Recent studies of the Fo motor have focused on the ion path through the membrane and the coupling Keywords atomic force microscopy; c ring stoichiometry; F-ATP synthase; Ilyobacter tarta
Trang 1in the sodium F-ATP synthase
Thomas Meier1, Jinshu Yu2, Thomas Raschle1, Fabienne Henzen1, Peter Dimroth1
and Daniel J Muller2
1 Institut fu¨r Mikrobiologie, Eidgeno¨ssische Technische Hochschule, Zu¨rich, Switzerland
2 Center of Biotechnology, University of Technology, Dresden, Germany
F-type ATP synthases are multisubunit protein
com-plexes located in the membrane of mitochondria,
chloro-plasts and bacteria These enzymes use an electrochemical
H+ or Na+ gradient across the host membrane for
the synthesis of ATP, the universal energy currency of
living cells [1] F-ATP synthases are built from two
subcomplexes: the water soluble F1 part, with the
composition a3b3cde, and the membrane-embedded Fo
part, which in the simplest case has the subunit
com-position ab2c10)14 In the crystal structure of F1, a3b3
forms a cylinder around the extended a-helical c
sub-unit [2] Part of the c subsub-unit protrudes from the
bottom of the cylinder and forms the central stalk
together with the e subunit A striking feature of the structure is an inherent asymmetry among the catalytic
b subunits In combination with the asymmetric loca-tion of the central c subunit, and in accordance with the binding change mechanism [3], this arrangement suggests a catalytic mechanism in which the c subunit rotates within the a3b3 cylinder Elegant experiments have subsequently visually verified this rotation [4] The Fo part, a rotary motor by itself, is fueled by the electrochemical H+ or Na+ gradient and, upon rota-tion, translocates these ions across the membrane Recent studies of the Fo motor have focused on the ion path through the membrane and the coupling
Keywords
atomic force microscopy; c ring
stoichiometry; F-ATP synthase; Ilyobacter
tartaricus; Propionigenium modestum
Correspondence
T Meier, Institut fu¨r Mikrobiologie,
Eidgeno¨ssische Technische Hochschule
Zu¨rich (ETH-Ho¨nggerberg),
Wolfgang-Pauli-Str 10, CH 8093 Zu¨rich, Switzerland
Fax: +41 44 6321378
Tel: +41 44 6325523
E-mail: meier@micro.biol.ethz.ch
(Received 27 June 2005, accepted 26
August 2005)
doi:10.1111/j.1742-4658.2005.04940.x
The Na+-dependent F-ATP synthases of Ilyobacter tartaricus and Propioni-genium modestum contain membrane-embedded ring-shaped c subunit assemblies with a stoichiometry of 11 Subunit c from either organism was overexpressed in Escherichia coli using a plasmid containing the corres-ponding gene, extracted from the membrane using detergent and then puri-fied Subsequent analyses by SDS⁄ PAGE revealed that only a minor portion of the c subunits had assembled into stable rings, while the major-ity migrated as monomers The population of rings consisted mainly of c11, but more slowly migrating assemblies were also found, which might reflect other c ring stoichiometries We show that they consisted of higher aggre-gates of homogeneous c11 rings and⁄ or assemblies of c11 rings and single
c monomers Atomic force microscopy topographs of c rings reconstituted into lipid bilayers showed that the c ring assemblies had identical diameters and that stoichiometries throughout all rings resolved at high resolution This finding did not depend on whether the rings were assembled into crys-talline or densely packed assemblies Most of these rings represented com-pletely assembled undecameric complexes Occasionally, rings lacking a few subunits or hosting additional subunits in their cavity were observed The latter rings may represent the aggregates between c11 and c1, as observed
by SDS⁄ PAGE Our results are congruent with a stable c11ring stoichiom-etry that seems to not be influenced by the expression level of subunit c in the bacteria
Abbreviations
AFM, atomic force microscopy.
Trang 2between ion flux and torque generation [5–7]
Addi-tionally, using various experimental approaches,
increasing evidence has accumulated on the overall
shape of the Fo domain, particularly the ring-shaped
c subunit assemblies The 2.4 A˚ resolution structure of
the Ilyobacter tartaricus c ring, reported recently,
imposes important restrictions on proposed models for
ion translocation and torque generation [8]
Further-more, a k ring structure from the V-ATPase of
Entero-coccus hirae shares features of ion binding with the
I tartaricusc ring, supporting a common ion
transloca-tion mechanism in the two types of ATPases [9] For
detailed insights into this mechanism, high-resolution
structural data of the Fosubunits a and b2are required
It has been shown that these subunits flank the c ring
on the outside, but details of their structures have not
yet been explored [10] The number of c subunits in the
rotor ring is not fixed but varies among species For
example, rings comprising 10, 14 and 11 subunits have
been found in yeast [11], chloroplasts [12] and the
bac-terium I tartaricus [13], respectively Recently, a ring
of 15 subunits was found in the alkaliphilic
cyanobacte-rium Spirulina platensis, demonstrating that a
sym-metry mismatch between the F1and Fomotor is not an
essential feature for function [14] The number of
binding sites on the c ring determines the ATP to
proton⁄ Na+ ratio, and therefore this stoichiometry is
an important bioenergetic parameter for the cell As
the number of subunits can vary among species, the
question was raised whether this stoichiometry could
also vary within one species in order to adapt to
speci-fic energetic requirements of the cells [15] This
proposi-tion seemed to be supported by an effect of the carbon
source on the expression level of subunit c in
Escheri-chia coli However, structural analyses of rotors from
I tartaricus and from chloroplasts showed that their
stoichiometry seems to be constrained by the nearest
neighbor interaction between the subunits [16] This
question has also been addressed with subunit c from
Escherichia coli [17], where annular shaped particles
were detected by electron microscopy after
reconstitu-tion from single c subunits In agreement with the
above conclusions, it has been suggested that the
primary protein structure determines the ability of
subunit c to form rings Furthermore, it was shown, by
gradient gel analysis, that the number of subunits in
the oligomer III isolated from the Chlamydomonas
reinhardtii chloroplast ATP synthase is not affected
by the metabolic state of the cells [18] However, to
date, no structural methods have been applied to
clarify whether the stoichiometry of the c rings is
influenced by variation of the expression level of
subunit c
In the present study we investigated whether the
c subunits from the sodium F-ATP synthase of
I tartaricus and of Propionigenium modestum assemble into uniform rings after heterologous overexpression in
E coli Atomic force microscopy (AFM) topographs showed that complete rings were composed exclusively
of 11 subunits and that defective rings exhibited the same diameter as intact ones Hence, intrinsic features
of the I tartaricus c subunits are responsible for the formation of c11oligomers in the fully assembled rings
Results and Discussion
Synthesis and assembly of subunit c from
I tartaricus in E coli
To investigate whether heterologously synthesized c subunits from I tartaricus assemble properly into rings, we used the E coli strain BL21(DE3) as a host From wild-type P modestum or I tartaricus cells, the
c11 ring is easily purified by extraction from mem-branes using lauroylsarcosine and subsequent ammo-nium sulfate precipitation As a result of their extreme stability, these rings are easily recognized by SDS⁄ PAGE The E coli BL21(DE3) cells transformed with recombinant plasmids harboring the c subunit genes of I tartaricus or P modestum under control of the strong T7 promoter produced large amounts of the appropriate c subunit in the monomeric state, but also sizeable amounts of oligomeric assemblies with a ratio
of 9 : 1 (c1: coligo) For further analyses, these assemblies were purified by sucrose density gradient centrifugation and subjected to SDS⁄ PAGE The results shown in Fig 1 indicate that the assemblies consisted not only of c11, but also of higher aggregates However, these aggregates are made up exclusively of
c subunits because they are converted completely into the monomeric form by treatment with trichloroacetic acid A similar pattern of bands was observed with the recombinant T67C mutant, with the exception of an additional band corresponding to (c11)2and aggregates
at the top of the gel For comparison, the c11ring pre-paration of wild-type I tartaricus cells is shown Here, the c11 ring formed the most prominent species, and higher aggregates or the c monomer were less abun-dant than in the preparations from recombinant E coli cells
Investigation of the aggregation status of c ring preparations by Blue Native PAGE
As shown above (Fig 1) our c ring preparations con-tained various amounts of the c monomer unit and a
Trang 3number of aggregates that resisted disassembly into c11
by SDS To further investigate the aggregation status
of these preparations, we performed Blue Native
PAGE in the first dimension followed by SDS⁄ PAGE
in the second dimension The results (Fig 2) indicate
that the c ring prepared from I tartaricus wild-type
cells in octylglucoside contained not only c11 and the
stable aggregates observed in Fig 1, but also a number
of supercomplexes corresponding to (c11)n, with n
ran-ging from two to approximately five The most
abun-dant species was c11, and each supercomplex of higher
order was present in two- to threefold lower quantities
than the previous one All of these supercomplexes
dis-assembled by SDS into the c11oligomer (and the
SDS-stable aggregates of c11, see below) as shown by the
equal mobility during SDS⁄ PAGE (the second
dimen-sion in Fig 2) The aggregation into supercomplexes
was prevented if the detergent octylglucoside was
replaced by Triton X-100 (Fig 2A,B) The formation
of supercomplexes was also investigated in the
recomb-inantly synthesized T67C mutant Here, in addition to
higher aggregates, a (c11)2 form was observed which
Fig 1 SDS gel electrophoresis of purified c ring preparations The
c rings from Ilyobacter tartaricus and Propionigenium modestum
were heterologously expressed in Escherichia coli and purified as
described in the Experimental procedures Two to three
micro-grams of each sample was subjected to SDS ⁄ PAGE and the gels
were stained with silver The positions of the monomeric c subunit
(c1), the c ring (c11) and the c ring dimer (c11)2are marked on the
left side Lanes 1 and 4, purified c rings from P modestum and
I tartaricus, respectively, isolated from the heterologous expression
cultures Lanes 2 and 5, disintegration of c rings to the c monomer
by treatment with trichloroacetic acid Lane 3, mutant T67C
harbouring an SDS-stable c 11 dimer Lane 6, c ring purified from
wild-type I tartaricus cells A molecular mass standard is shown.
A
B
C
Fig 2 Supercomplex formation of c 11 rings visualized by Blue Native gel electrophoresis Five micrograms of c ring in buffer con-taining 10 m M Tris ⁄ HCl, pH 8.0, and 1.5% (w ⁄ v) octylglucoside was loaded on a Blue Native gel (5–17% acrylamide gradient), as described in the Experimental procedures The samples contained purified c ring from wild-type Ilyobacter tartaricus cells without (A) and with (B) addition of 0.2% (v ⁄ v) Triton X-100 The c ring mutant T67C of Propionigenium modestum was used in (C) After the run
in the first dimension, the gel lane was loaded onto a SDS gel for the run in the second dimension The gels were subsequently stained with silver Intact c 11 ring and its supercomplexes were marked with c11with the indexed numbers (n ¼ 1–4) correspond-ing to the amount of complexed rcorrespond-ings (c11)n The monomeric c sub-unit is marked with c 1 A molecular mass standard is shown.
Trang 4did not disintegrate into the c11oligomer by SDS,
indi-cating that a covalent disulfide bond had been formed
by the newly introduced cysteine residues
Composition of the SDS-resistant c11aggregates
As described above, our c11ring preparations also
con-tained a distinct number of SDS-resistant complexes of
higher molecular weight, which might signify rings
with different amounts of tightly bound phospholipids
To investigate this possibility, the c ring preparation
was incubated with phospholipase C, phospholipase
A2 or lipase, and the products were analyzed by
SDS⁄ PAGE The results (Fig 3) indicate that none of
these enzymes significantly decreased the amount of
the stable c11 aggregates A similar observation was
made after incubating the sample for 5 min at 95C in
SDS-containing loading buffer; this result confirms the
extreme stability of these aggregates We conclude,
from these results, that the higher molecular weight of
the stable aggregates cannot be attributed to strongly
bound phospholipids This conclusion is in agreement
with those of previous experiments, which showed that
the detergent-purified c ring contained no bound
phospholipids and the ones observed on one side of
the rings originated from the reconstitution procedure
[19]
On SDS⁄ PAGE, the SDS-resistant aggregates migra-ted between c11and (c11)2 We therefore reasoned that these complexes might consist of c11with one or more
c monomers attached To investigate this possibility, the homogeneous c11 ring was isolated by electro-elution of the c11 band excised from the SDS gel (Fig 4) During storage for at least 1 month, no aggre-gates or monomeric c units were formed from pure
c11ring preparations However, after addition of isola-ted c monomers and incubation overnight, the stable aggregates were formed again This suggests that the c monomer assembled with other c subunits and rings to form a ladder of higher aggregates To test this hypo-thesis, higher aggregates were specifically electroeluted from the gel and subjected to SDS⁄ PAGE without heat treatment The results showed that some aggre-gates converted to c11and c1 It may therefore be con-cluded that c11and c1 form stable aggregates and that these aggregates are in dynamic equilibrium with c11 and c1 The addition of palmitoyl-oleyl-phosphatidyl-choline to pure c11 did not result in the formation of any stable aggregates, confirming our conclusion that these aggregates do not represent c11rings with bound phospholipid molecules
Further experiments showed that the aggregation of
c11 and c1 was faster at 25C or 37 C than at 4 C
Fig 4 In vitro aggregation of c11with c1to complexes resistant to SDS For the preparation of homogeneous c11, 1 mg of wild-type c ring from Ilyobacter tartaricus (lane 1) was applied onto a prepara-tive SDS gel After the run, the c11band was cut out with a scalpel and the protein was electroeluted from the gel pieces to obtain pure c ring, as described in the Experimental procedures (lanes 2 and 5) As a control, c-ring bands migrating more slowly were cut out and electroeluted (lane 3) Upon incubation of 2 lg of pure c11 with 2 lg of c 1 purified in detergent, the slower migrating band reappeared (lanes 4 and 8) Upon incubation of 2 lg of pure c 11
with 2 and 10 lg of c1purified in chloroform ⁄ methanol, the slower migrating c ring aggregates did not reappear (lanes 6 and 7) Lane
9, c 1 purified by extraction with chloroform ⁄ methanol Lane 10, c 1
purified by sucrose density gradient centrifugation with octylgluco-side as the detergent Lane 11, 2 lg of c ring after incubation with
5 lg of palmitoyl-oleyl-phosphatidylcholine A molecular mass standard is shown.
Fig 3 Incubation of c ring with phospholipases and lipase The
c ring samples isolated from Ilyobacter tartaricus wild-type cells
were incubated with phospholipase C (PLC), phospholipase A2
(PLA2) and lipase (Lip), as described in the Experimental
proce-dures, and 4 lg aliquots were loaded onto an SDS gel The
enzymes alone were applied to separate lanes, as indicated Also
shown is the nontreated c ring (–) and the c ring incubated at 95 C
for 5 min The gel was stained with silver A molecular mass
stand-ard is shown.
Trang 5and reached approximately 90% completion after
1 day Interestingly, the stable aggregates of c11and c1,
or of several c1 moieties, were formed with c1 isolated
in detergent (by sucrose density centrifugation) but not
after the extraction of c1 with chloroform⁄ methanol
and reconstitution into a water⁄ detergent mixture
These results suggest different structures for the two
different preparations of the c monomer
AFM of c subunit preparations
To investigate whether the heterologously expressed c
rings exhibited stoichiometries other than the
previ-ously observed undecameric composition, all purified
samples were reconstituted into lipid bilayers, as
des-cribed previously [20], and imaged by AFM
High-resolution AFM topographs of c ring preparations
from I tartaricus (Fig 5) and P modestum (Fig 6)
showed surveys of crystalline (A) and densely packed
(B) regions of the reconstituted c subunits The
undecameric subunit stoichiometry of the c rings was
more clearly visible in the densely packed regions of
the unprocessed topographs Those c rings that were assembled into a 2D crystal exhibited an upside-down orientation, with one oligomer neighbored by three oligomers showing an opposite orientation In agree-ment with previous results, the more elevated oligo-mers (bright white areas) protruded from the lower and wider c rings by about 1.1 ± 0.2 nm (n¼ 50) and thus partly prevented the AFM stylus from contouring the wider rings [13] However, for statistical analyses
we performed reference-free single particle analysis of the densely packed c rings All classes of complete c rings exhibited 11 subunits forming the donut-like oligo-mer (first image of Figs 5D and 6D) However, some rings were incomplete, missing one or more subunits Compared with AFM topographs of c rings isolated from wild-type I tartaricus ATP synthase [13,16], the reconstituted samples investigated in the present study showed more of these structural inconsistencies The presence of incompletely assembled c rings from
I tartaricus and spinach chloroplast F-ATP synthases was previously observed by AFM [16] As the dia-meter of the incomplete c rings did not change in any
Fig 5 Atomic force microscopy (AFM) topographs of c subunit oligomers from Ilyobacter tartaricus F-ATP synthase overexpressed in Escherichia coli The undecameric oligomers were reconstituted into the lipid bilayer and imaged in buffer solution (A) A survey of oligomers assembled into a 2D crystal The donut-shaped oligomers were inserted into the membrane exposing either one of their surfaces to the AFM stylus (B) A survey of densely assembled oligomers Arrows point out oligomers either missing one subunit or showing additional subunits inside their central cavities (C) A gallery of c rings observed from the densely packed arrangement The first topograph represents
a reference-free single particle average obtained from more than 300 c rings Most of the examples selected exhibit additional central pro-trusions (D) A gallery of c rings observed from the crystalline arrangement Examples selected exhibit additional central propro-trusions The dashed circles with a diameter of 5.7 nm demonstrate that the outer diameters of the c rings are very consistent with each other Topo-graphs exhibit a gray scale corresponding to a vertical height of 3 nm.
Trang 6preparation investigated, it was concluded that the
diameter of the c rings may be determined by the
struc-ture of the c monomer and not by the number of
assem-bled subunits This finding is in agreement with our
present analysis of the defective rings overexpressed in
E coli, which exhibited the same outer diameter
5.7 ± 0.3 nm (n¼ 300) as the complete rings
5.6 ± 0.3 nm (n¼ 280) within an experimental error of
0.1 nm This structural agreement did not depend on
the number of subunits missing to complete the c ring
The incompletely assembled c rings prepared from
chloroplasts and bacteria represented less than 5% of
all rings imaged [16] In contrast, c rings from I
tar-taricus (Fig 5) or P modestum (Fig 6) synthesized
recombinantly in E coli showed an increased amount
of incomplete c rings, exhibiting a total content of
8% (n ¼ 2000) Among these, c10, c9 and c8
assem-blies represented the most abundant species These
defective rings could probably not be observed on the
SDS gel because the detergent dissociates the less
stable c2 to c10 assemblies into monomeric units
Therefore, we assume that upon insertion of the last, 11th, c subunit, the assembly becomes resistant to SDS
or heat treatment The observed accumulation of the incomplete c10 complex in the recombinant c ring preparations suggests that the insertion of the last c subunit forms the limiting step in the assembly process
of a functional oligomer
Upon closer inspection, the occurrence of additional protrusions in the cavity, and sometimes at the side of some oligomers, became apparent (galleys of Figs 5 and 6) It may be assumed that these protrusions rep-resent one or more c subunits attached to the ring-shaped oligomer Such a finding is in agreement with the observation presented in Fig 4, in which the com-plete c subunit oligomers, hosting additional c sub-units, migrate at higher molecular weights in the SDS gel electrophoresis Furthermore, it also corresponds
to the recent observation that the analogous rotor from chloroplast F-ATP synthase may accommodate small transmembrane proteins within its central cavity [21]
Fig 6 Atomic force microscopy (AFM) topographs of c subunit oligomers from Propionigenium modestum F-ATP synthase overexpressed
in Escherichia coli The oligomers were reconstituted into the lipid bilayer and imaged in buffer solution (A) Survey of undecameric oligo-mers assembled into a 2D crystal The donut-shaped oligooligo-mers were inserted into the membrane exposing either one of their surfaces to the AFM stylus (B) A survey of densely assembled oligomers Arrows point out oligomers either missing one subunit or showing additional subunits inside their central cavities (C) A gallery of c rings observed from the densely packed arrangement The first topograph represents
a reference-free single particle average obtained from more than 200 c rings Most of the examples selected exhibit additional central protru-sions (D) A gallery of c rings observed from the crystalline arrangement Examples selected exhibit additional central protruprotru-sions The dashed circles with a diameter of 5.7 nm demonstrate that the outer diameter of the c rings is very consistent with each other Topographs exhibit a gray scale corresponding to a vertical height of 3 nm.
Trang 7Recently, the crystal structure of the rotor ring from
the I tartaricus F-ATP synthase has been solved and
provides striking details concerning the mechanism of
the Fo motor of ATP synthase [8] Of particular
interest in this structure is the architecture of the
Na+-binding site, which closely resembles that of the
k ring from the E hirae V-ATPase [9] As a result of
its extreme stability [22], the c11 rotor ring from the
Na+-translocating F-ATP synthase from I tartaricus
seems to be particularly suitable for structural
investi-gations For a more detailed characterization of this
system, and to increase experimental options, we have
now investigated the aggregation behavior of the c11
ring isolated from wild-type I tartaricus cells, and we
have explored the subunit c assembly of the protein
expressed heterologously in E coli Under all
investi-gated conditions, these assemblies were found to
con-sist exclusively of rings of uniform size, allowing tight
packaging of 11 monomeric units In accordance with
previous observations, some of these rings had gaps
indicative of the absence of, in most cases, one c
sub-unit [16] As these rings had the same diameter as
the c11 rings, they were regarded as incompletely
assembled In preparations derived from recombinant
E coli cells, the incomplete assemblies were more
abundant than in preparations derived from wild-type
I tartaricus cells In both wild-type and recombinant
preparations, the majority of the incomplete rings
lacked only one monomer It can therefore be
conclu-ded that the insertion of the last monomer is the
lim-iting step in the assembly of the ring Whether this
step, which is particularly demanding, requires a
spe-cific assembly factor, is completely unknown A
can-didate for such a factor is the membrane insertion
protein, YidC, which was recently shown to be
required for in vitro assembly of the c ring from
E coli [23] Irrespective of the assembly mechanism,
our results clearly show that the size of the ring is
not changed by massive overexpression of subunit c
in the E coli host cells, indicating that intrinsic
fea-tures of the monomeric unit determine the number of
subunits that can be packed into the ring These data
are fully compatible with the recent finding that the
stoichiometry of the subunit III cylinder within the
ATP synthase of the green algae, C reinhardtii, is
not affected by the metabolic state of the cells [18]
However, such findings are difficult to reconcile with
the proposed variation of c ring stoichiometries in
E coli, which are dependent on the expression level
or the nutritional status of the cells [15]
Knowledge on the aggregation behavior of the c ring has been of considerable value in exploring suitable crystallization conditions for structure determination Two types of aggregates had to be taken into account The first were supercomplexes of the (c11)n type The formation of these supercomplexes is dependent on the detergent because they are formed in octylglucoside, but not in Triton X-100 These supercomplexes dis-aggregate completely into the c11rings in the presence
of SDS The second type of aggregate appears as a ladder above the original c11band on SDS⁄ PAGE and consists of c11 rings hosting varying amounts of the c monomer Aggregates are particularly abundant in
c ring preparations from E coli expression clones where the c monomer is present in high amounts Once these aggregates were formed they remained stable and were minimally influenced by additives such as deter-gents, organic solvents, salts or lipids (like 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine) AFM topographs
of these samples showed an exclusively undecameric stoichiometry in the completely assembled rings This
is also observed in the noncrystalline areas of the reconstituted vesicles, demonstrating that it is not an artifact from the 2D crystallization
Slower migrating bands of c rings, as observed on SDS gels, suggest that a certain fraction of c rings may host additional subunits The AFM topographs indi-cate that these additional subunits may be hosted at the outer sides and within the central cavities of the rings That these bands are composed exclusively of c sub-units has been proven by biochemical analyses and, in addition, the formation of these aggregates from pure
c11and c1has been demonstrated in the present study
Experimental procedures
Materials Chemicals were purchased from Fluka (Buchs, Switzerland) including lipase from Aspergillus oryzae N-Lauroylsarcosine sodium salt and n-octyl-beta-d-glucopyranoside were pur-chased from Sigma (Buchs, Switzerland) and Glycon Biochemicals (Luckenwalde, Germany), respectively Primers were custom synthesized by Microsynth (Balgach, Switzer-land) Phospholipase A2 from hog pancreas, and phospho-lipase C from Bacillus cereus, were purchased from Sigma (St Louis, MO, USA)
Construction of plasmid pt7cIT The atpE gene from I tartaricus [24] was amplified with Pfu polymerase and the following two primers:
Trang 85¢-GGAGGAAATAAGCATATGGATATG-3¢ (forward),
containing an NdeI site, and 5¢-CCTTTCAGGAAGCT
TCCTCC-3¢ (reverse), containing a HindIII site The PCR
product and plasmid pt7-7 [25] were digested with these
two restriction enzymes and ligated before transformation
into E coli DH5a Plasmid pt7c [26] was mutagenized with
the Quick Change Site Directed Mutagenesis Kit
(Strata-gene, La Jolla, CA, USA) to yield the single mutation,
T67C, in the P modestum subunit c (plasmid pt7cT67C)
Synthesis and purification of c oligomers
from strain BL21(DE3) transformed with various
plasmids
E coli BL21(DE3) (Novagen, Madison, WI, USA) was
transformed with plasmids pt7c, pt7cIT and pt7cT67C, as
described above The transformed E coli cells were grown
in 2 L of Luria–Bertani (LB) medium to reach an
attenua-nce (D) of 0.6 at 37C in the presence of 200 lgÆmL)1
ampicillin After cooling on ice for 5 min, the expression
was induced with 0.7 mm isopropyl thio-b-d-galactoside
and allowed to continue for 6 h at 30C, to yield typically
2.5 g of cells per L of medium The cells (1 g wet weight)
were suspended in 8 mL of 50 mm potassium phosphate
buffer, pH 8.0, containing 1 mm 1,4-dithio-dl-threitol,
0.1 mm diisopropylfluorophosphate and a spatula tip of
DNaseI Preparation of membranes was performed at 4C
The cell suspension was passed twice through a French
pressure cell at 12 000 psi (8.3· 104kPa) After the
removal of cell debris by centrifugation at 15 000 g for
20 min, ultracentrifugation was performed at 200 000 g for
60 min The membrane pellet was washed once with 4 mL
of 20 mm Tris, 5 mm EDTA, and then adjusted to pH 8.0
with HCl Solubilization of the membranes was
accom-plished with 2 mL of 20 mm Tris⁄ HCl, pH 8.0, containing
5 mm EDTA and 1% (w⁄ v) N-lauroylsarcosine for 10 min
at 65C After ultracentrifugation at room temperature,
the pellet was discarded and contaminating membrane
pro-teins were precipitated with (NH4)2SO4 at 65% (w⁄ v)
sat-uration After 20 min of incubation at 20C, the sample
was centrifuged for 20 min at 39 000 g The filtrated
super-natant containing the c oligomer was dialysed against 5 L
of 10 mm Tris buffer, which was adjusted to pH 8.0 with
HCl using a dialysis membrane with a molecular cut-off of
6000 Da
The protein sample was concentrated by ultrafiltration
with Centricon tubes YM-10 (Millipore, Billerica, MA,
USA) to a concentration of 1 mgÆmL)1and applied to the
top of a density gradient (5 mL) of 5–30% sucrose
con-taining 20 mm Tris⁄ HCl, pH 8.0, 10 lm
1,4-dithio-dl-thre-itol and 1% (w⁄ v) octylglucoside After ultracentrifugation
(4C, 16 h, 150 000 g) in a Beckman SW55-Ti rotor
(Beckman, Coulter, Inc., Fullerton, CA, USA), fractions
of 0.5 mL were collected from the top and analysed by
SDS⁄ PAGE [27] The c ring-containing samples were
pooled and concentrated by ultracentrifugation (18 h,
200 000 g, 4C) The final protein concentration was typ-ically between 1.5 and 3 mgÆmL)1 Fractions containing the monomeric c subunit were also collected and used to study the association with c11 rings to stable c11(c1)n
aggregates
Reconstitution of densely packed and 2D crystalline c ring samples
The c rings purified from these expression cultures were crystallized in two dimensions by mixing octylglucoside-sol-ubilized protein with 1 mgÆmL)1 1-palmitoyl-2-oleyl-sn-gly-cero-3-phosphocholine at a lipid : protein ratio of 0.8 (w⁄ w) in a total volume of 50 lL, followed by dialysis for
24 h at 25C against 200 mL of buffer (10 mm Tris ⁄ HCl,
pH 7.5, containing 200 mm NaCl and 0.02% NaN3), then for another 24 h at 37C The crystals were stored at 4 C for further analysis Subunit c monomers solubilized in chloroform⁄ methanol were purified according to the proce-dure described previously [26]
Purification of pure c11without supercomplexes and attached monomers
The c ring was isolated from wild-type I tartaricus cells as previously described [20] One milligram of the protein was loaded onto a preparative SDS-containing gel, according to Scha¨gger et al [27], together with a prestained marker After the run, the c11ring was visible, without staining, as
a result of the high local protein concentration The band was excised from the gel with a scalpel and subjected to electroelution (at 25 mA) for 6 h at 4C
Phospholipase A2, phospholipase C and lipase digestions
Eighty micrograms of purified subunit c11ring from I tar-taricus [20] was incubated for 14 h at 37C with 10 U of phospholipase A2, 2 U of phospholipase C or 5 U of lipase, in the presence of 50 mm Tris⁄ HCl buffer with a pH adjusted to 8.0, 7.2 or 8.0, respectively, and 1.5% (w⁄ v) octylglucoside
Blue Native PAGE Blue Native PAGE was carried out as described previously [28] Separation gels with a linear gradient of 5–17% acryl-amide were prepared and overlayed with 4% sample gels Samples of 2–5 lg protein each were mixed with sample buffer [50 mm Tris⁄ HCl, pH 6.8, containing 12% (v ⁄ v) gly-cerol and 0.01% (w⁄ v) Serva blue G] After running for 1 h
at 100 V with cathode buffer (50 mm Tricine, 15 mm Bis-Tris⁄ HCl, pH 7.0) containing 0.02% (w ⁄ v) Serva blue G,
Trang 9the cathode buffer was replaced with buffer containing only
0.002% (w⁄ v) Serva blue G and the run continued at
400 V Native protein complexes were then analyzed by
SDS⁄ PAGE, as described previously [27], with the lanes
from the Blue Native PAGE embedded into a 4% stacking
gel
Atomic force microscopy
The samples were diluted to a concentration of 10
lgÆmL)1 in 200 mm NaCl, 10 mm Tris⁄ HCl, pH 7.5
To allow adsorption of the membranes, a drop of 30 lL
was placed onto freshly cleaved mica After an adsorption
time of 15 min, the sample was gently washed using the
above buffer solution containing no membrane proteins to
remove weakly attached material from the mica surface
Contact mode AFM topographs were then recorded in the
same buffer, at room temperature, at forces of < 100 pN
applied to the AFM stylus, and at scanning line frequencies
of typically 4–6 Hz The AFM used was a Nanoscope E
(Digital Instruments, Santa Barbara, CA, USA) equipped
with a 120 lm piezo scanner and a fluid cell Cantilevers
(Olympus, Tokyo, Japan) had oxide-sharpened Si3N4 tips
and a spring constant of 0.09 NÆm)1 No differences
between topographs recorded simultaneously in trace and
in the retrace direction were observed, indicating that the
scanning process did not influence the appearance of the
biological sample
AFM data analysis and image processing
Individual particles of the AFM topographs were selected
manually and subjected to reference-free alignment and
averaging using the SPIDER image processing system
(Wadsworth Laboratories, New York, NY, USA)
Refer-ence-free averages generated by translational and rotational
alignment of single particles enhanced common structural
features among the c oligomers (Figs 5 and 6) To assess
the rotor symmetry, the rotational power spectrum of the
averaged image was calculated using the semper image
processing system (Synoptics Ltd, Cambridge, UK)
Alter-natively, the rotational power spectrum of each individual
particle was calculated and then averaged (data not
shown) It appeared that all averaged classes showed a
stoi-chiometry of 11 subunits except for those of defect
parti-cles The diameter of intact and defective c rings was
determined as described previously [16]
Other methods
Gels were stained with silver [29] The protein
concentra-tion of samples was determined according to the
bicinchon-inic acid method [30] with bovine serum albumin as the
standard
Acknowledgements
The authors thank Marijke Koppenol for critically reading the manuscript This work was supported by the free state of Saxiona, the European community, and the Deutsche Forschungsgemeinschaft (DFG)
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