From the Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275-0376
Received for publication, December 5, 2002, and in revised form, January 8, 2003
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ABSTRACT |
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Interactions between subunit a and
the c subunits of the Escherichia coli ATP
synthase are thought to control proton translocation through the
Fo sector. In this study cysteine substitution mutagenesis was used to define the cytoplasmic ends of the first three
transmembrane spans of subunit a, as judged by
accessibility to 3-N-maleimidyl-propionyl biocytin. The
cytoplasmic end of the fourth transmembrane span could not be defined
in this way because of the limited extent of labeling of all residues
between 186 and 206. In contrast, most of the preceding residues in
that region, closer to transmembrane span 3, were labeled readily. The
proximity of this region to other subunits in Fo was tested
by reacting mono-cysteine mutants with a photoactivated cross-linker.
Residues 165, 169, 173, 174, 177, 178, and 182-184 could all be
cross-linked to subunit c, but no sites were cross-linked
to b subunits. Attempts using double mutants of subunit
a to generate simultaneous cross-links to two different
c subunits were unsuccessful. These results indicate that
the cytoplasmic loop between transmembrane spans 3 and 4 of subunit
a is in close proximity to at least one c
subunit. It is likely that the more highly conserved, carboxyl-terminal region of this loop has limited surface accessibility due to
protein-protein interactions. A model is presented for the
interaction of subunit a with subunit c,
and its implications for the mechanism of proton translocation are discussed.
The ATP synthase from Escherichia coli is typical of
the enzymes found in mitochondria, chloroplasts, and many other
bacteria that synthesize ATP (for a recent review see Ref. 1). It is composed of two subcomplexes: an F1 sector with subunits
that contain the catalytic sites and a membrane-bound Fo
sector with subunits that conduct protons across the membrane. In the
E. coli enzyme five different subunits are found in
F1: Evidence for rotation of subunits through 360 degrees in response to
ATP hydrolysis has been provided by direct observation of fluorescently
labeled actin filaments attached to Subunit b is thought to be embedded in the membrane via a
hydrophobic region near its N terminus. Studies of an
amino-terminal fragment of subunit b using NMR and
detection of disulfide formation have shown it to be Subunit c is a hydrophobic protein with a conserved aspartic
or glutamic acid that is thought to participate in proton translocation steps. NMR studies of the monomeric c subunit have shown it
to be an Subunit a has been analyzed by surface labeling of unique,
engineered cysteine residues. Such studies have established the number
of transmembrane spans (28-30) and have characterized the first
cytoplasmic loop (31) and the first periplasmic loop (32). These
results are summarized in Fig. 1, in
which five transmembrane spans are shown. The first cytoplasmic loop is
drawn to reflect the limited accessibility of its central region to the
reagent MPB.1 Residues
important for function (33-36) have been shown to reside in
transmembrane spans 4 (Arg-210 and Glu-219) and 5 (His-245). In
addition, Glu-196, appears to reside in the cytoplasmic loop preceding
transmembrane span 4. It is likely that Arg-210 of subunit a
interacts with the essential residue Asp-61 of subunit c
during coupled proton translocation since disulfide cross-linking
studies have shown that transmembrane span 4 of subunit a,
between residues 207 and 225, appears to be in contact with subunit
c (37).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
,
,
, and
with a stoichiometry of
3:3:1:1:1. In the Fo sector, three different subunits are
found: a, b, and c with a
stoichiometry of 1:2:9-12 (2).
or
(3-6). Therefore, it is
thought that the enzyme functions as a rotary motor. Other evidence has
been provided that a ring of c subunits is also part of the
rotor (7-12). Subunits that make up the stator include
,
a, and b in addition to
and
, which house
the catalytic sites. The mechanism by which the proton motive force
across the membrane drives rotation of the c oligomer of
Fo along with subunits
and
is not known. High
resolution structures of F1, primarily from bovine
mitochondria, have provided details about the catalytic sites and their
conformational changes (13-15), but less is known about the structure
of the Fo subunits.
-helical and
dimeric (16). Other studies have shown that a truncated, soluble form
of b is extended and dimeric (17-21). The amino terminus of
subunit b is thought to interact with subunit a,
and its carboxyl terminus is thought to interact with
(22).
-helical hairpin with two transmembrane spans connected by
a short polar loop (23-25), with the conserved Asp-61 residue near the center of the second helix. The conformation of subunit c appears to be pH-dependent, as indicated by
NMR studies (26). The number of c subunits that make up the
oligomer in Fo from E. coli is still uncertain
but is likely to be 10 (27).
View larger version (67K):
[in a new window]
Fig. 1.
The transmembrane model of subunit
a. The cytoplasmic loop between residues 64 and
100 is drawn to indicate that the central region has limited
accessibility to the reagent MPB, but the segments nearest the membrane
are highly exposed. The periplasmic loop between residues 124-146 has
been drawn to reflect its partial exposure throughout this
region.
The mechanism by which subunit a contributes to the proton
conducting path of the ATP synthase is not clear, but it is likely to
play such a role. Models have been presented in which subunit a contributes amino acids that make up two half-channels,
one opening to the periplasm and one to the cytoplasm, that allow access to the proton binding site on subunit c (38, 39).
Previously identified residues in subunit a (33-36),
thought to be important in proton access to subunit c, are
found within transmembrane spans, near the periplasmic surface (32). So
far, it is not clear how proton access might be controlled at the
cytoplasmic surface. These studies were undertaken to examine the
structure of the cytoplasmic loop between transmembrane spans 3 and 4 and to determine which other Fo subunits those residues
were near.
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EXPERIMENTAL PROCEDURES |
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Materials--
Restriction enzymes were obtained from New
England Biolabs. Synthetic oligonucleotides were obtained from Operon
Technologies. DNA sequencing was done by Lone Star Labs. MPB and
TFPAM-3 were obtained from Molecular Probes. Nickel-nitrilotriacetic
acid (Ni-NTA) resin and DNA miniprep kits were obtained from Qiagen.
Mouse anti-HA antibody was obtained from Roche Molecular
Biochemicals. Goat anti-mouse IgG-alkaline phosphatase
conjugate, goat anti-rabbit IgG-alkaline phosphatase conjugate,
avidin-conjugated alkaline phosphatase,
5-bromo-4-chloro-3-indoylphosphate p-toluidine salt, p-nitro blue tetrazolium chloride, SDS-PAGE, polyvinylidene
difluoride membranes, and low and broad range protein molecular weight
standards were obtained from Bio-Rad.
N-Octyl--D-glucoside was purchased from
Anatrace. All other chemicals were purchased from Sigma or Fisher. The
UV lamp was purchased from UVP, model UVL-56, with a wavelength of 365 nm. Anti-b antibodies and anti-c antibodies were
gifts of Dr. R. Capaldi, University of Oregon and Dr. K. Altendorf,
University of Osnabrück, Osnabrück, Germany, respectively.
Plasmids, Mutagenesis, Growth, and Expression-- The plasmids, pLN6HisHA (28), pLN7HisHA (31), pLN46HisHA (32), pTW1HisHA (28), pARP2HisHA (39), and pDP1018HisHA were used for the construction of mutants. Plasmid pDP1018HisHA was constructed by ligation of the 770-base pair BsaHI-AflIII fragment from pBJA1018 (39) and the 2592-base pair BsaHI-AflIII fragment from pLN7HisHA. These plasmids produce subunit a that includes an HA epitope (YPYDVPDYA), derived from the hemagglutinin protein of human influenza virus, and a His6 tag at the carboxyl terminus of the protein. These tags have no effect on function. These plasmids differ primarily in the placement of unique restriction sites that are necessary for cassette mutagenesis. Mutagenesis and growth of cultures were carried out as described previously (32).
Preparation of Inside-out Membrane Vesicles--
Inside-out
membrane vesicles were made from a 250-ml culture (per experimental
sample) in LB medium grown to A600 = 1.0. Cells were resuspended in 10 ml of 50 mM Tris-HCl, 10 mM MgSO4, pH 7.5, and passed through a French
press at 14,000 p.s.i. Cell debris and unbroken cells were removed by a
low speed spin at 8000 rpm for 15 min. The supernatant was then
centrifuged at 50,000 rpm for 1 h at 4 °C in a Beckman Ti-70
rotor. The pellet was resuspended and used in the experiments or stored
at 80 °C.
Labeling of Membrane Vesicles--
The inside-out membrane
vesicles were labeled in 200 mM Tris-HCl (pH 8.0), with 120 µM MPB at room temperature for 15 min. The reaction was
stopped by adding -mercaptoethanol to a final concentration of 20 mM. The vesicles were then centrifuged at 50,000 rpm for 45 min, and subunit a was purified by Ni-NTA resin as described below.
Cross-linking of Subunit a by TFPAM-3-- This cross-linker is expected to span 10-15 Å. The cross-linking was carried out by the methods described previously (31). The membrane vesicles were suspended in 50 mM MOPS (pH 7.0), 5 mM EDTA, and 10% glycerol. It was incubated with 200 µM TFPAM-3 for 60 min at room temperature, and the reaction was terminated by addition of 15 mM cysteine. After addition of 5 mM ATP, cross-linking was activated by UV light. The reactions were terminated after 2 h at room temperature.
Purification and Detection of Subunits and Molecular
Models--
After reaction, membrane vesicles were resuspended in
extraction buffer (200 mM Tris-HCl (pH 8.0), 1.5% octyl
glucoside, 0.1% deoxycholate, 0.5% cholate, 10 mM
-mercaptoethanol, 10 mM imidazole, and 1% Tween 20).
Subunit a was purified using Ni-NTA as described previously
(32). Samples of purified subunit a were subjected to
SDS-PAGE and transferred to a polyvinylidene difluoride membrane as
described previously (31). For detection of subunit a,
previously published procedures were followed (29). For detection of
subunit b, the blocked membrane was incubated at room
temperature for 2 h with b antibody at a dilution of
1:1000. After washing three times with TBS/Tween 20, it was incubated
with goat anti-mouse IgG-alkaline phosphatase conjugate at a dilution
of 1:1000 for 1 h. After another three washings with TBS/Tween 20, color was developed as described above. For detection of subunit
c, the blocked membrane was incubated at room temperature
for 2 h with c antibody at a dilution of 1:5000. After
washing three times with TBS/Tween 20, it was incubated with goat
anti-rabbit IgG-alkaline phosphatase conjugate at a dilution of 1:1000
for 1 h. After another three washings with TBS/Tween 20, color was
developed. The molecular models shown were created from the Protein
Data Bank file 1c99, model 1, using RasMol (40).
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RESULTS |
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A series of 46 monosubstituted cysteine mutants of subunit
a that are thought to be near the cytoplasmic surface were
constructed. All mutants grew in succinate minimal medium, indicating
the ability to carry out oxidative phosphorylation. Inverted inner
membrane vesicles were prepared from these mutants and were then
labeled with MPB to test the surface accessibility of each residue. The results for mutations between residues 167 and 174 are shown in Fig.
2, and the results for mutations between
residues 177 and 184 are shown in Fig. 3.
The results of all mutants are summarized in Table
I. For comparative purposes, each
labeling experiment contained G70C, a residue shown previously (29) to
label strongly in membrane vesicles, i.e. from the
cytoplasmic surface, and E131C, a residue that was shown to label
poorly in membrane vesicles (28) but strongly from the periplasmic
surface (29). In Fig. 2A, the results of MPB labeling in
membrane vesicles followed by Ni-NTA purification of subunit
a are shown. In B an immunoblot of the
corresponding samples is shown to confirm that the level of protein is
similar.
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The Proposed Cytoplasmic Loop between Transmembrane Spans 3 and 4-- In Fig. 2, the results of labeling of residues K167C, M168C, K169C, G170C, I171C, G173C, and F174C are shown. Only K167C, lane 4, shows no sign of labeling, while F174C, lane 10, labels as well as G70C, lane 2. Other residues show intermediate levels of labeling. In Fig. 3 the results of labeling of residues E177C, L178C, T179C, P182C, F183C, and N184C are shown. All of the residues label to an intermediate degree except T179C, which is near the background level. The results of H185C, W186C, A187C, F188C, I189C, V191C, and N192C are shown in Table I. None of them show significant labeling, expect for H185C and W186C. Other residues between 193 and 206 show only a trace of labeling, at most.
The Cytoplasmic Ends of Transmembrane Spans 1, 2, and 3-- Residues Phe-60, Arg-61, Val-63, and Ala-64 near the end of transmembrane span 1 were analyzed, and the results are presented in Table I. In this group, only A64C, lane 7, is labeled significantly when compared with the controls, G70C and E131C. The results of labeling I101C, A102C, and P103C, three residues near the cytoplasmic end of transmembrane span 2, are shown in Table I. Only I101C is labeled above a background level. The results of labeling residues near the cytoplasmic surface of transmembrane span 3, L160C, I161C, L162C, F163C, Y164C, and S165C, are also presented in Table I. None of these residues were labeled to a significant extent.
Cross-linking Analysis--
A series of cross-linking experiments
was conducted to determine if any of the residues in the cytoplasmic
loop between transmembrane spans 3 and 4 are near subunits b
or c. Monosubstituted cysteine mutants at the following
positions were reacted with the photoactivated cross-linker TFPAM-3:
165, 167-171, 173-175, 177-179, and 182-184. After UV activation,
nine residues showed evidence of cross-linking to subunit c,
and the results are presented in Fig. 4.
In A, samples S165C, K169C, G173C, and F174C are probed with
anti-HA for detection of subunit a, on the left
side, and the same samples are probed with anti-c
antibodies on the right side. The a-c cross-link
can be seen in both blots and is dependent upon UV activation,
designated by the "+" sign. Similarly, as shown in B,
samples E177C, L178C, P182C, and F183C are probed with anti-HA for
detection of subunit a on the left side and with
anti-c antibodies on the right side. Residue
N184C was also found to cross-link to subunit c in a similar manner (results not shown).
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The following double cysteine mutants were constructed and tested
for cross-linking using TFPAM-3: S165C/ P182C, K169C/P182C, G173C/P182C, F174C/P182C, E177C/P182C, L178C/P182C, S165C/F183C, K169C/F183C, G173C/F183C, F174C/F183C, E177C/F183C, L178C/F183C, S165C/N184C, K169C/N184C, G173C/N184C, F174C/N184C, E177C/N184C, and L178C/N184C. In each case only a single cross-linked product was
seen with no indication of an a-c2 product
(results not shown).
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DISCUSSION |
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The labeling results presented here provide further information
about the junctions of the first three transmembrane spans of subunit
a, and this information is summarized in Fig.
5. This model differs from that shown in
Fig. 1 in two respects. The junctions of the transmembrane spans at the
cytoplasmic surfaces have been adjusted to reflect the results of
labeling, and the periplasmic loop between spans 4 and 5 has been
adjusted, as discussed below. Previous work (29) had identified residue
T37C, at the amino-terminal end of transmembrane span 1, as accessible
by MPB labeling, but residues W39C and D44C were not. At the
carboxyl-terminal end of transmembrane span 1, residues 67-70 labeled
strongly, while residue 64 labeled weakly (29). In this work, residues
F60C, R61C, and V63C were shown not to label, relative to the weak
labeling of residue A64C. The results indicate a core transmembrane
segment of ~26 residues from 38 to 63. This matches well the
calculated hydropathy peak centered near residues 50-53 (41, 42). The carboxyl-terminal end of transmembrane span 2 was analyzed in previous
work (32). Residues D119C, L120C, and P122C were inaccessible to MPB
labeling, while residues D124C and P127C were labeled. Near the
amino-terminal end of transmembrane span 2, residues 92-98 were all
labeled by MPB (31). Here it was shown that residue I101C was labeled
to a small degree relative to residues A102C and P103C. These results
indicate a core transmembrane segment of ~22 residues from 102 to
123, which again matches closely the calculated hydropathy peak
centered near residues 109-111 (41, 42). The amino-terminal end of
transmembrane span 3 was analyzed in previous work (32), and residues
V142C, S144C, and D146C were all shown to be labeled with MPB. At the
carboxyl-terminal end of transmembrane span 3 residue K169C was shown
to be labeled with fluorescein-maleimide (30) and G172C was shown to be
labeled with MPB (28). Here, residues 160-165 and K167C were shown to be inaccessible to MPB, while M168C was labeled. Therefore, a core
transmembrane segment of ~21 residues exists from 147 to 167, which
again matches well the calculated hydropathy peak centered near
residues 156-158 (41, 42).
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In contrast, the results presented here give no indication of the amino-terminal end of transmembrane span 4. Previous studies had indicated that residue E196C could be labeled from the cytoplasmic surface, but those results occurred under slightly more strenuous labeling conditions (30), or the labeling was rather weak (28). In this study the labeling of all residues tested between 185 and 206 was seen to be very weak, and only a few showed a level of labeling that could be considered above background. While it is likely that absolute levels of labeling would differ under different conditions and with different maleimido-reagents, the lack of relative differences seen here makes it impossible to recognize the junction of transmembrane span 4 with the cytoplasmic surface. This also applies to the periplasmic ends of both transmembrane spans 4 and 5, where only three residues were found to be labeled by MPB (29). In studies of lac permease, single amino acid insertions (43) or deletions (44, 45) have been used as indicators of the ends of transmembrane spans. In previous work reported by this laboratory (39, 41), insertions of alanine after residues 202 and 225 did not seriously impair function of the ATP synthase, while those after positions 212, 217, and 222 did. These results are consistent with a hydrophobic core segment in transmembrane span 4 approximately between residues 202-225. Likewise, consideration of insertions of alanine (39) and MPB labeling (29) identifies a core segment in transmembrane span 5 between residues 238 and 259. The loop now identified between residues 226 and 236 is consistent with the marginal effects of alanine insertions after residues 225, 229, and 233. This is in contrast to the loss of function seen with insertions after residues 217, 222, 238 and 243 (37, 39).
The labeling of the residues in the putative loop between transmembrane
spans 3 and 4 showed a strikingly asymmetric pattern. Twelve of
thirteen residues tested between 168 and 184 were found to be labeled
by MPB, while none of the 20 residues tested between 185 and 206 showed
significant labeling. In consideration of the bulky nature of the
reagent MPB, it is likely that the amino-terminal half of this loop is
highly exposed, given the extensive labeling pattern. The lack of
labeling at the carboxyl-terminal end of the loop is consistent with
significant protein-protein interactions. This asymmetry correlates
with amino acid sequence conservation, in which conservation for
residues 190-225 in subunit a is high (46), while for
residues prior to 190 conservation is low. Evidence for a surface of
interaction between transmembrane span 4 of subunit a and
the carboxyl-terminal transmembrane span of subunit c has been provided by studies of engineered disulfides by Jiang and Fillingame (37). In the structural model of subunit c (26) determined at pH 8, shown in Fig. 6,
these residues form a curved surface, suggesting that transmembrane
span 4 of subunit a wraps around that surface. If Glu-196 of
subunit a were part of an -helical extension of
transmembrane span 4 then it would be found near the loop of subunit
c (residues 41-43) since L207C of subunit a
forms a disulfide with I55C of subunit c and residues 43-55 in subunit c are all
-helical in the structural model at
pH 8. This possibility is illustrated in Fig. 6 in which residues
190-225 of subunit a are modeled as an
-helix.
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The labeling pattern of residues 168-184 is consistent with a
connection from transmembrane span 3 to the top of a ring of c subunits with high surface accessibility. The
accessibility of these residues to MPB resembles that of the residues
near the cytoplasmic surface of transmembrane spans 1 and 2 of subunit a. The results of cross-linking with TFPAM-3 indicate that
this region is near subunit c, i.e. at a distance of 10-15
Å. The range of sites that can be cross-linked indicates that both the
end of transmembrane span 3 and the center of the second cytoplasmic loop are near subunit c. It is likely that the efficiency of
cross-linking was too low to detect a doubly cross-linked product.
Therefore, it remains uncertain if all positions cross-link to the same
subunit c. Although this loop is not predicted to be
entirely -helical (42), the pattern of labeling and cross-linking
from residues 173-179 (GFTKELT) is consistent with an
-helix.
The proposed interactions between subunit a and subunit
c discussed above have important implications for the
mechanism of proton translocation of which several proposals have been
described recently (26, 47). As shown in Fig. 6,
a-Arg-210 (blue) would be near the
c-Asp-61 (red). That would place
a-Glu-219 pointing into the bundle of transmembrane spans of
subunit a (30) and a-Glu-196 at the top near the
loop of subunit c. The residues in subunit a that
cross-link to subunit c are likely to be near the adjacent
subunit c in the ring. It has been proposed that the proton
motive force causes protonation of a network of residues in subunit
a near the periplasmic surface (32), including
a-Glu-219 and a-His-245. This could cause a
twisting or bending of transmembrane span 4 of subunit a
allowing protonation of c-Asp-61 from the periplasmic side.
This would cause a-Arg-210 to move away from c-Asp-61, as subunit c changes to its protonated
conformation (26) and the c-Asp-61 rotates to its new
position. The conformational changes in the transmembrane span 4 of
subunit a and in the loop region of subunit c,
would be transmitted to the Glu-196 region of subunit a.
This could promote the deprotonation of c-Asp-61 of the
adjacent subunit c, perhaps by the exposure of
a-Glu-196 and the introduction of water. At this point, the
a-Arg-210 would now be in position to be attracted
electrostatically to c-Asp-61, but since it would be to the
adjacent subunit c, one step of rotation will have occurred.
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ACKNOWLEDGEMENTS |
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We thank Dorothy Peprah and Jessica DeLeon-Rangel for assistance with plasmid construction and mutagenesis, Dr. R. Capaldi (University of Oregon) for supplying anti-b antibodies, and Dr. K. Altendorf (Universität Osnabrück, Osnabrück, Germany) for supplying anti-c antibodies.
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FOOTNOTES |
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* Support for this study was provided by National Institutes of Health Grant GM40508 and The Welch Foundation Grant N-1378.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: BioPort Corporation, Lansing, MI 48906.
§ To whom correspondence should be addressed: Department of Biological Sciences, 6501 Airline Rd., Southern Methodist University, Dallas, TX 75275-0376. Tel.: 214-768-4228; Fax: 214-768-3955; E-mail: svik@mail.smu.edu.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M212413200
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ABBREVIATIONS |
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The abbreviations used are: MPB, 3-N-maleimidyl-propionyl biocytin; HA, hemagglutinin; Ni-NTA, nickel-nitrilotriacetic acid; TBS, Tris-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; TFPAM-3, N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimido-propionamide.
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