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J Virol, January 1998, p. 358-365, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Potato Leafroll Virus Binds to the Equatorial
Domain of the Aphid Endosymbiotic GroEL Homolog
Saskia A.
Hogenhout,1
Frank
van der Wilk,1
Martin
Verbeek,1
Rob W.
Goldbach,2 and
Johannes
F. J. M.
van den
Heuvel1,*
Department of Virology, DLO Research
Institute for Plant Protection (IPO-DLO), 6700 GW
Wageningen,1 and
Department of Virology,
Wageningen Agricultural University, 6709 PD
Wageningen,2 The Netherlands
Received 1 August 1997/Accepted 5 October 1997
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ABSTRACT |
A GroEL homolog with a molecular mass of 60 kDa, produced by the
primary endosymbiotic bacterium (a Buchnera sp.) of
Myzus persicae and released into the hemolymph, has
previously been shown to be a key protein in the transmission of potato
leafroll virus (PLRV). Like other luteoviruses and pea enation mosaic
virus, PLRV readily binds to extracellular Buchnera GroEL,
and in vivo interference in this interaction coincides with reduced
capsid integrity and loss of infectivity. To gain more knowledge of the nature of the association between PLRV and Buchnera GroEL,
the groE operon of the primary endosymbiont of M. persicae (MpB groE) and its flanking sequences were
characterized and the PLRV-binding domain of Buchnera GroEL
was identified by deletion mutant analysis. MpB GroEL has extensive
sequence similarity (92%) with Escherichia coli GroEL and
other members of the chaperonin-60 family. The genomic organization of
the Buchnera groE operon is similar to that of the
groE operon of E. coli except that a
constitutive promoter sequence could not be identified; only the heat
shock promoter was present. By a virus overlay assay of protein blots, it was shown that purified PLRV bound as efficiently to recombinant MpB
GroEL (expressed in E. coli) as it did to wild-type MpB
GroEL. Mutational analysis of the gene encoding MpB GroEL revealed that the PLRV-binding site was located in the so-called equatorial domain
and not in the apical domain which is generally involved in polypeptide
binding and folding. Buchnera GroEL mutants lacking the
entire equatorial domain or parts of it lost the ability to bind PLRV.
The equatorial domain is made up of two regions at the N and C termini
that are not contiguous in the amino acid sequence but are in spatial
proximity after folding of the GroEL polypeptide. Both the N- and
C-terminal regions of the equatorial domain were implicated in virus
binding.
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INTRODUCTION |
Potato leafroll virus (PLRV; genus
Luteovirus), a positive-stranded RNA virus, mainly
replicates in the phloem tissue of a plant and is transmitted by aphids
in a persistent and circulative manner (28, 41, 46). When
they feed on the phloem sap, aphids ingest virus particles, which are
subsequently transported from the digestive tube into the hemolymph
(24) and from there across the basal lamina that surrounds
the accessory salivary cells into the salivary gland (25).
Virus particles that reach the salivary gland are eventually released
in the phloem sap of the plant as the aphid feeds (25). The
hemolymph acts as a reservoir in which PLRV is retained in an infective
form during the aphid's lifespan without replication (19).
It has previously been demonstrated that the primary endosymbiotic
bacterium (a Buchnera sp.) of Myzus persicae, the
principal vector of PLRV, plays a crucial role in determining the
persistent nature of PLRV in the aphid hemolymph (50).
Buchnera spp. abundantly produce a protein which is highly
homologous to the Escherichia coli chaperonin GroEL (5,
23, 35, 50). GroEL of the Buchnera sp. of M. persicae (MpB GroEL) was found to be released in the hemolymph,
most likely as a result of the lysis of endosymbiotic bacteria
(50). After antibiotic treatment of the aphid, MpB GroEL
could no longer be detected in the hemolymph and PLRV transmission was
greatly reduced due to degradation of virus capsid proteins (50). Since in vitro studies have previously shown that PLRV exhibits specific affinity for MpB GroEL, it was suggested that virus
particles associate with MpB GroEL in the hemolymph of the aphid to
retard proteolytic breakdown of virus particles (50).
Buchnera spp. are common to all major aphid groups but the
Phylloxeridae (9). These intracellular bacteria are
gram-negative and closely related to members of the
Enterobacteriaceae family (36, 48).
Buchnera spp. are harbored in specialized cells, mycetocytes, localized in the abdomen of the aphid (50) and are maternally inherited (9). Comparisons of rRNA sequences of Buchnera spp. and morphological features of aphid hosts
provide strong evidence that a single aphid ancestor was infected by
the bacterium about 250 million years ago (37).
GroEL of E. coli is a heat shock protein (Hsp60) with 60-kDa
subunits; it is involved in intracellular folding and assembly of
nonnative proteins in an ATP-dependent manner (18). Hsp60s are common to prokaryotes, mitochondria, and chloroplasts (18, 26). Crystallography of E. coli GroEL demonstrated
that the protein forms a homo-oligomer of 14 subunits, which are
arranged in two heptameric rings stacked back to back, and that each
subunit consists of the following three domains: the equatorial domain, the apical domain, and the small intermediate domain (7). In general, the apical domain of GroEL has previously been implicated in
polypeptide binding (22), a process which may require ATP hydrolysis. The ATPase activity of GroEL is regulated by GroES (34, 53), a single heptameric ring of 10-kDa subunits also encoded by the groE operon (14, 47). The
structural and functional characteristics of Buchnera GroELs
are highly similar to those of E. coli GroEL (23, 27,
38). However, unlike E. coli GroEL, Buchnera GroEL is not restricted to the cytosol of the
bacterium; it also occurs extracellularly in the hemolymph of an aphid
(23, 50, 51).
In this study, the nucleotide sequence of the gene encoding MpB GroEL
was determined and structural and functional domains were identified by
sequence comparison to the other GroELs. In addition, the regions
upstream and downstream of this gene were sequenced and compared with
the corresponding regions of E. coli. To gain a better
understanding of the molecular basis of the association between PLRV
and MpB GroEL, the protein was expressed in E. coli and
mutational analysis was carried out to identify the domain of MpB GroEL
implicated in PLRV binding.
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MATERIALS AND METHODS |
Isolation of genomic DNA from the Buchnera sp. of
M. persicae.
Approximately 1 g of M. persicae aphids was collected and surface sterilized with 70%
ethanol containing 0.5% Tween 20 and 0.5% hypochlorite. Sterilized
aphids were rinsed with water and homogenized in 3 ml of isolation
medium (8). Subsequently, the homogenate was filtered
through cheesecloth and centrifuged at 5,000 × g for
15 min. Either bacterial genomic DNA was isolated directly from the
resulting pellet (lysis buffer method) or further purification steps
were undertaken to enrich for bacterial cells (Ficoll procedure). In
the lysis buffer method, the pellet was incubated for 1 h at
56°C in 0.7 ml of lysis buffer (150 mM Tris-HCl [pH 8.0] containing
150 mM EDTA, 3% sodium dodecyl sulfate [SDS], and 1.5 to 2% sodium
lauroyl sarcosine). After 5 min of incubation on ice, 0.5 ml of
Tris-EDTA buffer was added, the suspension was gently mixed, and the
debris was allowed to precipitate. Genomic DNA was extracted with
phenol-chloroform from the supernatant. In the Ficoll method, the
pellet was resuspended in 2 ml of 100-fold-diluted isolation medium and
layered on a 2 to 10% Ficoll gradient in 0.01 M phosphate buffer (pH
7.2). After centrifugation at 400 × g for 10 min, the
fraction containing bacterial cells was collected. To this fraction, 5 volumes of saline-EDTA (0.15 M sodium chloride, 0.1 M EDTA [pH 8.0])
was added and the mixture was centrifuged at 1,000 × g
for 12 min. The pellet was resuspended in 1 ml of saline-EDTA
containing 8% SDS and incubated at 60°C for 10 min, and DNA was
extracted as mentioned above.
PCR amplification procedure.
PCR amplification was performed
in a final volume of 100 µl of 10 mM Tris-HCl (pH 8.3) containing 0.4 mM (total) deoxynucleoside triphosphates, 3 mM MgCl2, 50 mM
KCl, 1 µg of DNA, 0.25 µM (each) primers, and 2.5 U of
Taq polymerase (Boehringer Mannheim). Mixtures were
incubated for 2 min at 94°C, followed by 35 cycles of 1 min at
94°C, 1 min at 55°C, and 2 min at 72°C, with a final incubation of 10 min at 72°C. Samples were stored at 4°C until used. PCR products were analyzed on agarose gels.
Sequencing strategy.
Clones containing the MpB
groEL sequence were generated by PCR with primers F1 and R1
(Table 1). The primer sequences were based on the N-terminal amino acid sequence of MpB GroEL
(50) and the 3'-terminal nucleotide sequence of the
Buchnera groEL gene of Acyrthosiphon pisum
(38). The resulting 1,732-bp PCR product was cloned by using
a TA cloning kit (Invitrogen), yielding plasmid
pCR[Buchnera GroEL]. Overlapping restriction fragments were subcloned into pBluescript KS (Stratagene), and their nucleotide sequences were determined at the sequence facilities of the Department of Molecular Biology, Wageningen Agricultural University, with a
sequencing kit and AmpliTaq DNA polymerase (Applied Biosystems), universal and sequence-specific primers, and an automated sequencer (model 373; Applied Biosystems).
To determine the sequence of the entire MpB groE operon, a
genomic DNA library was constructed by using a 
ZAP II cloning kit
and Gigapack III Gold packaging extract (Stratagene) according to the
manufacturer's instructions. Genomic DNA from the Buchnera sp. of M. persicae was isolated by the lysis buffer method
and digested with XbaI. Fragments were ligated into
XbaI-digested ZAP II vector arms. Two radiolabeled
probes of 569 and 521 bp, corresponding to the 5' and 3' ends of the
open reading frame (ORF) coding for MpB GroEL, respectively, were used
to screen for recombinant clones. After the excision of positive
plaques, the nucleotide sequences of phagemids pSK2500 and pSK3500 (see Fig. 2) were determined.
Southern blot analysis.
Genomic DNA from the
Buchnera sp. of M. persicae was isolated by the
Ficoll method (see above), and 5 µg of DNA was digested with either
PstI, XbaI, or XhoI. Samples were run
on a 1% agarose gel and transferred to HybondN (Amersham). The
1,732-bp PCR product containing the MpB groEL gene mentioned
above was radiolabeled and used as the probe for hybridization.
GroEL isolation from the Buchnera sp. of M. persicae and from E. coli.
GroEL was isolated from the
endosymbiotic bacteria of 6-day-old M. persicae nymphs and
from heat-shocked E. coli cells as described before
(51).
Cloning and expression of Buchnera GroEL deletion
mutants.
Full-length MpB GroEL and deletion mutants of MpB GroEL
in fusion with glutathione S-transferase (GST) were
expressed in E. coli with plasmid pGEX-2T (Pharmacia). GST
fusion proteins were affinity purified with glutathione-Sepharose
(Pharmacia) according to the manufacturer's recommendations. To remove
the GST moiety, fusion proteins were incubated with thrombin for 3 h at 10°C. Cleaved products were analyzed on SDS-polyacrylamide gel
electrophoresis (PAGE) gels and by Western blot analysis with anti-MpB
GroEL immunoglobulin G (IgG). To ensure that similar quantities of
deletion mutants were tested for their virus-binding capacities
(described below), they were diluted to yield bands of similar
intensities, as assessed by amido black staining after
electroblotting. Each mutant was named after the positions of the
first and last amino acids bordering the included fragment.
Full-length MpB GroEL was obtained by digesting
pCR[
Buchnera GroEL] with
BamHI and cloning the
BamHI fragment containing the
MpB
groEL gene into
the
BamHI sites of pGEX-2T, resulting in
pGEX[
Buchnera GroEL]. Constructs for the expression of MpB
GroEL(1-121) and
MpB GroEL(1-314) were derived by digesting plasmid
pGEX[
Buchnera GroEL] with
SmaI (located
downstream of the
BamHI site in the
multiple cloning site of
pGEX-2T) and
ClaI or
XbaI. Protruding
5' ends
were filled in with the Klenow fragment of DNA polymerase
I and by
religation of constructs. pGEX-2T constructs for the
expression of all
other truncated mutants of GroEL were generated
by PCR. The primers
used were complementary or identical to the
border sequences of the
three domains recognized in MpB GroEL
and included additional
restriction sites (
BamHI,
EcoRI, or
HindIII
sites) for cloning purposes (Table
1). Plasmid
pCR[
Buchnera GroEL]
served as the template. All PCR
products were first cloned into
the pCRII vector (TA cloning kit;
Invitrogen), digested with
BamHI
or
BamHI/
EcoRI, and subsequently religated into the
BamHI or
BamHI/
EcoRI
sites of pGEX-2T.
For the expression of MpB GroEL(122-408/475-548),
a pGEX-2T construct
was synthesized with primer pair F2 and R3
and primer pair F6 and R1
(Table
1). The amplified fragments
of 850 (F2 and R3) and 225 (F6 and
R1) bp were cloned into pCRII
and digested with
BamHI/
HindIII and
HindIII/
EcoRI, respectively.
The
HindIII-cleaved ends of both fragments were ligated, and
the
ligated product was cloned into the
BamHI/
EcoRI sites of pGEX-2T.
All constructs were
verified by nucleotide sequence analysis.
The pGEX constructs mentioned above were introduced into
E. coli JM101, DH5
, or protease-deficient BL21 (Stratagene). For
expression, overnight cultures were diluted 1:10 in Luria broth
containing ampicillin (100 µg/ml) and incubated at 37°C for 3
h. Subsequently, 1 mM isopropyl-
-
D-thiogalactosidase was
added
to induce protein synthesis of the pGEX plasmid and cultures were
allowed to grow at room temperature. After 7 h, cells were
pelleted
at 4,000 ×
g for 10 min and resuspended in 50 mM Tris-HCl (pH
7.5) containing 10 mM MgCl
2. Cells were
lysed by one cycle of
freeze-thaw and sonication. Insoluble debris was
removed by centrifugation,
and the supernatant containing the soluble
protein was collected.
Virus overlay assay.
PLRV (52) was maintained on
Physalis floridana as previously described and purified from
leaf material by a modified enzyme-assisted procedure (49).
The virus overlay was performed essentially as described before
(50). Similar amounts of various MpB GroEL polypeptides were
run on denaturing polyacrylamide gels for SDS-PAGE. After
electrophoresis, gels were conditioned in 10 mM
3-[cyclohexylamino]-1-propanesulfonic acid (pH 11.0) containing 10%
methanol for 1 h and proteins were electrotransferred onto
nitrocellulose. Protein blots were incubated overnight with purified
PLRV (10 µg per ml), after which immunodetection with anti-PLRV IgG
and alkaline phosphatase-conjugated anti-rabbit IgG was carried out
(50).
Nucleotide sequence accession number.
The sequence data of
the groEL operon and its flanking regions have been
submitted to the GenBank database under accession no. AF003957.
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RESULTS |
Characterization of the groE operon of the
Buchnera sp. of M. persicae.
The nucleotide
sequence of the groE operon of the primary endosymbiont (a
Buchnera sp.) of M. persicae (MpB
groE) and its flanking sequences were determined both by PCR
and with clones derived from a genomic library. Southern blot analysis
revealed that one copy of the MpB groEL gene was present on
the genome (Fig. 1). The genomic
organization of the MpB groE operon (Fig. 2) is similar to that of the
groE and sym operons of E. coli and the intracellular symbiont of A. pisum, respectively
(29, 38). The operon accomodates two ORFs encoding 10- and
60-kDa proteins, which have 72 and 73% homologies at the nucleotide
level with E. coli groES and groEL, respectively.
The MpB groE genes are also highly homologous to
symS (89%) and symL (91%) from A. pisum. However, sequence comparisons of the promoter regions of
the groE operons of various Buchnera spp. with
that of E. coli revealed the only conserved element to be
the heat shock promoter. A constitutive promoter similar to the one in
the E. coli groE operon (and reported to be present in the
A. pisum groE operon) could not be identified. A terminator
sequence comparable to the one in the E. coli groE operon
was not present either. Most likely, the GC-rich inverted repeat at the
end of a Buchnera groE operon performs this function.
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FIG. 1.
Southern blot analysis of the Buchnera sp. of
M. persicae to determine the copy number of the
groEL gene. DNA from the Buchnera sp. of M. persicae was digested with XbaI, PstI, or
XhoI. XbaI recognizes a single restriction site
within the MpB groEL gene, whereas PstI and
XhoI restriction sites are present only outside this gene. A
radiolabeled PCR fragment comprising the gene encoding MpB GroEL was
used as the probe.
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FIG. 2.
Schematic representation of the MpB groE
operon and comparison of the chromosomal arrangements of the regions
flanking the groE operons of the Buchnera sp. of
M. persicae (a) and E. coli (b) (11,
12). Identical shading indicates high amino acid sequence
similarity of gene products. The percentage noted above a box indicates
the similarity of the MpB ORF product to the E. coli
homolog. #, percentage of similarity of the C-terminal 295 amino acids
of the 60-kDa (60 K) gene; *, percentage of similarity of the
N-terminal 186 amino acids of the 20-kDa (20 K) protein. The arrow
below each gene indicates the direction of translation.
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To determine the degree of conservation of the genomic region flanking
the MpB
groE operon, the regions upstream and downstream
were compared with those of
E. coli (Fig.
2), the only
well-characterized
free-living relative of
Buchnera spp.
(
36). Upstream of the
MpB
groE operon, three ORFs
which show similarities to genes on
the
E. coli genome were
identified. The ORF immediately adjacent
to the
groE operon
shows homology to the tRNA
Phe gene (
11). The
other ORFs display 69% similarity to the
E. coli 50-kDa
thiophene and furan oxidation protein (ThdF) and 83%
similarity to the
C-terminal part of the gene that encodes the
60-kDa inner membrane
protein of
E. coli (
2,
11). These genes
are
present at similar sites on the genome of
Buchnera
aphidicola,
the primary endosymbiont of
Schizaphis
graminum (
4), although
the tRNA
Phe gene has
not been previously reported. Interestingly, on the
E. coli
genome, the
groE operon is separated by approximately
500 kbp from the genes encoding ThdF and the inner membrane protein
(
11,
12). Downstream of the MpB
groE operon, two
genes which
display 70% similarity with a 37.8-kDa protein of
E. coli of unknown
function and 78% similarity with the N-terminal
sequence of elongation
factor P of
E. coli (
3,
12) were identified. In
E. coli,
an additional segment
of approximately 2 kbp harboring two ORFs
with unknown functions is
located between the terminator sequence
of the
groE operon
and the gene encoding the 37.8-kDa protein
(
12).
Analysis of the groEL gene of the Buchnera
sp. of M. persicae.
To ascertain whether MpB GroEL has
structural and functional similarities to E. coli GroEL, the
deduced amino acid sequence of the MpB groEL product was
compared with those of E. coli and other Buchnera
spp. GroELs (Fig. 3). This disclosed that
MpB GroEL is 98 to 99% similar to the GroELs of Buchnera
spp. of the aphids Sitobion avenae, Rhopalosiphum
padi, and A. pisum and 92% similar to E. coli GroEL. A comparison of the MpB GroEL sequence with conserved
residues in 50 prokaryotic Hsp60/GroEL homologs (22) showed
that all of these residues except for the alanine at position 294 (Ala294) are identical. In all of the Buchnera spp.
analyzed, Ala294 is replaced by serine (Fig. 3). Since the
Buchnera GroEL of A. pisum has previously been
demonstrated to fully complement E. coli GroEL in
groE mutants of E. coli (38), this
substitution seems to be of minor importance to GroEL's functioning as
a molecular chaperon in vivo. Moreover, in vitro experiments showed
that the replacement of Ala294 by glutamic acid in E. coli
GroEL did not affect polypeptide binding, folding, or its ATPase
activity (22). The highly conserved amino acid residues of
E. coli and Buchnera spp. GroELs are shown in
Fig. 3. They are evenly distributed over the three domains of GroEL and
are involved in polypeptide binding and folding (mainly located in the
apical domain), ATP binding and hydrolysis (equatorial domain),
maintaining inter- and intrasubunit interactions, and movement of the
GroEL domains relative to each other (13, 22, 30). Amino
acid residues in less-conserved regions which are known to mediate
polypeptide binding (Leu238 and Val264) (22) or which have
previously been reported to be essential for ATP binding in E. coli GroEL (Ala482 and Asp497) (6) are also identical
in MpB GroEL.
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FIG. 3.
Amino acid sequence alignment of the GroELs of E. coli (29) and Buchnera spp. from M. persicae (Mp), S. avenae (Sa)
(23), R. padi (Rp) (23),
and A. pisum (Ap) (38). Conserved
regions in other GroEL/Hsp60 chaperonins (7) are boxed.
Amino acids that are involved in polypeptide binding are indicated by
arrow heads, and those that are involved in ATP binding are indicated
by asterisks. Identical residues and gaps are indicated by periods and
dashes, respectively. The equatorial domain is indicated by a dashed
line, the intermediate domain is indicated by a continuous line, and
the apical domain is indicated by dots. The sequence alignment was
carried out by using the program PILEUP (Genetics Computer Group,
Madison, Wis.) (17).
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Binding of PLRV to Buchnera GroEL deletion
mutants.
To determine which of the three domains of the MpB GroEL
molecule are implicated in the interaction with PLRV in vitro, MpB GroEL was expressed in fusion with GST and affinity purified. After the
GST moiety was removed by thrombin, the recombinant protein was tested
for its PLRV-binding capacity by a virus overlay assay of protein blots
which had previously been used to show that PLRV displayed a high and
specific affinity for the 60-kDa subunit of MpB GroEL (50).
The in vitro binding assay clearly established that full-length
recombinant MpB GroEL bound PLRV as readily as wild-type MpB GroEL did
(data not shown).
By utilizing the sequence similarity between
Buchnera and
E. coli GroELs in areas that are relevant for intrasubunit
interactions,
the first set of deletion mutants, based on the primary
structure
of the different domains on the GroEL molecule, was generated
(Fig.
4a). The crystal structure of GroEL
shows that the individual
subunits are folded into three distinct
domains (
7), of which
only the apical domain is continuous
on the primary structure.
The equatorial and intermediate domains are
discontinuous, with
regions located in both N- and C-terminal halves of
the molecule
(Fig.
4a). Testing similar amounts of MpB GroEL deletion
mutants
in virus overlay assays revealed that purified PLRV displayed
affinities for all mutants containing the N- or C-terminal region
of
the equatorial domain (Fig.
4). Extending the N-terminal equatorial
domain [MpB GroEL(1-121)], but not the C-terminal equatorial domain,
with sequences of the intermediate and apical domains [MpB
GroEL(1-314)
and MpB GroEL(1-374)] improved the efficiency of virus
binding
(Fig.
4). Strikingly, PLRV binding to polypeptides containing
the apical domain alone [MpB GroEL(189-374)] or the entire region
between the
ClaI site (amino acid residue 122) and the C
terminus
of the intermediate domain (amino acid residue 408) did not
occur
(Fig.
4). The smallest deletion mutants that showed binding to
PLRV harbored the N-terminal 121 amino acid residues [MpB
GroEL(1-121)]
or the C-terminal 139 residues [MpB
GroEL(409-548)] (Fig.
4).
The presence of at least one of these
regions is required for
the virus-binding capacities of MpB GroEL
deletion mutants. The
virus overlay assay also showed that PLRV
interacted with
E. coli GroEL.
E. coli GroEL was
copurified with mutants expressed in
the protease-deficient strain
E. coli BL21. As
E. coli GroEL was
insensitive to
the thrombin treatment (Fig.
4b; compare lanes
2 and 3) and its
presence did not interfere with the migrations
of MpB GroEL mutant
polypeptides during SDS-PAGE, no steps were
undertaken to remove
endogenous GroEL from suspensions.
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FIG. 4.
PLRV binding to deletion derivatives of MpB GroEL. (a)
Schematic representations of MpB GroEL deletion mutants. The numbers in
parentheses correspond to the positions of amino acid residues of MpB
GroEL (Fig. 3) and mark the borders of the deletion mutants. The
ClaI and XbaI restriction sites are indicated by
arrowheads. N-eq, N-terminal region of the equatorial domain; N-int,
N-terminal region of the intermediate domain; Ap, apical domain; C-int,
C-terminal region of the intermediate domain; C-eq, C-terminal region
of the equatorial domain. (b) Virus overlay assays. Lanes: 1, wild-type
MpB GroEL; 2, GroEL of E. coli treated with thrombin; 3, GroEL of E. coli. All other lanes contain the indicated
deletion mutants of MpB GroEL, as depicted in panel a. The positions of
GroEL (60 kDa), GST (28 kDa), and the smallest truncated MpB GroEL
fragments that bind PLRV [(409-548) and (1-121)] are indicated by
arrowheads.
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It is noteworthy that all MpB GroEL constructs harboring the C-terminal
region of the equatorial domain produced smaller fragments
for which
PLRV showed affinities [MpB GroEL(122-548), MpB GroEL(189-548),
MpB
GroEL(376-548), and MpB GroEL(409-548)] (Fig.
4). These truncated
products were approximately 8.5 kDa smaller than the corresponding
mutants. Protein microsequencing by automated Edman degradation
of the
truncated products MpB GroEL(376-548) and MpB GroEL(409-548)
revealed
that the N-terminal residues are identical to those expected
in the
corresponding full-length polypeptides. This implies that
the 8.5-kDa
fragment was cleaved from the C terminus and that
this fragment is
dispensable for PLRV binding. Based on the relative
molecular masses of
the truncated products, the approximate position
of the truncation
mapped between amino acid residues 471 and 476
of MpB GroEL. The region
N terminal of the truncation site, which
is involved in PLRV binding,
is characterized by the presence
of three
-helices (
7).
To investigate more firmly the role
of these structural elements in
PLRV binding, the following two
additional mutants were constructed:
MpB GroEL(122-474), which
contained the
-helices (between residues
408 and 475), and MpB
GroEL(122-408/475-548), from which these elements
were deleted
(Fig.
5a). Purified PLRV
clearly demonstrated an in vitro binding
affinity for MpB
GroEL(122-474) but not for MpB GroEL(122-408/475-548).
Thus, the
determinant for PLRV binding is located between amino
acids 408 and 475 (Fig.
5b) of the C-terminal region of the equatorial
domain.
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FIG. 5.
Localization of the PLRV-binding site in the C-terminal
part of the equatorial domain of MpB GroEL. (a) Schematic
representations of the C-terminal deletion mutants of MpB GroEL. The
numbering and abbreviations used are explained in the legend to Fig. 4.
(b) Virus overlay assay of MpB GroEL deletion mutants. Lane 1, wild-type MpB GroEL; all other lanes contain the indicated MpB GroEL
mutants, as depicted in panel a. The positions of GroEL (60 kDa),
Buchnera GroEL(122-408/475-548), MpB GroEL(122-474), and MpB
GroEL(409-548) are indicated by arrowheads.
|
|
| |
DISCUSSION |
In the present study, the groE operon of the
Buchnera sp. from the major aphid vector of PLRV, M. persicae, was characterized and the PLRV-binding domain of MpB
GroEL was identified by deletion mutant analysis. PLRV-binding studies
revealed that virus particles exhibited in vitro affinities for all
deletion mutants of MpB GroEL containing parts of the N-terminal (amino
acid residues 1 to 121) (Fig. 3) or C-terminal (amino acids 409 to 474)
(Fig. 3) regions of the equatorial domain (Fig. 4 through
6). Computer-generated structural
predictions of the monomer of MpB GroEL (39) showed that
these two regions assemble in the tertiary structure. It is therefore
suggested that the residues involved in PLRV binding from either region
join to compose a single PLRV-binding site. These results are
remarkable, as previous single amino acid replacement studies of
E. coli GroEL have demonstrated that residues in the apical
domain are generally involved in polypeptide binding and folding
(7, 22). Thus far, the equatorial domain has been implicated
only in the in vitro binding of two multimeric proteins, ribulose-1,5-biphosphate carboxylase-oxygenase and malate dehydrogenase (54). Apparently, protein-binding sites are not necessarily located in the apical region of the central cavity of the GroEL cylinder but may be located in the equatorial domain as well. Large
multimeric proteins and luteoviruses may employ these sites to overcome
the size limitations (50 to 80 Å wide [15]) imposed by the central cavity of the GroEL molecule. The equatorial domain also
accommodates the ATP-binding site on its external envelope (7, 22,
42). Like the putative PLRV-binding site, this site is composed
of amino acid residues from both C- and N-terminal regions of the
discontinuous equatorial domain (Fig. 3) (6). All of the MpB
GroEL mutants in this study contain deletions known to impair
intersubunit interactions (13, 22, 30) and are unable to
assemble into the multimeric form of GroEL which prevails in the aphid
hemolymph (51). Single amino acid replacements in the
PLRV-binding regions which do not affect GroEL assembly are required to
verify the role of the equatorial domain of the native molecule in the
interaction with PLRV.
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FIG. 6.
Summary of the PLRV-binding regions in MpB GroEL. The
PLRV-binding regions in the N- (amino acids 1 to 121) and C- (amino
acids 409 to 475) terminal parts of the equatorial domain (N-eq and
C-eq, respectively) are shaded. N-int, N-terminal region of the
intermediate domain; Ap, apical domain; C-int, C-terminal region of the
intermediate domain.
|
|
Based on the differential binding of subgroup I and II luteoviruses and
pea enation mosaic virus to Buchnera GroELs from vector and
nonvector species and to GroEL of E. coli (23,
51), it was concluded that the basic virus-binding capacity
resides in a conserved part of the GroEL molecule (51).
Indeed, the regions in the equatorial domain of MpB GroEL which mediate
PLRV binding are highly conserved among Buchnera GroEL
homologs. However, regions of variability in these or other parts of
the GroEL molecule may potentially influence the efficiency of binding,
thus explaining the observed differences in the affinity of
luteoviruses for GroEL homologs (23, 51).
While GroEL is abundantly produced by Buchnera spp. in
aphids (5, 27), Buchnera GroES is difficult to
detect (5, 32). Undoubtedly, Buchnera GroES is an
important cofactor for cellular protein folding (38), but
its potential role in extracellular protein interactions in the
hemolymph of an aphid is yet to be investigated. Bacterial symbionts
and pathogens, like Rhizobium meliloti, Pseudomonas
aeruginosa, the X bacteria of Amoeba proteus, and
Agrobacterium tumefaciens, have adopted different strategies at the transcriptional and translational levels to overproduce GroEL
homologs relative to GroES production (1, 16, 21, 43, 45).
P. aeruginosa and Rhizobium meliloti employ three groEL genes, of which there are one and two copies,
respectively, on operons that also encode GroES (43). In the
symbiotic bacteria of Amoeba proteus, GroEL is overproduced
by an additional promoter in the GroES-encoding part in front of the
groEL gene (1); in Agrobacterium
tumefaciens, two mRNA fragments are produced and the mRNA fragment
containing the gene encoding GroES is rapidly degraded (45).
We detected only one copy of the gene encoding MpB GroEL (Fig. 1),
located on the same operon that harbors the groES gene (Fig.
2). This suggests that overproduction of MpB GroEL occurs through the
mechanisms found in the X bacteria of Amoeba proteus or
Agrobacterium tumefaciens. Alternatively, it may well be
that Buchnera GroEL is more stable than is GroES and readily
accumulates while GroES is rapidly degraded.
To further identify potential genetic elements that may explain the
high level of GroEL accumulation, sequences upstream of the coding
regions were compared with the consensus sequences involved in
transcription and translation of the groE operons of
E. coli and the Buchnera sp. of A. pisum (38, 44, 55). This comparison disclosed the
presence of sequences highly homologous to the E. coli heat
shock promoter and Shine-Dalgarno sequences (Fig.
7). Although the MpB groE
operon sequence is nearly identical to that of the Buchnera
sp. of A. pisum in this region, we were not able to identify
the constitutive promoter sequence of the groE operon of
E. coli, which was previously reported to be present on the
groE operon of the Buchnera sp. from A. pisum (38). Our observation corroborates recent
findings that the only conserved promoter sequences of the
groE operons of the Buchnera spp. of A. pisum and Schizaphis graminum are those recognized by
32, a factor involved in the heat shock response
(5), and that the heat shock promoter alone is responsible
for transcription of the groE operon of the
Buchnera sp. from A. pisum (44). An AT-rich nucleotide sequence upstream of this promoter, which may enhance promoter activity (10), is present in the
groE operons of both E. coli and
Buchnera spp. (Fig. 7). These observations are of interest,
since GroEL expression in Buchnera spp. is similar to that
in E. coli cells growing under stress (5).
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FIG. 7.
Sequence comparison of regions involved in transcription
and translation of the groE operons of E. coli
(29, 55) and the Buchnera spp. of A. pisum (Ap) (38) and M. persicae
(Mp). Conserved regions of the putative heat shock promoter
and Shine-Dalgarno (SD) sequences are boxed, and the inverted repeats
of putative transcription terminator sites are indicated by arrows. The
localization of the constitutive promoter within the groE
operon of E. coli is underlined. Gaps are indicated by
dashes, stop codons are indicated by asterisks, and start codons are
indicated by M.
|
|
In vivo interference with the interactions among extracellular MpB
GroEL, PLRV, and beet western yellows luteovirus led to the suggestion
that a transient association is required to protect luteoviruses in the
hemolymph of an aphid from proteolysis (50, 51). Clearly,
these interactions differ from the usual intracellular polypeptide-GroEL interactions; the mechanisms and potential roles of
cofactors, including that of GroES, have not yet been revealed. It
should be noted, however, that a functional extracellular GroEL was
also observed in Helicobacter pylori, a gram-negative
bacterium which causes chronic gastritis. It produces a GroEL homolog
(HspB) which protects against inactivation of urease outside the
bacterial cell in the hostile environment of the stomach of a
vertebrate host (20, 40). Urease and HspB are released,
probably by cell autolysis, and adhere to the surfaces of intact
bacteria (40). Moreover, surface-associated Hsp60 fractions
were also found in P. aeruginosa and Legionella
pneumophila (31, 33). In this respect, it is
interesting that GroEL proteins of Buchnera spp. were found
to be more related to Hsp60s of pathogenic bacteria, such as L. pneumophila, than to E. coli GroEL (26).
| |
ACKNOWLEDGMENTS |
This research was financed in part by Priority Program Crop
Protection grant 45.014 from the Ministry of Agriculture, Nature Management and Fisheries (LNV) and the Netherlands Organisation for
Scientific Research (NWO).
Our discussions with K. E. Richards were greatly appreciated.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Virology, DLO Research Institute for Plant Protection (IPO-DLO), P.O. Box 9060, 6700 GW Wageningen, The Netherlands. Phone: 31 317 476141. Fax: 31 317 410113. E-mail:
J.F.J.M.vandenHeuvel{at}IPO.DLO.NL.
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Abstract
Introduction
