °C (Table II). The
Q36R substitution is located two amino acid residues C-terminal to the
conserved hydrophobic tripeptide and may bypass the GroEL44 block by a
different mechanism than the L35I substitution of Gp31.
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ACKNOWLEDGEMENTS |
---|
We thank Eric Morency and "La science appelle les jeunes" for their catalytic role in isolating the RB49 cocOamE45 mutant; Françoise Schwager for excellent technical assistance; and Luli Billecchi-Mestre for cheerful editing of the manuscript.
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FOOTNOTES |
---|
* This work was supported by FN Grant 31-47283-96 and the Canton of Geneva. Research in Toulouse was financed by the CNRS and by grants from the Association pour la Recherche sur le Cancer and the Midi-Pyrénées Regional Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF303100.
§ To whom correspondence should be addressed. Tel.: 41-22-702-5514; Fax: 41-22-702-5502; E-mail: deborah.ang@medecine.unige.ch.
¶ Present address: Inst. for Biochemistry and Molecular Biology, Albert-Ludwig University of Freiburg, Herman-Herder-Str. 7, D-79104 Freiburg, Federal Republic of Germany.
Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M008477200
2 F. Keppel and C. Georgopoulos, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: tetR, tetracycline-resistant; HPLC, high pressure liquid chromatography.
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