From the
Institute of Molecular Biology and Biotechnology, FORTH and Department of
Biology, University of Crete, P.O. Box 1527, GR-711 10 Iraklio, Crete, Greece,
M.E. Mueller Institute for Structural Biology,
Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel,
Switzerland, the ¶Department of Biological
Sciences, Imperial College at Wye, University of London, Ashford, Kent, United
Kingdom TN25 5AH, the ||Laboratory of Biological
Chemistry, Medical School, University of Ioaninna, Ioannina 45110 Greece, and
the **Molecular Genetics Unit CNRS FRE 2364, Institut
Pasteur, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France
Received for publication, February 24, 2003 , and in revised form, April 21, 2003.
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ABSTRACT |
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INTRODUCTION |
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TTS translocases contain an inner membrane core
(4,
12) and a putative ATPase that
is essential for secretion and is related to F1-ATPase subunits
(1315).
However, whereas F1-ATPase is functional as an
3
3 hexamer, flagellar FliI was proposed to
be monomeric (16,
17). FliI
(18) and Salmonella
InvC (15) have been shown to
hydrolyze ATP, albeit at much lower levels than the F1. Genetic and
in vitro studies have suggested physical interactions between the
ATPase and other TTS components
(16,
17,
19,
20). Nevertheless,
ultrastructural studies have failed to detect the ATPase associated with the
TTS translocase (7,
8,
12,
21), and the structure,
topology, and function of these proteins remain elusive.
To understand the molecular mechanism of the TTS ATPase, we purified and characterized HrcN of Pseudomonas syringae pathovar phaseolicola. HrcN is present in four quaternary forms. Of these, Form III (575 kDa) is a highly activated ATPase, a peripheral membrane protein, and may catalyze protein translocation.
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EXPERIMENTAL PROCEDURES |
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The hrcNPsph coding sequence was PCR-amplified (Deep Vent DNA polymerase; New England Biolabs) using cosmid pPL6 (carrying the P. syringae phaseolicola TTS regulon) (26) as a template and oligonucleotide primers (forward) 5'-TGAGGTGAACATATGAACGCTGCACTGAGC-3' and (reverse) 5'-GCGGATCGGATCCCAGTGTGTCTTCC-3' tailed with NdeI and BamHI restriction endonuclease sites, respectively. The NdeI-BamHI fragment was cloned in the corresponding sites of pET16b, giving rise to pIMBB240.
Metal Affinity
ChromatographyHis10HrcNPsph was purified by
metal affinity chromatography. E. coli cells expressing HisHrcN
(36 g) were grown in LB medium (40 liters) supplemented with ampicillin
(0.1 mg/ml) and IPTG (0.3 mM) for 3 h at 22 °C. Cells were
harvested, resuspended in Buffer A (450 ml; 50 mM Tris-HCl, pH 8,
50 mM NaCl, 10 mM
-mercaptoethanol, 50
mM imidazole, 20% glycerol), and broken by sonication (8 x 30
s; 10-µm setting; 4 °C; Soniprep 150). Insoluble debris and membranes
were removed by ultracentrifugation (100,000 x g; 4 °C)
Cytosolic extracts were loaded at 1.5 ml/min on a
Ni2+-nitrilotriacetic acid Fast Flow (Qiagen) resin (30
ml; equilibrated with Buffer A). The resin was washed with Buffer A (4.5
liters), followed by 150 ml of Buffer A, 50 mM imidazole.
Immobilized HisHrcN was eluted with two imidazole steps of 150 mM
(
6 mg) and 350 mM (
24 mg).
Hydrodynamic AnalysesSEC was as described (22), using Superose 6HR 10/30 or Superdex 200HR 10/30 prepacked FPLC columns (Amersham Biosciences). Stoke's radii (R) were determined from a plot of Kav versus log R of standards (see Table I) as described (27). Elution volumes were converted to Kav by the equation Kav = (Ve Vo)/(Vt Vo), where Vo represents the void volume (7.2 ml), Vt is the total bed volume (24 ml), and Ve is the elution volume. Sedimentation through sucrose gradients was carried out in a benchtop ultracentrifuge (Optima TLX; Beckman) (28). Typically, purified protein (2040 µg or 200 µl of 10 mg/ml total cytoplasmic extract) was layered on a 2-ml 2040% sucrose gradient prepared in 50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 10 mM EDTA, 5 mM dithiothreitol. After centrifugation (14 h; 50,000 rpm; 210,000 x g; TLS-55 rotor), fractions (100 µl) were collected. Proteins separated by SDS-PAGE were visualized by Coomassie Brilliant Blue staining or by immunostaining. s20,w values were determined by their sedimentation relative to protein standards (see Table I).
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Mr values were calculated as described
(27). Mr =
[(s20,wfN)/(1
v)], where f (frictional coefficient) = 6
R,
N is Avogadro's number,
is the solvent viscosity (of water)
(1.0019 centipoise), R is the Stokes radius (meters), v is
the protein's partial specific volume (0.74 ml/g) calculated from its amino
acid composition (available on the World Wide Web at
pbil.ibcp.fr),
and
is the density of water (0.99823 g/ml).
AUC was performed on an Optima XL-A analytical ultracentrifuge (Beckman) equipped with a 12-mm Epon double-sector cell in an An-60 Ti rotor. Sedimentation equilibrium runs were performed at 20 °C. Average molecular masses were evaluated using a floating base-line computer program (SEGAL) that adjusts the base-line absorbance to obtain the best linear fit of ln(absorbance) versus the square of the radial distance as described (available on the World Wide Web at www.biozentrum.unibas.ch/personal/jseelig/AUC/software00.html). A partial specific volume of 0.73 ml/g was used.
Transmission Electron MicroscopyHisHrcN particles were adsorbed for 20 s to glow-discharged carbon-coated copper grids, washed three times in double-distilled water, and negatively stained with 0.75% uranyl formate. Images were recorded on Eastman Kodak Co. SO-163 film (nominal magnification x 50,000), using a Hitachi H-8000 transmission electron microscope operating at 200 kV and employing low dose conditions (5 electrons/A2 per image).
To examine Hrp pilus production, E. coli strains were grown on TEM grids overnight in LB at 37 °C and washed twice in warmed LB, and cell density was adjusted to 0.2 A600 (29). IPTG (2 mM final concentration) was added, and 20-µl drops of the suspension were applied to TEM grids and incubated (5 h, 30 °C). Cells for sectioning were prepared as above except that they were grown in 10-ml flasks (5 h), pelleted, and fixed (1 h; 2% formaldehyde, 0.5% glutaraldehyde, 50 mM sodium cacodylate, pH 7.2), dehydrated in a graded ethanol series, and embedded in LR white resin. Ultrathin sections (90 nm) were mounted on 300-mesh gold grids and immunogold-labeled (29).
Chemicals, Biochemicals, and Miscellaneous MethodsChemicals were from Sigma, DNA enzymes were from MINOTECH, oligonucleotides were from MWG, dNTPs were from Promega, Sequenase was from Amersham Biosciences, cross-linkers were from Pierce, Centricon ultrafiltration concentrators were from Millipore, and detergents were from Anatrace. Protein purification, manipulations, and detection were as described (22, 30), using Amersham Biosciences FPLC systems and columns. Blue native electrophoresis was as described (31). Inner membrane vesicles were prepared as described (32, 33).
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RESULTS |
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HrcN synthesized with an amino-terminal decahistidinyl tag in E.
coli was purified to homogeneity (Fig.
1C, lane 5), and a polyclonal antiserum was
raised against it (see "Experimental Procedures"). HisHrcN yield
was poor (1 mg/liter); an expressed band of the expected molecular weight (48
kDa) was clearly detectable by -HisHrcN immunostaining (lanes
8 and 10) and poorly by Coomassie Brilliant Blue staining
(lane 2). A unique band of 48 kDa was also detected by
-HisHrcN immunostaining in extracts from P. syringae
phaseolicola cells that had been induced for TTS expression
(Fig. 2D, lane
6) (25) but not in
extracts from uninduced cells (lane 5). The
-HrcN antiserum
was used to subcellularly localize HrcN.
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HrcN Associates with MembranesTo determine the subcellular
localization of HrcN, we produced cytosolic extracts of P. syringae
phaseolicola (Fig.
2A) grown under TTS regulon-inducing (lanes
24) or noninducing (lane 1) conditions and examined them
by immunostaining. HrcN was only detected after growth under inducing
conditions (lane 2), whereas two control proteins, DnaK and SecY,
were expressed under both conditions. TTS-expressing cells were disrupted, and
polypeptides were separated into soluble (lane 4) and
membrane-associated (lane 3) material by ultracentrifugation (see
"Experimental Procedures"). Most of HrcN (90%) was detected
in the membrane-containing fraction that also contains the polytopic membrane
protein SecY (37). As
expected, the cytoplasmic DnaK protein was almost exclusively detected in the
soluble fraction. Similar results were obtained with E. coli MC4100
harboring the complete P. syringae phaseolicola TTS regulon on the
low copy number cosmid pPPY430 and the hrpL
factor under
lac control on a separate plasmid
(23,
38). MC4100/pPPY430/pHrpL
grown in the presence (Fig.
2B, lane 2) but not in the absence (lane
1) of IPTG expressed HrcN. Intracellular HrcN production is coincident
with biosynthesis of the extracellular TTS pilus
(Fig. 2E,
arrows) (9) and with
the acquisition of pathogenic potential (data not shown)
(23). TTS pili were not
present in noninduced cells (Fig.
2D). As expected, expression of control proteins DnaK and
SecY remained largely unaltered under both conditions
(Fig. 2B).
Fractionation of cellular material from induced cells revealed that HrcN is
found predominantly in the insoluble membrane fraction together with SecY
(lane 3), whereas DnaK is found in the soluble fraction (lane
4). To exclude the possibility that HrcN co-sediments with membranes
through nonspecific aggregation, MC4100/pPPY430/pHrpL was grown in the
presence (Fig. 2C,
lane 2) and in the absence (lane 1) of IPTG, and inner
membrane vesicles were prepared
(32,
33). HrcN was isolated bound
to inner membrane vesicles from cells expressing the TTS regulon (lane
2) but not from the nonexpressing cells (lane 1), whereas the
inner membrane protein SecY was present on both vesicle preparations. In
summary, our data indicate that HrcN appears to specifically associate with
the inner membrane of the cell.
To further study HrcN membrane association, we performed -HrcN
immunogold labeling (Fig. 2, F and
G). On whole cell thin sections, most (16-fold more) gold
particles were localized specifically to the inner face of the cell envelope
of E. coli MC4100 expressing the P. syringae phaseolicola
TTS (Fig. 2G) rather
than to the cytosol. Interestingly, most of the gold particles were clustered
(Fig. 2F). Labeling
was largely specific to HrcN, since 5-fold fewer gold particles localized to
the cell envelope of cells that did not express the TTS regulon
(Fig. 2F). Immunogold
labeling of the major outer membrane protein A (OmpA) revealed a similar
distribution of gold particles (95% of total label) to the cell periphery of
both induced (Fig. 2I)
and uninduced cells (Fig.
2H). 7580% of the label localized specifically to
the outer membrane leaflet. We conclude that HrcN associates with the inner
membrane.
HrcN Is a Peripheral Membrane ProteinTo examine the nature of HrcN association with the membrane, we employed a number of reagents used routinely to differentiate integral membrane proteins from peripherally associated polypeptides (Fig. 3). Even at modest concentrations, the chaotropes urea (Fig. 3A) and guanidine (Fig. 3B), efficiently removed HrcN (Fig. 3, lanes 4 and 6), indicating that the protein is only peripherally associated with the membrane. Other agents removed membrane-bound HrcN less efficiently (Na2CO3; Fig. 3C, lane 8) or not at all (NaCl; lanes 4 and 6), suggesting that HrcN interaction with the membrane is strong. All of the detergents tested extracted and solubilized significant amounts of HrcN from the membrane (Fig. 3, D (lanes 4 and 6) and E (lanes 4, 6, and 8)), although dodecyl maltoside was less efficient (Fig. 3D, lane 8).
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Finally, trypsinolysis was used to probe accessibility of HrcN at the
membrane. Soluble HrcN is particularly sensitive to even low amounts of
trypsin and appears by immunostaining with the polyclonal -HrcN
antiserum to be completely digested at trypsin concentrations above 0.1
µg/ml (Fig. 3F,
lanes 3 and 4). Protease resistance of an equivalent amount
of membrane-bound HrcN (Fig.
3G, lane 1) was significantly enhanced (compare
lanes 24 of G with lanes 24 of
F). Nevertheless, no immunostaining of the membrane-bound protein was
detectable at trypsin concentrations above 3 µg/ml
(Fig. 3G, lane
5), suggesting extensive degradation. Therefore, HrcN bound to the
membrane is only partially protected and remains largely protease-accessible.
We conclude that HrcN is a peripheral membrane protein that tightly associates
with an unknown membrane component(s).
HisHrcN Is a Functional ATPaseIs HisHrcN a structurally
intact and enzymatically active ATPase? Far UV CD showed that recombinant
HisHrcN is folded and extensively -helical (>40%;
Fig. 4A) and melts in
three distinct transitions (Tm1app = 41.3,
Tm2app = 46, and Tm3app = 62.7 °C;
Fig. 4B). This
indicated that HisHrcN is structurally intact, and its ATPase function was
tested below. HisHrcN hydrolyzes ATP in a linear time-dependent manner
(Fig. 4C). Increasing
the ATP concentration reveals that HisHrcN displays apparent Michaelis-Menten
saturation kinetics (Fig.
4D and see below), yielding a Vmax of
40 (µmol of Pi/mg of HisHrcN/min) and an apparent
Km of 1.3 mM. HisHrcN ATP hydrolysis
was optimal in the presence of Mg2+ but not of other
divalent metal ions (Fig.
4E), at pH 8 (Fig.
4F), and at 28 °C
(Fig. 4G).
Interestingly, HisHrcN ATPase activity was stimulated by increased protein
concentration in a nonlinear fashion (Fig.
4H), suggesting that the enzyme may be cooperatively
activated by self-association (see below). We conclude that HisHrcN is a
highly active ATPase that may be activated by homo-oligomerization.
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HisHrcN OligomerizationF1- and
V1-ATPases are hexameric
(39,
40). To examine whether
HisHrcN also oligomerizes, we investigated its hydrodynamic behavior using
size exclusion chromatography (SEC; Fig.
5A) and rate zonal centrifugation (RZC;
Fig. 5B). SEC analysis
on a Superose 6 resin (exclusion limit >5 MDa;
Fig. 5C) revealed four
HisHrcN populations with distinct Stokes radii
(Table I): Form I (fractions
3948) presumably monomeric and the most prominent species (82%);
Form II (fractions 3639); Form III (fractions 2634); and Form IV
(fractions 1921). Form II (see Fig.
5A; 150 mM elution) was not always detectable
and could not be studied further. Form III, the second most prominent
(
13%), migrated as a broad peak, suggesting a dynamic assembly state.
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We next examined HisHrcN quaternary organization using RZC
(Fig. 5B; see
"Experimental Procedures")
(28). HisHrcN Form IV and a
mixture of Forms IIII were analyzed separately in 2040% sucrose
gradients. The four forms migrated as distinct species with defined
sedimentation coefficients (Table
I). Sedimentation coefficients (determined by RZC) were combined
with Stokes radii (determined by SEC) to provide molecular weights
irrespective of shape (Table I;
see "Experimental Procedures")
(27). These indicate that Form
I corresponds to a HisHrcN monomer (Mr(app) = 46,700).
Form II (Mr(app) = 300,000) could represent hexamers,
and Form III (Mr(app) = 570,000714,000) could
represent dodedamers or slightly larger homo-oligomers
(Table I). Form IV is a very
large species (Mr(app)
3.8 MDa).
To accurately measure the masses of the HisHrcN forms, we employed sedimentation equilibrium analytical ultracentrifugation. Scaling up of HisHrcN purification yielded amounts sufficient for analytical ultracentrifugation analysis only of Forms I and III (Fig. 5C; Table I). A single exponential curve optimally fitted to the available data revealed calculated masses for Form I and III of 43.5 kDa (monomer) and 575.8 kDa (11.9 subunits), respectively.
HisHrcN Form III from the peak SEC fraction of Fig. 5A was also examined by negative stain transmission electron microscopy (Fig. 5D). Peak fraction 30 contained a distribution of fairly uniform particles (white arrows). Image averaging (inset) reveals a round particle with 13 ± 1-nm outer diameter. Additional less uniform particles are also seen in the micrograph (black arrows). These particles are very large and presumably represent material aggregated during adsorption on the grid. We conclude that HisHrcN oligomerizes into homo-hexa- and dodecameric assemblies in solution in the absence of ligands or other TTS proteins.
HisHrcN Form III Is a Highly Active ATPaseTo determine whether all forms of HisHrcN were active in ATP hydrolysis, SEC fractions of HisHrcN were assayed for ATPase activity (Fig. 6A). Strikingly, the bulk of the high level ATPase activity was associated with Form III despite the fact that Form III represents only a minor physical population of the enzyme. In contrast, the activity of Form I, the major HisHrcN population, was measurable albeit low. ATPase activities of Forms II and IV were very low and were not studied further.
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To compare the activities of Forms I and III, we characterized the chromatographic peak fractions by detailed kinetics (Table II). Form I had a Km of 0.1 mM, whereas that of Form III was 10-fold higher. Form I had a low ATP turnover similar to that of other TTS ATPases (15, 18). Strikingly, the specific activity (i.e. ATP turnover per protomer) of Form III was more than 700 times that of Form I (Table II).
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To exclude the possibility that the ATPase activity associated with Form III was the result of a highly active contaminant present in trace amounts, we sought to determine whether Form III could be assembled de novo from isolated monomeric HisHrcN. To this end, highly purified HisHrcN Form I (see "Experimental Procedures") largely devoid of Form III (<2%) was concentrated (as in Fig. 4H), and samples at increasing concentrations were analyzed by SEC (Fig. 6B). Concentration caused visible reduction of Form I and concomitant increase of Form III. Similarly, the ATPase of Form III generated in this experiment is also enhanced significantly in a concentration-dependent manner (Fig. 6C) and displays a specific ATP turnover (33 min1/protomer) similar to that of unconcentrated Form III (Table II).
We conclude that the HisHrcN ATPase is hyperactivated upon dodecamerization, implying that Form III is a physiologically relevant form of the enzyme and does not arise from nonspecific aggregation. Form III may be the functional form of the enzyme during TTS protein translocation.
Native Soluble P. syringae phaseolicola HrcN Forms
OligomersTo test whether native HrcN in P. syringae
phaseolicola is also oligomeric, we first characterized HrcN from the
small cytoplasmic pool. Cytosolic polypeptides from P. syringae
phaseolicola expressing the TTS translocase were cross-linked in
vivo (Fig. 7A).
The addition of the homobifunctional amine-specific cross-linker DSP led to
the appearance of two novel slow migrating species with approximate masses of
300 and
600 kDa (lane 2; filled arrows) not
present in the untreated sample (lane 1). Both cross-linked species
disappeared upon reduction of the cross-linker (lane 3). Two
nonspecific immuno-reacting cytosolicpolypeptides that are not affected by DSP
were used as internal controls (lane 2; open arrows).
Similar results were obtained with formaldehyde (data not shown).
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We next determined whether the cross-linked species
(Fig. 7A) derived from
preexisting HrcN oligomers. Polypeptides from TTS-expressing P. syringae
phaseolicola cells were separated by SEC
(Fig. 7, B and
C) and RZC (Fig. 7,
D and E), analyzed by SDS-PAGE, and visualized
by either Coomassie Brilliant Blue (Fig. 7,
B and D) or by -HrcN immunostaining
(Fig. 7, C and
E). Four distinct HrcN forms were identified: Form I,
monomeric HrcN (Fig.
7E, fractions 48); Form II of
300
kDa (Fig. 7E;
fractions 11 and 12), Form III of
629 kDa
(Fig. 7C,
fractions 3335, and Fig.
7E, fractions 14 and 17), and Form IV
of
3.5 MDa (Fig.
7C, fractions 1821, and
Fig. 7E, fractions
19 and 22). The ratio between the four forms varied in different
cell extracts, perhaps implying a dynamic assembly state.
We conclude that, like recombinant HisHrcN, native HrcN forms oligomeric assemblies in vivo in the P. syringae phaseolicola cytoplasm. These assemblies have apparent masses similar to the four forms of purified HisHrcN (Fig. 5, Table I) and may represent hetero- or homo-oligomers.
Native HrcN at the Membrane Is DodecamericTo determine
which of the HrcN forms is membrane-bound (Figs.
2 and
3), isolated membranes from
TTS-expressing E. coli were treated with the homobifunctional
cysteine-specific cross-linker bismaleimidohexane (BMH;
Fig. 8A) to promote
the formation of interprotomer covalent links between the nine HrcN cysteinyl
residues. A gradual increase of the cross-linker concentration from 0.03
mM (lane 2) to 0.36 mM (lane 5) led to
the appearance of discreet higher order species of 300 and
600 kDa
followed by the disappearance of the monomeric form. Similar results were
obtained using the cross-linker DSP (Fig.
8, lanes 68).
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To examine oligomerization of membrane-bound HrcN under more
"native" conditions and to determine its mass, BN-PAGE
(31) was employed
(Fig. 8B). Isolated
membranes from TTS-expressing E. coli were extracted with the
nonionic detergent -octyl glucoside, and solubilized polypeptides were
separated by BN-PAGE and immunostained with
-HrcN antibodies. A single
broad band of
600 kDa was detected in these extracts
(Fig. 8B, lane
2) but was not present in extracts from noninduced cells (lane
1). Since the same migration behavior was seen at different detergent
concentrations (0.252%), including those below the critical micellar
concentration, the detergent is unlikely to contribute excessively to the
apparent mass. This
600-kDa membrane-extracted HrcN species migrates
similarly to the soluble dodecameric HisHrcN Form III
(Fig. 8B, lane
3). In addition, BN-PAGE analysis reveals the presence of a distinct
150-kDa species (filled arrow) not seen previously with the
other methods. These results indicate that the dodecamer is the predominant
membrane-bound HrcN form.
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DISCUSSION |
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HisHrcN was purified in a folded form and in amounts sufficient for
biochemical and structural analysis. The protein melts in three transitions,
which could indicate that HrcN, like the F1-ATPase subunits
(40), is a three-domain
protein. Hydrodynamic, cross-linking, and ultrastructural analyses revealed
that both native HrcN and recombinant HisHrcN assemble in distinct quaternary
forms (Fig. 5,
6,
7,
8;
Table I): (a) a
monomeric Form I of 48 kDa; (b) a rather unstable Form II of
300 kDa that could be a transient intermediate (Form II may be similar to
the X-300 cross-linked species (Figs.
7A and
8A); (c) the
dodecameric 575-kDa Form III that comprises an organized round particle with
an outer diameter of
13 nm (Fig.
5D) (this particle, with an average protein density of
0.8 Da/Å3 and an assumed height of 5.6 nm, would have the
mass of Form III in solution (
575 kDa)); and (d) Form IV, a very
large aggregate (Table I). An
additional form of 150 kDa was only detected by BN-PAGE
(Fig. 8B).
Concentration of HisHrcN Form I leads to de novo formation of Form
III (Fig. 6, B and
C) in a reversible
reaction.3 Therefore,
since oligomerization does not require any detectable auxiliary factors, it is
an intrinsic property of the HrcN polypeptide. The findings that Form III is
active in ATP hydrolysis (Fig.
6), is obtained in the presence of reducing agent, forms canonical
particles (Fig. 5D),
and is also generated natively in wild type P. syringae phaseolicola
cells (Fig. 7) argue strongly
against the possibility that the dodecameric particle is the result of
nonspecific aggregation or misfolding. Nevertheless, it will be important to
accurately determine the kinetics of assembly-disassembly of the
"ATPase-inactive" monomer to the "ATPase-active"
dodecameric particle. Modulation of this reaction may be a fundamental element
of the catalytic cycle.
Immunogold localization (Fig.
2G) and biochemical fractionation (Figs.
2, AC, and
3) experiments revealed that
90% of HrcN associates with the membrane. Importantly, membrane-bound
HrcN is almost exclusively a dodecamer
(Fig. 8B). It was
recently suggested that the homologous FliI ATPase may associate with the
phospholipid component of the cell membrane
(41). Earlier studies failed
to demonstrate a direct physical link of the TTS ATPase with the membrane
where the putative translocation "conduit" resides. Other ATPases
that catalyze protein translocation through membranes (e.g. Tim44
(42), PulE
(43), and SecA
(37)) are also soluble
proteins that associate peripherally albeit very tightly with membranes. We
are currently examining the potential role of lipids or proteinaceous factors
(e.g. other TTS subunits) in HrcN membrane assembly. Such composite
interactions would explain three distinct properties of HrcN observed here:
(a) it is relatively recalcitrant to extraction from the membrane
with agents (other than nonchaotropes) that usually remove peripheral membrane
proteins (Fig. 3C);
(b) its ATPase activity in solution is unregulated
(Table I); and (c)
immunoelectron microscopy experiments reveal a significant degree of
clustering at defined membrane locations
(Fig. 2G). Such sites
could represent associations of dodecameric HrcN with the membrane-embedded
TTS translocase. Other soluble, peripheral ATPase subunits of protein
translocases (e.g. SecA)
(37) are similarly tightly
bound to the membrane with complex lipid and protein interactions and appear
to even become integrated in the bilayer plane.
Mutagenesis experiments and sequence conservation between TTS
(14,
15) and flagellar ATPases
(13) and the
F1/V1-ATPase subunits suggested that these proteins may
link metabolic energy to secretion through the TTS translocase. HisHrcN has at
least two distinct ATPase activities: the low level basal activity of
monomeric HisHrcN (Kcat = 0.052
s1) (Table
II) that is similar to the ATPase activities of a
Shigella InvC-glutathione S-transferase fusion
(Kcat = 0.2 s1)
(15) and of the flagellar FliI
(Kcat = 0.16 s1)
(18) and the novel high level
activity of HisHrcN Form III (Kcat = 37.3
s1) (Table
II). In fact, there is a cooperative induction of the ability of
the enzyme to hydrolyze ATP as it assembles from Form I into Form III (Figs.
4H and
6, B and C).
Similarly, assembled F1 hexamers detached from the membrane
component (F0) are highly active in ATP hydrolysis
(40), whereas individual
F1 or
monomers are not
(44). In F1 and
several other ATPases,
/
dimers form the minimal mononucleotide
binding and ATPase unit (44).
Our data indicate that oligomerization of the TTS ATPase may act as an
intramolecular regulatory activation mechanism.
Aditional intermolecular factors may control the activation of HrcN ATPase by regulating its oligomerization. One such factor could be the FliH protein (HrpE in P. syringae phaseolicola; YscL in Yersinia), shown to bind to the flagellar TTS ATPase FliI (19, 20) in vitro and to repress the basal ATPase activity of monomeric FliI (16). Thus, FliI, which, like HrcN, interacts with itself (19), may be prevented from forming large oligomeric assemblies (16, 17) active in ATP hydrolysis. Such interactions would ensure that the active dodecameric form of the TTS ATPase is only assembled in a controlled and ordered manner during the catalytic cycle at the membrane.
The observation that different HrcN forms can coexist (Figs.
5A,
7E, and
8B, lane 3)
suggests that HrcN oligomerization is highly dynamic. This could explain why
stable complexes of TTS machineries that include the ATPase have not yet been
isolated. In this sense, TTS ATPases may be more similar to the dynamically
assembling V1 (39)
rather than to the more stably membrane-bound F1
(39,
40).
F1/V1-ATPases function as hexamers of alternating
3
3 subunits
(39,
40) and, like HrcN, display
ATPase activity in the absence of any other subunits
(45). However, our experiments
suggest that HrcN Form III has a dodecameric or two-hexamer organization. HrcN
organization is therefore distinct from that of the F1-ATPase.
Other dodecameric traffic ATPases homologous to F1 are known and
comprise double hexameric stacks (AAA family)
(46). HrcN Form II of
300
kDa (Figs. 5A,
7, A and E,
and 8A) that is barely
active in ATP hydrolysis (Fig.
6A) could represent a hexameric assembly
intermediate.
Collectively, our data lead us to formulate the hypothesis that HrcN Form III may be the active enzyme species in the cell during TTS protein secretion. Clearly, additional experimentation using a functionally reconstituted in vitro system will be required to directly test this and to determine the physiological relevance of the other TTS ATPase forms. Determination of the molecular features that allow the apparently similar F1 and TTS ATPases to participate in the transmembrane pumping of such divergent substrates (i.e. protons for the F1/V1 and polypeptides for the TTS) promises to be an exciting future challenge.
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FOOTNOTES |
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* This work was supported by European Union Directorate of Science and
Technology Grants Biotech2-BIO4-CT97-224 (to A. Ec., N. J. P., and J. M.) and
RTN1-1999-00149 (to A. Ec., A. E., and A. P. P.), the a grant from the BBSRC
(to J. M.), and the Maurice Mueller Foundation and Swiss National Foundation
for Scientific Research Grant 31-59415.99 (to A. E.). The costs of publication
of this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
To whom correspondence should be addressed. Tel./Fax: 30-2810-391166; E-mail:
aeconomo{at}imbb.forth.gr.
1 The abbreviations used are: TTS, type III secretion; BN-PAGE, blue native
PAGE; DSP, dithiobis(succinimidyl propionate); SEC, size exclusion
chromatography; RZC, rate zonal centrifugation; TEM, transmission electron
microscopy; IPTG, isopropyl-1-thio--D-galactopyranoside;
FPLC, fast protein liquid chromatography; MOPS, 4-morpholinepropanesulfonic
acid.
2 A. P. Tampakaki and N. J. Panopoulos, unpublished results.
3 C. Pozidis and A. Economou, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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