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J Biol Chem, Vol. 273, Issue 51, 34075-34086, December 18, 1998
andFrom the Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, Maryland 21201
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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The bacteriophage T4 GroES homologue, gp31, in
conjunction with the Escherichia coli chaperonin GroEL, is
both necessary and sufficient to fold the T4 major capsid protein,
gp23, to a state competent for capsid assembly as shown by in
vivo expression studies. GroES is unable to function in this role
as a productive co-chaperonin. The sequencing and characterization of
mutations within gp23 that confer GroEL and gp31 chaperonin-independent
folding of the mutant protein suggest that the chaperonin requirements
are due to specific sequence determinants or structures in critical
regions of gp23 that behave in an additive fashion to confer a
chaperonin bypass phenotype. Conservative amino acid substitutions in
these critical regions enable gp23 to fold in a GroEL-gp31
chaperonin-independent mode, albeit less efficiently than wild type,
both in vivo and in vitro. Although the
presence of functional GroEL-gp31 enhances folding of the mutated gp23
in vivo, GroEL-GroES has no such effect. Site-directed
mutagenesis experiments suggest that a translational pausing mechanism
is not responsible for the bypass mutant phenotype. Polyhead reassembly
experiments are also consistent with direct, post-translational effects
of the bypass mutations on polypeptide folding. Given our finding that
gp31 is not required for the binding of the major capsid protein to
GroEL and that active GroES is incapable of folding the gp23
polypeptide chain to native conformation, our results suggest
co-chaperonin specificity in the folding of certain substrates.
GroEL and its co-chaperonin, GroES, are stress-inducible proteins,
synthesized from the groE operon of Escherichia
coli (1, 2). GroEL and GroES are members of the chaperonin or
Cpn60/Cpn10 family of proteins and are thought to be general catalysts
unrestricted in ability to fold proteins by interacting reversibly with
polypeptides to passively prevent or deter incorrect protein folding
and aggregation (3, 4). The mechanism of chaperonin-mediated
polypeptide folding involves the interaction of GroEL with non-native
polypeptide, subsequent GroES association, ATP hydrolysis, and release
of the native protein. Both GroEL and GroES are required for E. coli growth at all temperatures, and GroEL has been shown to bind
up to 50% of all E. coli proteins in vitro
suggesting that the chaperonins have the ability to interact with a
wide array of polypeptide substrates (5, 6).
GroEL is initially synthesized as a 60-kDa monomer, which oligomerizes
into a decatetramer double-donut structure, two stacked rings each with
7-fold symmetry (7, 8). The GroEL double donut structure has the
dimensions 14.5 × 16 nm with an inner diameter of 6 nm which
enable it to accommodate a 35-kDa protein in the central cavity prior
to GroES binding (9). The GroEL co-chaperonin, GroES, forms a single,
seven-membered, domed ring structure consisting of identical 10-kDa
subunits (10-13). Mutational analysis of the GroEL apical domain,
which faces the opening of the central cavity in the GroEL toroid,
indicates an overlap in polypeptide GroES-binding sites (14), and
cryoelectron microscopy structural studies show major conformational
changes associated with the addition of Mg-ATP and GroES that
effectively increase the size of the GroEL folding chamber to
accommodate the folding of proteins approximately 60 kDa in size (12,
15). These experiments suggest a critical role for GroES as an
intricate player in the binding and release of the polypeptide
substrate. However, in vitro experiments demonstrate
GroEL-mediated folding of some proteins in the absence of the
co-chaperonin (16-18), thus in vitro assessment of
the co-chaperonin requirement for folding a particular substrate might
be misleading as compared with in vivo determination. In fact, the significance of the co-chaperonin in GroEL-mediated protein
folding remains to be fully understood.
In addition to their role in the folding of E. coli
proteins, GroEL and GroES are important components in phage assembly
pathways. Unlike bacteriophages lambda (1), T5 (19), Mu (20), HK97 (21), and PRD1 (22) which require both GroEL and GroES for growth,
bacteriophage T4 is not affected by any of the known missense mutations
in the gene encoding the E. coli GroES co-chaperonin. During
bacteriophage T4 infection the major capsid protein is synthesized as
56-kDa monomers that form a lattice structure shown to be composed of
hexameric arrays of gp23. Before 1970, it was determined that
bacteriophage T4 synthesizes a bacteriophage protein that is required
to assemble its major capsid protein, gp23, into head structures;
without this factor what would now be called inclusion bodies of the
major capsid protein accumulate in vivo precluding the
formation of proheads (23). This factor, identified as gp31,
participates together with the E. coli Cpn60 protein, GroEL,
in the correct folding of gp23 (24-28). Several lines of evidence
support the interaction of GroEL, gp31, and the T4 major capsid protein
during head assembly. A gp23-gp31 interaction is suggested by
T4bypass31 phage, which contain mutations in the gene
encoding the major capsid protein that appear to obviate the gp31
requirement (29). Specific missense mutations in genes 31 and groEL block T4 propagation at the same point
in the head morphogenesis pathway; no head structures are assembled and
the immature, uncleaved form of gp23 is found to aggregate. Also, certain missense mutations in gene 31 are able to suppress
groEL mutations that normally block T4 capsid assembly in an
allele-specific manner. Thus, during T4 infection, gp31 appears to
replace GroES as the co-chaperonin for GroEL (30, 31).
The nature of the interaction between chaperonin and target protein
remains to be determined. The literature suggests this interaction is
both indiscriminate and sequence-independent (4). The unique adaptation
of the E. coli chaperonin system by bacteriophage T4 might
provide an alternative route to study the role of the co-chaperonin in
chaperonin-mediated protein folding. However, the specificity
requirement for T4 gp31 in conjunction with GroEL to fold gp23 has not
in fact been established. T4 infection inactivates E. coli
RecA and Rec BCD and replaces these proteins with others of apparently
equivalent catalytic properties, gpuvsX and
gp46/47. Other E. coli enzymes (e.g.
DNA polymerase, DNA ligase, valyl-tRNA synthetase) are bypassed or
altered for phage enzymes of comparable catalytic properties, sometimes
for unknown reasons, whereas phage enzymes of novel specificity
(e.g. RNA ligase) are also synthesized (59). We thus
proposed to determine by in vivo expression studies the
basis for the phage T4 gp31 requirement as follows: whether this is a
consequence of T4-imposed changes to the infected cell (e.g.
GroES inactivation during infection, limiting functional groES due to host protein synthesis shut-off, increased
folding requirements during viral assembly requiring enhanced
catalysis, etc.) or whether there is indeed specificity in the
GroEL-gp31-mediated folding of the substrate T4 major capsid protein
that requires a special factor despite evidence that the E. coli Cpn60-Cpn10 system is capable of generic protein folding.
In this paper, we use expression vectors to show that co-expression of
gene 31 with gene 23, in the presence of
wild-type GroEL, is necessary and sufficient to form polyhead
structures in vivo and that GroES is apparently unable to
function in this role. Polyheads, which are open tubes formed
exclusively of gp23, require proper folding and oligomerization of the
major capsid protein and are thought to display the same structure
found in active T4 proheads (Fig. 2E; cf. Ref.
32); polyhead formation is thus indicative of folded gp23. Although
folding and polymerization of the wild-type major capsid protein
strictly require GroEL and gp31, we show that polyheads are produced in
the absence of both proteins by T4 phage containing two types of known
bypass31 mutations in the major capsid protein, both
in vivo and in vitro. Characterization and
analysis of T4bypass31-2 mutant phage, which are able to
grow in the absence of chaperonins at high temperature (>40 °C),
identify three missense mutations that confer chaperonin-independent
folding to the mutant gp23 protein in an additive fashion. These data suggest that the ability of the gp23BY protein to fold
independently of chaperonin assistance is tightly associated to certain
localized sequence determinants within the major capsid protein.
Mutagenesis of one of these sites allows us to investigate the
specificity of amino acid replacements that confer the phenotype,
determine its chaperonin independence, and investigate a proposed
mechanism of translational pausing (33, 34) which might promote protein
folding in the absence of chaperone assistance.
Materials--
Molecular biology reagents were purchased from
either Boehringer Mannheim, New England Biolabs, or Life Technologies,
Inc. Antibodies against GroEL and GroES were purchased from Sigma; antisera against T4 gp31 and gp23 were described previously (24, 32). Tran35S-label was purchased from NEN Life
Science Products.
Bacterial and Phage Strains--
The bacteria and phage strains
used are listed in Table I. E. coli B40 su+I, B40 su+II, CR63, LE392,
Be supo, and P301 supo were used as
permissive and non-permissive hosts for T4 amber mutants. Other
suppressor strains, which were kindly provided by J. Miller (see Ref.
35), were used in the analysis of the bypass31-2 site
presented in this work. pTrc99A (Amersham Pharmacia Biotech) and
pET3a-derived plasmids (Novagen) were transformed and expressed in
HMS174(DE3), BL21(DE3), and their non-DE3 derivatives (Novagen).
T4D+ was the T4 wild-type strain. A phage mutant is
described by its gene number, followed by the type of mutation and the
name of the specific mutation. Multiple mutations are denoted the same way with a hyphen between mutations. Other phage strains used in these
experiments include the following: Plasmid Construction and Site-directed
Mutagenesis--
Constructs were made by inserting the target T4 gene
downstream of the T7 promoter in either the pET3a (Novagen) expression vector using the NdeI and BamHI restriction sites
or the pTrc99A (Amersham Pharmacia Biotech) expression vector using the
XbaI restriction endonuclease site.
PCR1 primers, synthesized by
the Biopolymer Center at the University of Maryland, Baltimore, School
of Medicine, complementary to upstream and downstream sequences of T4
gene 23 were used to introduce AseI and
BamHI restriction enzyme sites and to isolate the gene from
T4+ or T4bypass31-1-2 mutant DNA. Constructs
were made by inserting the target T4 gene into the pET3a (Novagen)
expression vector, creating pET23 and pETBY, respectively. Isolating an
AseI-BglII fragment containing gene 31 from M13mp11-TR5 and directly cloning into the pET3a vector as
described above (30) constructed pET31. An XbaI fragment
from this vector, containing gene 31 and its Shine-Delgarno
sequence, was then directly cloned into pTrc99A (Amersham Pharmacia
Biotech), creating pTrc31. The double construct was made by inserting a
BglII-EcoRI fragment-containing gene
31 from pET31 into pET23 previously cleaved by
BamHI and EcoRI. Although the T7 promoter is used
to initiate transcription, the natural Shine-Dalgarno site is
maintained for each gene. Individual plasmids were transformed into the
E. coli groEL44 strain CG2241, which is non-permissive for
T4 growth at 37 oC or above and whose target plasmids can
be induced by Protein Expression and Isolation of Polyhead Structures--
For
plasmid expression and polyhead isolation experiments,
plasmid-containing bacteria were grown to an
A600 of 0.6 in M9S media supplemented with
ampicillin (150 µg ml In Vitro Denaturation and Refolding of the T4 Major Capsid
Protein--
Once isolated, polyheads consisting of either wild-type
gp23 or gp23BY were dialyzed against buffer B (1 mM KPO4, pH 7, 1 mM
MgSO4) at 4 °C. Polyheads are destabilized under these
conditions and depolymerize (40). Samples were then centrifuged at
18,000 rpm in an SS34 rotor for 20 min at 4 °C. The purpose of this
step was to pellet any intact polyhead structures and to take only the
soluble proteins in the supernatant for subsequent analysis. The
supernatant was observed by electron microscopy and was found not to
contain any polyhead structures. Half of the sample was used for
reassembly and half for denaturation/renaturation experiments. Urea was
added to the depolymerized gp23 (wild type and gp23BY) to a
concentration of 6 M. The sample was incubated for 45 min, and the urea was then slowly dialyzed away with buffer B to initiate polypeptide renaturation. Once the urea content reached micromolar concentrations, the dialysis buffer was diluted 1:10 with fresh buffer
B. Polyhead reassembly was initiated by dialyzing the sample against 25 mM KPO4, pH 7, 1 mM
MgSO4 at room temperature for 1 h. Over a period of
3 h the KPO4 concentration in the dialysis buffer was
increased to a final concentration of 100 mM. Samples were
applied to previously prepared electron microscope grids, stained with
either 1% phosphotungstic acid or uranyl acetate, and observed in the
electron microscope to determine whether polyheads were formed.
Electron Microscopy--
Methods for the fixation, dehydration,
embedding, and thin sectioning of bacteria are a slight modification of
previously published procedures (41). Polyhead-containing samples were preserved in 1% final concentration of glutaraldehyde and negatively stained with 1% uranyl acetate for observation.
Complementation Experiments--
Bacteriophage growth on the
groES strain, JZ483, and the suppressor minus strain,
Be, was determined under various conditions by calculating
lambda, T4, or site-directed mutant phage burst sizes. The burst size is given as the number of viable phage progeny produced per infected bacterium. JZ483 bacteria containing either pEGS1 or pTrc31 plasmids were induced by the addition of 4 mM IPTG at an
A600 of 0.4 for 1 h at 37 °C. Bacteria
were then infected with either lambda or T431(AmNG71) phage at an m.o.i. (multiplicity of
infection) of 0.1. The phage were allowed to adsorb to the bacteria for
7 min after which antibody directed against either lambda or T4 phage was added. Bacteria were diluted and incubated for 90 min followed by
the addition of chloroform to release progeny phage. T4 phage were then
titered on the suppressor minus strain Be, and the
suppressor plus strain CR63 and lambda phage were titered on LE392 to
determine the phage yield per infected cell.
Immunoprecipitation and Radiolabeling Experiment--
E.
coli Be were grown at 37 °C in M9 medium (39) to an
A600 of 0.3 which corresponds to ~2 × 108 bacteria/ml and infected with one of the following
phage: T423 (AmB2), T4+,
T431(Am NG71)31(AmN54), or
T4bypass31(1+2)-31(AmN54)31(AmNG71) at an m.o.i. of 5, or with no phage. Bacteria were
superinfected with the same m.o.i. at 8 min.
Tran35S-cysteine-methionine label (NEN Life Science
Products) was added to the media at 10 µCi/ml just prior to infection
to label GroEL protein in the uninfected bacteria and at 10 min
post-initial infection to label late T4 structural proteins in
T4-infected bacteria. After 15 min post-initial infection (gp31
synthesis ceases by 15 min (24)), bacteria were placed in an ice-water bath, and excess non-radioactive methionine and cysteine were added
together with a casein amino acid (CAA) mixture (0.2 ml 20% CAA/ml
infected cell culture). Infected bacteria were then pelleted in a
Sorvall centrifuge (5,000 rpm in a SS-34 rotor for 8 min at 4 °C).
Cell pellets were then stored in a Immunoprecipitation of Uninfected Extracts of Expression
Vector-containing pET23, pET2331, and pETBY--
The procedures
described for immunoprecipitation of radioactive, infected bacterial
extracts were followed after induction of the T4 proteins for
3.5 h, 15 min, or 12 min with IPTG, except that bacterial pellets
were first lysed with egg white lysozyme for 10 min at room
temperature, and ADP was used rather than ATP in the lysis and
immunoprecipitation solution. Immuno-detection employed the ECL
technique (Amersham Pharmacia Biotech).
Identification of Essential Components Required for the Proper
Folding and Oligomerization of the T4 Major Capsid Protein--
In
order to study the role of gp31 as a T4 co-chaperonin, T4 genes
23 and 31 were cloned independently into the
pET3a expression vector. Four plasmids were created as follows: one
containing the wild-type gene 23 sequence (pET23), one
containing the mutant T4bypass31(1+2) gene 23 (pETBY), one containing gene 31 (pET31), and one plasmid
containing both wild-type genes 23 and 31 in
tandem (pET2331)(cf. "Experimental Procedures").
Individual plasmids were transformed into HMS174(DE3), a
groEL wild-type strain, and CG2241, a groEL44
strain which is non-permissive for T4 growth at 37 °C. The bacteria
were induced with IPTG or
Expression of gene 23WT and gene
23BY in HMS174(DE3) and CG2241 bacteria from
pET23, pET2331, and pETBY constructs is shown in Fig.
1A and was confirmed by
Western blot analysis with anti-gp23 antibody (data not shown).
Polyheads isolated by differential centrifugation of cell lysates (40)
were analyzed by SDS-PAGE and Western blot analysis (Fig. 1,
B and C). Lanes 2 and 3 of Fig. 1, B and C, show that the formation of gp23
polyhead structures in a wild-type host (i.e. bacteria
containing normal levels of GroEL and GroES) requires the co-expression
of gene 31. In contrast, polyheads are formed by the mutant
gp23, gp23BY, in a wild-type host, even in the absence of
gene 31 co-expression (Fig. 1, B and C,
lane 4).
When pET23, pET2331, and pETBY plasmids were induced to express their
target genes in the GroEL-deficient strain, CG2241, at the
non-permissive temperature, only gp23BY expressed from the
pETBY plasmid was able to form polyhead structures in vivo
(Fig. 1, B and C, lanes 5-7). Gene expression
from all three of the plasmids was far lower in the groEL44
strain CG2241 at 37 °C, probably both because of the
Aggregation analysis (data not shown) (23, 42) and electron microscopic
observations of the induced plasmid-containing bacteria were in
complete agreement with the conclusions drawn from PAGE. Electron
micrographs of thin sectioned bacteria expressing gene
23WT from pET2331, pET23, or gene
23BY from pETBY in groEL+ and
groEL
The abundance of polyheads was markedly lower in HMS174(DE3) bacteria
expressing gene 23BY when compared with bacteria
co-expressing genes 23WT and 31.
Also, bacteria expressing gene 23BY appear as an
intermediate between bacteria expressing gene
23WT with and without the T4 co-chaperonin gp31;
they contain polyheads as well as inclusion bodies. This suggests that
folding and/or polymerization of gp23, synthesized from the
bypass31(1+2) mutant DNA, is less efficient than that of the
wild-type protein, and this is consistent with the proportionate
decrease in the T4bypass31 phage yield when compared with T4
wild type (Table IV (29)).
The T4bypass31 mutant phage appear to have simultaneously
lost the gp31 requirement and to have gained the ability to grow on the
E. coli groEL44 mutant CG2241 (29, 43). Table
II shows a more extensive analysis of
T4bypass31 phage growth on several groEL and
groES mutant strains. As determined previously, all of the
groEL and groES mutations affect lambda phage
viability, whereas wild-type T4 phage growth is only affected by some
mutations in the groEL gene (Table II) (1, 30, 31). The T4
phage containing a double amber mutation in gene 31 and
wild-type gene 23 is unable to grow in any of the mutant
strains because of the lack of gp31, confirming the T4 co-chaperonin
specificity requirement for gp31. Unlike wild-type T4, the
T4bypass31 single and double mutants are unaffected by any
of the groEL or groES mutations tested. This
suggests the T4bypass31-1 and T4 bypass31-2
mutations each confer changes in the structure of the major capsid
protein which allow it to fold independently of GroEL, GroES, and gp31. In vitro refolding studies using purified proteins also
support our in vivo conclusion that the bypass31
mutations confer chaperonin-independent folding of the major capsid
protein (cf. "Experimental Procedures). As expected, both
wild-type gp23 and gp23BY proteins form polyheads from a
depolymerized state (40, 42). However, gp23BY appeared to
more readily dissociate from polyheads in low ionic strength, low
temperature buffers (42). Although the yield was low, multilayered
polyhead structures, previously observed exclusively and
intracellularly in vivo, were observed upon renaturation of the purified gp23BY, but not the wild-type gp23 protein,
from 6 M urea in the absence of chaperonins (Fig.
2F).
Functional Differences of Cpn10 Homologues GroES and
gp31--
While our work was in progress, it was shown that gp31 can
act as a functional surrogate for GroES and as a general co-chaperonin during the reassembly of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) protein and during lambda and T5 infection (19). Thus it is a
distinct possibility that GroES and gp31 are functionally or
catalytically interchangeable and that gp31 is produced because available GroES protein is inactivated or limiting during T4 infection (cf. Introduction). If gp31 and GroES can both interact with
GroEL as well as with the same substrates with comparable efficiency, one might expect the overproduction of GroES protein to compensate for
a defective 31 protein during T4 infection. Complementation testing was
used to determine whether gp31 and GroES as over-produced from pTrc31
and pEGS1, respectively, are functionally interchangeable in
vivo. Both pTrc31 and pEGS1 were transformed into the
groES42 mutant JZ483 and induced with IPTG to express their
target genes. The bacteria were then infected with lambda or
T431(AmNG71) phage, and the burst size for each
phage was determined. Fig. 3A
shows lambda phage growth is rescued by the over-expression of
groES from pEGS1, approximately 92% of lambda phage growth
on a fully permissive strain. In agreement with previous findings (19), over-expression of gene 31 can also rescue lambda phage
growth although less effectively than GroES, approximately 8% of
control. In the case of T4 phage growth, however, over-expression of
groES does not rescue T431(AmNG71)
phage growth, approximately 0.002% of control (Fig. 3B).
Burst size measurements were also carried out in another
groES mutant, groES619, with similar results, and both expression vectors produced the cloned gp31 and GroES
co-chaperonins at very high levels following induction (42). These data
demonstrate that GroES and gp31 are not equally interchangeable.
Although gp31 can at least partially compensate for a defective GroES, it does not appear to rescue lambda phage growth as effectively as
GroES. Furthermore, overproduction of GroES does not compensate for a
gp31 deficiency, demonstrating that T4 specifically requires gp31 for
the proper folding and oligomerization of its major capsid protein,
gp23, consistent with the in vivo expression studies.
Interaction of Wild Type and bypass31 Mutant Major Capsid Protein
with GroEL--
Co-immunoprecipitation experiments using anti-GroEL
polyclonal antibody were designed to determine whether the wild-type
gp23 and the mutant gp23BY both interact with GroEL during
T4 infection and to determine whether gp31 is involved in the initial
binding of the gp23 polypeptide to GroEL. E. coli
Be bacteria were grown to an A600 of
0.3 in labeling media and either left uninfected or infected with one
of the following phage at an m.o.i. of 5:
T421(AmH29), a head assembly proteinase-deficient strain; wild-type T4; T431(AmNG71)-
31(AmN54); or
T4bypass31(1+2)-31(AmNG71)-31(AmN54). Bacteria were labeled with Tran35S-label at 10 min
post-infection for 5 min to specifically radiolabel T4 structural
proteins. Fig. 4A (lanes
1-5) shows abundant T4 late proteins labeled in whole
cell extracts including gp23 and gp23*, which is the N-terminal
processed form of the major capsid protein found in the mature prohead
structure. Immunoprecipitation of cell lysates with GroEL antibody
identified the major capsid protein as a T4 late protein that interacts
with the Cpn60 (Fig. 4A, lanes 6-10).
Co-immunoprecipitation experiments done in parallel without the
addition of GroEL antibody showed some nonspecific precipitation;
however, the amount of nonspecific intact gp23, wild-type or mutant,
was negligible (Fig. 4A, lanes 11-15). As host protein
synthesis ceases upon infection, bacteria were labeled prior to
infection to show precipitation of endogenous GroEL together with some
E. coli proteins possibly associated with GroEL (Fig. 4A, lane 10). Fig. 4A (lanes 7 and
9) shows that both the wild-type gp23 and the mutant
gp23BY co-immunoprecipitate with GroEL from the infected
cell lysates. Thus, the bypass mutations do not prevent binding of
gp23BY to GroEL, as expected from the better folding of
gp23BY in the presence of GroEL (42). Although this
gp23BY-GroEL interaction is not necessary for functional
folding of the mutant major capsid protein, the presence of functional
GroEL and gp31, but not GroES, enhances gp23BY folding
(42). The co-immunoprecipitation of wild-type gp23 with GroEL even in
the absence of gp31 (Fig. 4A, lane 8) suggests that gp31 is
not necessary for binding to GroEL but that it is required for
functional protein folding. Immunoblotting of the cell extracts and
co-immunoprecipitates with GroES and gp31 antisera shows that both
GroES and gp31 are present and are found to interact with GroEL during
T4 infection (Fig. 4B, lanes 6-15, and data not shown).
Immunoprecipitation experiments using extracts prepared following
induction of uninfected bacteria containing the pET23, pET22331, and
pETBY expression vectors yielded comparable results (data not shown),
i.e. gp23, and gp23BY were associated equally
with GroEL in the presence or absence of gp31, and GroES continued to
be associated with GroEL following induction of gp31 synthesis.
Our work clearly demonstrates the specificity of the gp31 requirement
for the folding of the T4 major capsid protein, gp23. As GroEL and
GroES intracellular levels are comparable, it is interesting to
speculate as to what occurs in vivo during T4 infection when
both GroES and gp31 are present. Electron micrographs of negatively
stained purified gp31 show a ringed structure with a central hole that
is similar in dimension to GroES (42). Assuming that gp31 forms a 7-mer
oligomeric structure, the number of gp31 molecules synthesized during
T4 infection can be calculated from immunoquantitation measurements
(24) to be approximately 7,800 gp31 septemers/cell. Intracellular
concentrations of GroEL and GroES have been determined previously to be
approximately 1,600 GroEL 14-mers/cell and 3,000 GroES 7-mers/cell
(44). Thus, it is possible that this difference in co-chaperonin
availability in the infected host increases the likelihood that gp31
will interact with GroEL and thus, favors gp23 folding.
Identification and Characterization of Mutations in the T4 Major
Capsid Protein That Confer Chaperonin-independent Protein
Folding--
The pETBY plasmid was used to sequence the mutations in
gene 23 that confer chaperonin-independent folding to the
major capsid protein. The T4bypass31-1 mutation is a
temperature-sensitive, conservative single amino acid replacement
located near the carboxyl terminus of gp23 and converts alanine 455 to
valine 455 in agreement with UV mapping data (43). The
T4bypass31-2 mutation, which resisted genetic mapping (43),
was found to consist of three single base mutations in the middle of
gene 23 which convert glycine 292 to serine 292, valine 306 to isoleucine 306, and valine 307 to isoleucine 307. The effect of each
of these amino acid changes in conferring the bypass mutant phenotype
could be determined by introducing the changes singly and in
combination into wild-type gene 23 followed by scoring for
growth on groEL mutant E. coli strains and in the
absence of functional T4 gene 31.
Overlap PCR mutagenesis was used to introduce site-directed mutations
into genomic wild-type gene 23 to determine whether all
three mutations at the bypass31-2 site are required for the bypass phenotype. The various mutated gene 23 sequences were
cloned into the pET3a vector and subsequently recombined into
bacteriophage T431(AmNG71)-31(AmN54),
which does not produce intact gp31 in a suppressor minus host
bacterium. By using this technique several combinations of the three
mutations were tested for the ability to grow on groEL and
groES mutant bacterial strains at a temperature that was
non-permissive for the groE mutations (Table
III). As expected, the wild-type gene
23-containing phage are unable to grow in the absence of
either gp31 or GroEL, and the T4bypass31(1+2) phage, also
containing double amber mutations in gene 31, grow well on
all strains tested. All of the site-directed mutant phage recombinants
grow on CR63, the fully permissive strain. The inability of T4136VV,
T415S-I1, and T415S-I2 to grow on the suppressor-negative Be strain demonstrates the inability of the single site
mutated phage to bypass the gp31 requirement. Analysis of phage growth on the groE mutant strains, which are also
suppressor-negative, shows that the two isoleucine mutations are
essential to conferring chaperonin-independent folding to the gp23
polypeptide. Although the presence of the mutant serine at position 292 is not essential for the bypass phenotype, the plaque size of T4156GI2
was significantly smaller than that of the serine containing T4136SB
(42), suggesting that serine 292 does enhance the ability of these
phage to grow in the absence of functional GroEL or gp31. As
demonstrated by phage growth experiments, the additive contribution of
all four bypass mutations (bypass31-1 [V] and
bypass31-2 [S-II]) is required for optimal gp23 folding in
the absence of gp31 and GroEL. Analysis of the three mutations found in
the T4 bypass31-2 mutant phage indicates that these
mutations also behave in an additive fashion, the contribution of the
Gly Examination of a Translational Pausing Mechanism for
Chaperonin-independent Folding--
The bypass31-2
mutations are of particular interest because they confer
chaperonin-independent folding even at high temperatures where
chaperonins are especially required for most proteins. A curious
observation is that all of the bypass mutations (the single bypass31-1 and multiple bypass31-2) are missense
mutations that cause a reiteration of a preceding amino acid (Ser-Ser,
Ile-Ile, and Val-Val). The introduction of an infrequently used T4
codon by the missense mutation that introduces a serine at position 292, AGU, not found in gene 23 and rarely in T4 structural
genes (45), suggested a role for translational pausing in the
chaperonin-independent folding of the bypass31-2 mutant
major capsid protein due to low charged tRNA abundance. Such a
mechanism has been proposed to account for codon usage differences
within protein domains and interdomain regions (34, 46), and lowered
translation has, in fact, been shown to account for enhanced folding of
a yeast protein (33). In addition, some gene 31 mutations
suppress an E. coli rho gene mutant defective growth
phenotype by an unknown mechanism which could be interpreted to reflect
coupling of protein folding to concurrent transcription-translation
(27, 47).
The introduction of an amber codon at position 292 of the wild-type
major capsid protein enabled translational pausing to be examined as a
potential mechanism in mediating chaperonin-independent protein
folding, since under suppression conditions amber codons should be
translated with a significant delay as compared with other codons (48).
This approach simultaneously allowed the influence of the amino acid
substitution at the am position to be assessed by using tRNA
suppressors to incorporate amino acids of various sizes and chemical
attributes. The TAG codon was introduced either as the sole mutational
change (AmB2) or with the addition of the two isoleucine
residues at positions 306 and 307 (AmB2-II). These
amber-containing genes (AmB2 and AmB2-II) were recombined into an otherwise wild-type T4 phage, resulting in
T423(AmB2), or into a phage containing a
temperature-sensitive mutation in gene 31, resulting in
T423(AmB2)-31(tsA70) and
T423(AmB2-II)-31(tsA70). Fig.
5 summarizes the effects of amino acid
substitutions at position 292 of the major capsid protein tested by
infecting various tRNA suppressor strains with the recombinant phage at
30 and 41 °C. Analysis shows that the bulky aromatic amino acids
phenylalanine and tyrosine are detrimental to the folding of the gp23
polypeptide chain and that all other amino acids substitutions tested
allowed for growth of the T423(AmB2) phage. Tests with
T423(AmB2)-31(tsA70) are
similar to that described above at the permissive temperature and
demonstrate that none of the amino acid substitutions is able to confer
a bypass phenotype in the absence of the isoleucine residues at
positions 306 and 307. Growth of
T423(AmB2-II)-31(tsA70) at the non-permissive temperature shows a wide variety of amino acid
substitutions (including charged, uncharged, and small apolar substitutions) allow for growth in the absence of gp31 when present in
conjunction to the two isoleucine residues. An interesting and
initially surprising finding was that phage which incorporate a leucine
at position 292 grow normally at 30 and 41 °C in the presence of
wild-type gp31 and when the temperature-sensitive gp31 protein is
present and functional at 30 °C. However, when tested at the
non-permissive temperature, leucine has an apparent dominant negative
effect on the isoleucine-induced bypass phenotype, reconverting the
polypeptide to a chaperonin-dependent state. These data do
not support a translational pausing mechanism for the
chaperonin-independent folding phenotype but rather indicate that the
amino acid substitutions themselves are critical post-translationally and that this region of the polypeptide is critical for the specific chaperonin-mediated folding requirement. This again demonstrates the
importance of the specific amino acid sequence in this particular region of the major capsid protein on polypeptide folding and on
chaperonin dependence; however, the specific differences are difficult
to interpret since subtle, conservative amino acid replacements yield a
mutant phenotype.
Our work shows that a bacteriophage T4 GroES homologue is required
to fold the major capsid protein because of specific structure(s) in
the gp23 molecule. Despite these GroES-refractory structures, mutations
in the gp23 polypeptide can confer chaperonin-independent folding. This
interpretation of the bypass mutations is supported by the following
observations. Genetic testing of a number of groES- and
groEL-deficient E. coli shows that the
T4bypass31 mutants are able to form phage independently of
specific GroEL, gp31, and GroES mutations (Tables II and III). In these
experiments, only gene 31 contains null mutations;
consequently, only the absence of its function in the Cpn10-Cpn60
chaperonin-dependent folding pathway can be rigorously
assessed in vivo. However, the fact that all of the GroEL
and GroES mutants are bypassed by the same T4 mutants makes the
possibility that the T4bypass31 mutations modify the major
capsid protein so as to allow productive interactions with specific
partially functional groEL or groES mutants
unlikely. Moreover, it appears that gp23 cannot be mutated so as to be
assisted in folding by GroES. The fact that the bypass31
mutants continue to grow significantly better in the presence of gp31
and GroEL (but not GroES-GroEL) than in their absence (Table III) also
suggests that the mutant polypeptide chains are not shifted to a
different chaperonin folding pathway, rather continue to be acted on by the gp31-GroEL system, if available. Moreover, the GroEL and other chaperone (e.g. DnaK) independence in the folding of the
gp23BY protein is also addressed by in vitro
renaturation experiments of purified gp23 which demonstrate enhanced
chaperonin-independent folding of the mutant gp23BY protein.
The chaperonin family is well known for high levels of homology across
mitochondria, chloroplast, and bacterial homologues (4). Although the
co-chaperonins GroES and gp31 both interact with GroEL, there is no
significant sequence identity between the two co-chaperonins at the
primary or predicted secondary structural levels (30, 31). However,
mobile loop domains, believed to be involved in the Cpn10-GroEL
interaction, have been identified in the amino-terminal region of both
proteins (49, 50). Also, analogous to GroES (10 kDa), gp31 monomers are
12 kDa (10, 30) and appear to form oligomeric ring structures when
over-expressed in vivo (42). However, the fact that gp31 was
identified in a monomeric state in T4-infected bacteria may reflect
weaker gp31 oligomerization as compared with groES (24, 37). In fact, in view of these monomer-multimer equilibria, it is not excluded that
hetero-oligomeric gp31-GroES, and/or "symmetrical" gp31-GroES-GroEL complexes could form with GroEL in vivo. And it appears that
T4bypass31 phage assembly in the presence of core proteins,
approximately 50% (Table IV), is more efficient than
gp23BY polyhead formation in their absence, less than 10%
(Fig. 1, lane 4). This difference might explain the
observation that in the absence of prohead core proteins, gp23
aggregates rather than forming polyheads in an infected bacterium (52,
61).
Although it might be supposed from experiments demonstrating a general
co-chaperonin role of gp31 (19) that gp31 together with GroEL actively
fold a wide spectrum of T4-coded proteins in the infected bacterium,
the existence of the T4bypass31 mutations shows that such
interactions with T4 substrates other than gp23, if they exist, are
non-essential. Western blotting of cell lysates shows that both GroES
and gp31 co-chaperonins are present simultaneously in phage-infected
bacteria (Fig. 4) and calculations of the quantities of each Cpn10
suggest that the GroEL-gp31 interaction may be the favored Cpn60-Cpn10
interaction. Also, we have analyzed several members of the T-even
bacteriophage family and have found through PCR assay that gene
31 is maintained (42). More extensive analysis (53) has
revealed that gene 31 is not only maintained in 49 members
of the T-even family but that the primary sequence is also highly
conserved. Collectively, the data suggest gp31 has evolved to
specifically address the folding needs of the T4 major capsid protein
that are beyond the means of the GroEL-GroES system and challenges the
notion of an absence of specificity in target protein-GroES-GroEL
chaperonin interactions.
The T4bypass31-2 mutation displayed unexpected complexity,
consisting of three closely linked contributing missense mutations; this reconstruction (Tables III and IV) shows why this mutation could
not be mapped (43). Thirteen independently isolated
T4bypass31 mutations have been localized as repeats of or
very close to the bypass31-1 and bypass31-2
mutation sites suggesting that only a very limited number of such
mutant sites exist in gene 23 (29, 43). These bypass regions
display sequence homology to the T4 head vertex protein gp24 (54) (Fig.
6). It has been previously noted that, in
contrast to the amber and bypass24 mutations, head size
determining mutations (pt and ptg) and
temperature sensitivity mutations (trb, ts, and
cs) found in gene 23 cluster in these gp24
homology boxes (32, 54, 55). Since all of these mutant sets are thought
to approach saturation, this clustering should be significant. These
regions, therefore, also appear to be especially critical for the
proper folding of the major capsid protein.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
; 21
,
T421(AmH29); 23,
T423(AmB2); T431(AmNG71);
31=,
T431(AmNG71)-31(AmN54);
BYP1, T4bypass31-1 -31(AmN54); BYP2, T4bypass31-2-31(AmNG71)-31(AmN54);
and the double 31 bypass, BYP1+2, T4bypass31-1-2 mutants
were used in complementation experiments. DNA from the double
T4bypass31 strain was used as the DNA source for the
construction of the pETBY plasmid.
Bacteriophages, bacteria, and plasmids used in this work
CE6 and HMS174(DE3), which is a lysogenic strain
carrying an IPTG-inducible gene for T7 polymerase (36). The plasmid
pEGS1, a generous gift from E. Eisenstein, contains the gene encoding
GroES and was transformed into the strain JZ483 and used in GroES
over-expression studies (37). Transformants were screened by marker
rescue experiments, and protein expression was confirmed by 12.5%
SDS-PAGE or, in the case of gp31, 16% Tricine-polyacrylamide gel
electrophoresis, followed by Western blot analysis (data not shown).
The sequence of gene 23 from both wild-type T4 and
T4bypass31-1-2 was determined by automated sequencing at the
Biopolymer Laboratory Core Facility, University of Maryland at
Baltimore. Site-directed mutagenesis of gene 23 (38) used
wild-type and mutagenic primers, synthesized at the University of
Maryland Biopolymer Laboratory at University of Maryland, Baltimore, to
introduce site-directed missense and amber mutations into wild-type
gene 23. The mutated gene 23 was then cloned into
the pET3a expression vector as described previously and recombined into
bacteriophage T4. The mutations were subsequently confirmed by
automated sequencing.
1) and induced for 4 h at
37 °C with the addition of IPTG to a final concentration of 0.4 mM, for pET-derived plasmids, or of 5 mM, for
pTrc31 and pEGS1. The groEL mutant strain CG2241 containing pET23, pET2331, or pETBY was grown in M9S (39) media supplemented with
0.2% maltose and ampicillin (150 µg ml1) to an
A600 of 0.6 at 30 °C. Expression was induced
by CE6 infection at 37 °C for 4 h (36). For Western blot
analysis, samples were electrophoresed in a 12.5% SDS-polyacrylamide
gel and electroblotted onto Immobilon-P (Milipore) and probed with
antiserum. Rabbit antibodies directed against gp23 were used in
conjunction with the chemiluminescent ECL technique (Amersham Pharmacia
Biotech) for detection of gp23 protein. The procedure for polyhead
isolation has been previously published (40). HMS174(DE3) bacteria
containing either pET2331 or pETBY were grown to an
A600 of 0.6. Bacteria were induced for 4 h
at 37 °C and centrifuged at 5,000 rpm in a Sorvall SS-34 rotor at
room temperature for 10 min. The cell pellets were resuspended in (0.01 original culture volume) 100 mM
KPO4, pH 7, 10 µg/ml pancreatic DNase I
(Sigma), 5 mM EDTA. Cell number was normalized by
A600 readings. All subsequent steps were
performed at room temperature. 100 µg/ml lysozyme was added to the
bacteria to begin lysis, completed by the addition of 1-2 drops of
chloroform, and confirmed visually with the light microscope. Following
addition of MgSO4 to 10 mM and incubation,
polyhead-containing lysed bacteria were pelleted by centrifugation at
low speed (5,000 rpm in a Sorvall SS34 rotor) for 8 min. The
first supernatant was discarded. The pellet was repeatedly centrifuged
and washed four times in buffer A (100 mM KPO4,
pH 7, 10 mM MgSO4), and the supernatants were
pooled. The pooled supernatants were centrifuged at the above speed to
remove any particulate debris. The clarified supernatant was then
centrifuged at high speed (18,000 rpm in a Sorvall SS34 rotor)
for 60 min to pellet the polyhead structures. Repeated low and high
speed centrifugations were used to remove cellular debris, including
contaminating vesicles, to yield a relatively pure gp23 polyhead sample.
80 °C freezer until use or
directly used for immunoprecipitation with anti-GroEL or anti-GroES
antibody. The following procedure is based on a 2-ml original culture
volume. 0.4 ml of lysis buffer containing 1 mM ATP was
added to the cell pellets. Lysis buffer contained 20 mM
Tris-Cl, pH 7.5, 80 mM KCl, 5 mM
MgCl2, 1% Triton X-100, 1% Nonidet P-40, and 1% bovine
serum albumin. The bacteria were incubated at 37 °C for 1 h to
allow for lysis to occur. The samples were centrifuged to remove the
bacterial debris (16,000 rpm in a microcentrifuge or 5,000 rpm in a
Sorvall SS34 rotor at room temperature). All subsequent steps were
performed at room temperature unless otherwise indicated, and 1 mM neutralized ATP was added along with each manipulation
of the cell lysate. A 1:100 dilution of normal rabbit serum was added
to the supernatant, and the samples were incubated for 1 h on an
orbital shaker. 0.02 ml of a 50% protein A-Sepharose slurry (Amersham
Pharmacia Biotech) was then added to the lysate and incubated for
1.5 h. The protein A-Sepharose was gently pelleted at 1,000 rpm in
a microcentrifuge or by gravity, and the pre-cleared supernatant was
used in subsequent steps. 0.5 ml of dilution buffer (20 mM
Tris-Cl, pH 7.5, 5 mM MgCl2 and 1 mM ATP) was added to dilute the KCl and detergent
concentrations that might destabilize the tertiary
(Cpn60-Cpn10-polypeptide) complex. The lysates were split into two
groups as follows: one which was not treated with antibody, and one
which was treated with a 1:1000 dilution of GroEL antibody or a 1:1000
dilution of GroES antibody. Antibody was not added to one of the
fractions to account for nonspecific interactions or the pelleting of
large aggregates and phage that might occur during the procedure. The three fractions (no antibody, GroEL, or GroES antibody treatment) were
incubated for 1.5 h, followed by addition of 0.05 ml of 50% protein A-Sepharose, followed by another 1.5 h incubation. The Sepharose conjugated to the anti-GroEL antibody was pelleted by centrifugation at 1,000 rpm in a microcentrifuge for 1 min. The Sepharose was then washed three times in 20 mM Tris-Cl, pH
7.5, 5 mM MgCl2, 20 mM KCl, and 1 mM ATP. The fourth wash was in 20 mM Tris-Cl,
pH 6.8. The bound proteins were then eluted from the Sepharose by
addition of 1× SDS-PAGE sample buffer containing 50 mM
Tris-Cl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1%
bromphenol blue, and 10% glycerol. Radiolabeled proteins that
co-immunoprecipitated with GroEL antibody were observed after overnight
exposure of x-ray film. Precipitation of GroEL protein was confirmed by
Western blot analysis of 8% SDS-PAGE of cell lysates after
immunoprecipitation using anti-GroEL antibody. Similarly, precipitation
of GroES was confirmed by Western blot analysis of a 17% SDS-PAGE.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
CE6 and then either lysed for polyhead
isolation or prepared into thin sections.
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Fig. 1.
Polyhead formation in vivo
following expression vector synthesis of the major capsid protein
gp23, but not of the gp23BY mutant capsid protein, requires
synthesis of the co-chaperonin gp31 and functional GroEL chaperonin as
determined by SDS-PAGE. HMS174(DE3) and the mutant
groEL44 strain CG2241 contained expression vectors pET23
(expresses gene 23), pET2331 (expresses genes 23 and 31), and pETBY
(expresses mutant gene 23 chaperonin bypass). Coomassie Blue-stained
SDS-PAGE of cell lysates before (A) and after (B)
polyhead isolation (cf. "Experimental Procedures");
C, Western blot of B with anti-gp23 antibody.
Lanes 2-4 are 2/3 the concentration of lanes
5-7. Molecular mass markers are indicated in kDa. Lane
1, partially purified gp23 control (gp23 (arrow)
is 56 kDa); lane 2, pET23/HMS174(DE3); lane 3,
pET2331/HMS174 (DE3); lane 4, pETBY/HMS174(DE3); lane
5, pET23/CG2241groEL44; lane 6,
pET2331/CG2241groEL44; and lane 7,
pETBY/CG2241groEL44.
CE6
induction and the GroEL deficiency of the mutant host at a temperature
semi-permissive for growth. Comparable amounts and stabilities of gp23
resulted from all three constructs (Fig. 1A, lanes
5-7, and Western blotting, data not shown); however, only
expression of gene 23BY yields polyheads both in
the absence of gp31 and in the groEL44-deficient host, as
judged by SDS-PAGE, Western blotting (Fig. 1, B and
C, lanes 5-7), and by electron microscopy (Fig.
2, D and E). Thus, production of polyheads is not closely related to the amount of gp23
accumulation since polyheads can be formed when expression levels are
low as well as high (Fig. 1, A and C,
cf. lanes 7 and 3).
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Fig. 2.
Polyhead formation in vivo
following expression vector synthesis of the major capsid protein
gp23, but not of the gp23BY mutant capsid protein, requires
synthesis of the co-chaperonin gp31 and functional GroEL chaperonin as
determined by electron microscopy of thin sectioned E. coli. HMS174 (DE3) and the mutant groEL44 strain
CG2241 contained expression vectors pET23 (expresses gene
23), pET2331 (expresses genes 23 and
31), and pETBY (expresses mutant gene 23 chaperonin bypass, gp23BY). A-D, thin sections
of pET2331/HMS174 (DE3) (A); pET23/HMS174 (DE3)
(B); pETBY/HMS174 (DE3) (C); and
pETBY/CG2241groEL44 (D). E, negatively
stained polyhead isolated from CG2241groEL44 bacteria
expressing pETBY at the non-permissive temperature. F,
negatively stained gp23BY multilayered polyhead reassembled
after in vitro renaturation from 6 M urea in the
absence of chaperone proteins. Polyhead tubules and cross-sections of
tubules are shown in A, C, and D. Only inclusion
bodies can be seen in HMS174 (DE3) bacteria expressing the wild-type
gp23 protein in the absence of gp31 (B) whereas both
polyheads and inclusion bodies are observed in bacteria producing the
gp23BY protein (C and D).
Magnification of electron micrographs in A-D. × 50,000. Magnification of E and F, × 200,000.
hosts are shown in Fig. 2, A-D. A
comparison of Fig. 2, A and B, shows
multi-layered polyhead tubules are abundant in bacteria-expressing gene
23WT in the presence of functional GroEL and the
T4 co-chaperonin gp31 (an average of 44 polyheads/section). Fig.
2A shows both a longitudinal view of polyheads in the lower
cell, and a cross-section of polyheads found as bundles of concentric
rings, indicative of multilayered polyheads, in the upper cell. The
latter structures are expected to accumulate because inner gp23 layers
provide an assembly template for unassembled major capsid protein in
the absence of accessory T4 prohead core proteins (60). As seen in Fig.
2B, bacteria expressing only gene
23WT were never observed to produce polyheads
but rather form large inclusion bodies presumably containing aggregated
23 protein. Multi-layered polyheads were also found in bacteria
expressing gene 23BY even in the absence of
functional gp31 and GroEL proteins (Fig. 2, C and
D). Fig. 2E shows an electron micrograph of a
negatively stained polyhead isolated from the groEL44 mutant
CG2241 bacteria expressing gene 23BY.
Structurally these gp23BY containing polyheads appear
morphologically indistinguishable as judged by optical diffraction from
those found in T4 mutant infection (32, 42).
The effect of various groEL and groES mutant strains on T4bypass31
phage growth
. Phage tested are as follows: lambda; T4D+;
T431=,
T431(AmN54)31(AmNG71);
BY1, T431(AmN54)bypass 31-1; BY2,
T431(AmN54)31(AmNG71)bypass31-2;
BY1+2,
T4bypass31-(1+2)-31(AmNG71)-31(AmN54).
Bacteria were grown overnight at 30 °C in M9S media, normalized for
cell number, and used to make lawns on LB plates. Phage were diluted to
105 and directly spotted onto the test strain. Phage growth
was evaluated by plaque formation after 14 h growth at 37 °C
for all strains except for CG2246, which was analyzed at 41 °C.
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Fig. 3.
The effect of GroES and gp31 expression on
bacteriophage (A) and bacteriophage T431
(AmNG71) (B) growth on a
groES-deficient strain. The groES42 strain
JZ483 (JZ) was transformed with the parental plasmid pTrc99A (JZ99),
pEGS1 (JZES), or pTrc31 (JZ31). gpES or gp31 expression was induced by
the addition of 4 mM IPTG at an A600
of 0.4 for 1 h at 37 °C. Bacteria were then infected with
either lambda or T431 (NG71) phage. The phage
were allowed to adsorb to the bacteria for 7 min after which antibody directed
against either or T4 phage was added. Infected bacteria were then
diluted to 104 and plated immediately to determine number
of infected bacteria. The remainder of the bacteria was incubated 90 min at 37 °C, lysed with chloroform, and titered to determine the
burst sizes (the number of viable phage progeny produced per infected
bacterium). Burst measurements reported for are averaged from six
independent experiments, for T431 (AmNG71) from
seven independent experiments.
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Fig. 4.
gp23 and GroES interact with GroEL following
T4 infection in the presence or absence of gp31 co-chaperonin
synthesis. Immunoprecipitation of the major capsid protein gp23 in
T4 mutant infected E. coli Be bacteria with
GroEL antibody (A) or GroES antibody (B).
A shows autoradiograms of 8% SDS-PAGE of
35S-amino-acid-labeled cell lysates (WCX) and
cell lysates immunoprecipitated with GroEL antibody
( 
GroEL) or without antibody (ab). Lanes
indicate infection of Be bacteria as follows: lane
a, T421 (AmH29); lanes 1, 6, and
11, T423 (AMB2); lanes 2, 7, and 12, wild-type T4; lanes 3, 8, and
13, T431
(AmNG71)-31(AmN54); lanes 4, 9, and 14, T4bypass31
(1+2)-31(AmNG71)-31(AmN54);
lanes 5, 10, and 15, uninfected
bacteria. The positions of GroEL (65 kDa) and gp23 (56 kDa) are
indicated. B, 17% SDS-PAGE of cell lysates that were
immunoprecipitated with GroES antisera and immunoblotted with either
GroES antiserum or GroEL antiserum, as indicated. Lane 1,
purified gp31; lane 2, purified GroES-GroEL mixture;
lane 3, T423 (AMB2); lane
4, wild-type T4; lane 5, T431
(AmNG71)-31(AmN54); lane 6,
T4bypass31
(1+2)-31(AmNG71)-31(Am54); lane
7, uninfected bacteria; lane 8, uninfected bacteria run
in parallel without GroES antiserum addition during the
immunoprecipitation.
Ser change most apparent when accompanied by the two isoleucine
mutations (Table III). The results of burst size determinations of
site-directed T4 mutant phage infections of the suppressor minus strain
Be provide another measure of the ability of these phage to
grow in the absence of gp31 and is in complete agreement with the
conclusions drawn from plaque formation and morphology (Table
IV). In summary, characterization of the
three mutations at the bypass31-2 site show that all three
mutations (S-II) contribute to optimal growth in the absence of GroEL
and gp31 and that the two isoleucines are essential for
chaperonin-independent protein folding.
Characterization of T4 phage modified by site-directed mutagenesis on
groEL- and groES-defective strains
,
poor phage growth with small plaque size (<1 mm); and , no phage
growth.
Burst size determinations of site-directed T4 mutant phage on the
nonsuppressor E. coli strain Be
4 and incubated at 37 °C
for 90 min. T4 phage was titered on Be (supo) and CR63
(supD). The results of three independent experiments were
averaged and S.E. is indicated. The burst size is given as the number
of viable phage progeny produced per infected bacterium. pfu,
plaque-forming units.
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Fig. 5.
Amino acid substitutions at residue 292 of
the major capsid protein gp23 which confer or block gp23 assembly and
the gp31-GroEL bypass phenotype. Growth of T423
(AMB2), T423
(AMB2)-31(tsA70), and T423
(AMB2-II)-31(tsA70) bacteriophage on
E. coli tRNA suppressor strains for
sup+phe, sup+tyr,
sup+glu, sup +gln,
sup+ala, sup+leu,
sup+ser, and sup+gly
was at the permissive (30 °C) and nonpermissive (40 °C)
temperature for the gene 31 ts mutant. Serial dilutions of
bacteriophage T4 strains were spotted onto tRNA suppressor host lawns
on LB plates and incubated at the indicated temperature for 16 h
to determine growth by plaque formation.
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 6.
Location of the GroEL-gp31 chaperonin bypass
mutations on the major capsid protein gp23 genetic and polypeptide map
(32, 55). The locations of gene 24bypass (byp24),
temperature range of gene 24bypass (trb), petite
(pt), petite and giant phage (pt and
ptg), bypass31-1 and bypass31-2
mutations are shown. The amino acid changes resulting from the
trb and bypass31 mutations are indicated
below the polypeptide map. The dark boxes on the
polypeptide map represent regions of homology to the capsid vertex
protein, gp24. Note the clustering of size, temperature, and chaperonin
sensitivity determining mutations in the homology box regions.
Arrows indicate the transfer of epitopes from the inside to
outside ( 
), outside to inside (
), or lack of such transfer (
),
upon expansion-rearrangement of the gp23 monomers following assembly
into the polyhead-like (cf. Fig. 2) hexameric surface
lattice.
The specificity of the T4 major capsid protein for the gp31 co-chaperonin may be explained in several ways according to current models for GroEL-catalyzed protein folding and chaperonin structures (7-9, 14, 56, 57). One trivial explanation is that gene 23 expression has a toxic effect on GroES forcing T4 to synthesize a "gp23 immune" GroEL-Cpn10 complex. We find this unlikely since we are able to express high levels of gene 23 constitutively from expression vectors without lethal effects on bacteria (data not shown). In another model, GroES could bar entry of gp23 to the central folding cavity of GroEL, whereas GroEL engaged with the gp31 co-chaperonin might admit gp23. However, our co-immunoprecipitation experiments of infected or uninfected cell lysates with anti-GroEL antibody show that gp23, with and without gp31 present, can be co-immunoprecipitated with GroEL. This suggests that gp31 is not required for gp23 "targeting" or binding to GroEL but that it is required for the productive folding of the protein. A model in which the binding of gp31 to GroEL creates a larger folding cavity than when GroES is bound to GroEL could also explain the GroEL-gp31-specific requirement. The slightly larger gp31 might complex with GroEL while gp23 is within the folding cavity, whereas GroES would be prevented from complex formation by the gp23. This model would be consistent with the relatively large (56 kDa) T4 major capsid protein and selective pressure against its size reduction. A recent crystallographic determination of the gp31 structure establishes this size increase and proposed this upper-limit size mechanism (51). However, it appears uncertain that 56 kDa is an upper limit to the GroES-GroEL complex. The ability of the GroEL-GroES complex to fold proteins larger than gp23, such as sigma 70, the bacteriophage Lambda B protein (60 kDa) or Mu H protein (64 kDa) suggests that the size of the folding cavity alone may not adequately explain the gp31 requirement (1, 20). Indeed, the fact that the lambda gpB folding requirements are less well catalyzed by the larger T4 gp31-GroEL chaperonin system (Fig. 3) argues against the folding chamber size hypothesis. If this size hypothesis is incorrect, then the gp23-gp31-GroEL chaperonin specificity results demonstrated in this paper appear to place constraints on models for GroEL catalyzed folding, favoring "in chamber" folding.
An alternative explanation for these specificity results is that GroES
may allow the premature release of partially folded gp23 from
GroEL, whereas gp31 may stabilize the polypeptide-GroEL interaction
resulting in more complete folding in the chamber. We can further
speculate that GroEL bound by gp31 makes specific associations with
wild-type gp23 that do not form with the GroEL-GroES complex and that
these interactions enable the polypeptide to achieve native state, a
view consistent with the possibly exclusive targeting of gp23 folding
by gp31 in a T4 infection. In this view, the major capsid protein
sequence alone may not contain all of the folding information required.
Just as the bypass31 mutations impart new folding
information to the gp23 polypeptide enabling chaperonin-independent
folding, gp31 may engage the GroEL chaperonin in such a way that it
provides some specific folding information to the wild-type gp23
molecule, e.g. by producing a more energetically folded
intermediate. In fact, it is tempting to speculate that the additivity
of the contributing tightly clustered bypass amino acid changes to a
chaperonin-independent folding mode corresponds to efficient localized
GroEL chaperonin action on these portions of the polypeptide substrate.
In this view, the role of the GroEL-gp31 chaperonin system may not only
be to shift the folding equilibrium away from aggregation but through
specific GroEL-gp31-gp23 interactions to foster an environment that
allows for proper folding of specific refractory sequences or local
structures. Although the three-dimensional structure of the T4 major
capsid protein is not determined, it has been shown to undergo a
remarkably large structural change which extends to the secondary
level, following its assembly preparatory to DNA packaging (Fig. 6);
thus, it is assembled in a metastable state in the polyhead (32, 58).
Whether this is related to the specific co-chaperonin assembly
requirement remains to be determined.
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ACKNOWLEDGEMENTS |
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We thank Marcus H. Hagogue III for expert assistance with thin section preparation; Dr. Martin Kessel for assistance with microscopy of purified gp31 ring structures; and Dr. Zhao Jun Ren for technical assistance with the initial sequencing of the T4bypass31-1 mutation. We also thank Dr. Lee Simon for the T4bypass31 mutants; Dr. Ed Eisenstein for the GroES over-expression plasmid; Dr. Costa Georgopoulos for the groE mutants; Dr. Henry Krisch for interesting discussion and for providing us with helpful information prior to publication; and Drs. Julienne Mullaney, Gerry Barcak, Kim Collins, and Terry Rogers for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was supported by Grant AI11676 from the National Institutes of Health and by a grant from the University of Maryland at Baltimore Graduate School for travel to present a timely version of this work at the Conference on Stress Proteins in Medicine, February 27 to March 5, 1995, Sante Fe, NM.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.
Present address: Center for Bio/Molecular Science and Engineering,
Naval Research Laboratory, Washington, D. C., 20375.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD 21201-1503. Tel.: 410-706-3510; Fax: 410-706-8297; E-mail: lblack{at}umaryland.edu.
The abbreviations used are:
PCR, polymerase
chain reaction; IPTG, isopropyl-1-thio-
-D-galactopyranoside; WT, wild type; m.o.i., multiplicity of infection; PAGE, polyacrylamide gel
electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
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Abstract
Introduction
Procedures
Results
Discussion
References
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