University of Groningen
µH+ and ATP Function at Different Steps of the Catalytic Cycle of Preprotein
Translocase
Schiebel, Elmar; Driessen, Arnold; Hartl, Franz-Ulrich; Wickner, William
Published in:
Cell
DOI:
10.1016/0092-8674(91)90317-R
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Publication date:
1991
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Citation for published version (APA):
Schiebel, E., Driessen, A. J. M., Hartl, F-U., & Wickner, W. (1991). µH+ and ATP Function at Different
Steps of the Catalytic Cycle of Preprotein Translocase. Cell, 64, 927-939. DOI: 10.1016/00928674(91)90317-R
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Cell, Vol. 64, 927-939,
March
6, 1991, Copyright
0 1991 by Cell Press
&&i+ and ATP Function at Different Steps
of the Catalytic Cycle of Preprotein Translocase
Elmar Schiebel, Arnold J. M. Driessen,’
Franz-Ulrich
Hartl,t and William Wickner
Molecular Biology Institute
and Department of Biological Chemistry
University of California
Los Angeles, California 90024-l 5
Summary
Preprotein
translocation
in E. coli requires ATP, the
membrane electrochemical
potential ASH+, and translacase, an enzyme with an ATPase domain (SecA) and
the membrane-embedded
SecYIE. Studies of translocase and proOmpA reveal a five-step catalytic cycle:
First, proOmpA binds to the SecA domain. Second,
SecA binds ATP. Third, ATP-binding
energy permits
translocation
of ~20 residues of proOmpA.
Fourth,
ATP hydrolysis
releases proOmpA.
ProOmpA
may
then rebind to SecA and reenter this cycle, allowing
progress through a series of transmembrane
intermediates. In the absence of ASH+ or association
with
SecA, proOmpA passes backward through the membrane, but moves forward when either ATP and SecA
or a membrane electrochemical
potential is supplied.
However, in the presence of API++ (fifth step), proOmpA rapidly completes translocation.
ApHf -driven
translocation
is blocked by SecA plus nonhydrolyzable ATP analogs, indicating that ASH+ drives translocation when ATP and proOmpA are not bound to SecA.
Introduction
The availability of all the isolated genes(Biekeret
al., 1990)
and pure proteins (Brundage et al., 1990) involved in bacterial secretion makes this an attractive system for mechanistic studies. SecB is a soluble protein that stabilizes
many preproteins in translocation-competent
conformations. It has a major role as a chaperone in vivo (Kumamoto
and Gannon, 1988; Kumamoto, 1989) and in vitro (Collier
et al., 1988; Leckeret al., 1989,199O; Randall et al., 1990).
ProOmpA, a precursor protein with a typical leader domain, forms a complex with SecB in the cytosol. SecB has
a dual function, both keeping the precursor competent
for translocation as well as supporting its targeting to the
membrane at SecA (Hart1 et al., 1990), the peripheral component of the membrane-bound
translocase. The proOrnpA-SecB
complex binds with high affinity to membrane-bound SecA, which is the second receptor of the
export pathway (Hart1 et al., 1990). The affinities of SecA
for the SecB protein and for the leader and mature regions
of IproOmpA (Lill et al., 1990) allow it to serve in this recept Present address: lnstitut fur Physiologische
Chemie, Goethestrasse
33, 6000 Munchen 2, Germany.
* Present address:
Department
of Microbiology,
University
of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
tor capacity. SecA itself is bound to the membrane by its
high affinities for acidic lipids (Lill et al., 1990; J. P. Hendrick and W. W., unpublished
data) and for the membrane-embedded
SecYlE protein (Hart1 et al., 1990).
SecA, bound to its high-affinity receptor, is dramatically
activated as a “translocation
ATPase” (Lill et al., 1989)
when it binds a precursor protein. While proOmpA that is
in complex with SecB is in a folded structure (Lecker et al.,
1990), protein translocation
generally requires an unfolded state (Wolfe and Wickner, 1984; Eilers and Schatz,
1986; Pfanner et al., 1987; Liu et al., 1989) and can occur
via defined translocation intermediates (Tani et al., 1989,
1990).
Though these steps, which target preproteins to their
high-affinity binding site on SecA, are understood in some
detail, little has been known of the purpose of the ATP
hydrolysis, the catalytic events during translocation, or the
energetics of each stage of translocation. We now present
data that define a multistep cycle of translocation and explore the energetics and catalysis of each step of this process. The first stage, which encompasses just enough
translocation to allow removal of the leader sequence by
leader peptidase, requires the energy of ATP binding without hydrolysis. The second stage is a series of transmembrane preprotein intermediates.
Progress through these
intermediates requiresenergy
input from the translocation
ATPase or Ap,++, the membrane electrochemical
potential. Translocation proceeds by a series of defined steps,
illustrated in our working model (Figure 1): First, the binding of proOmpA, or a translocation intermediate of proOmpA, to SecA, activating its ATP-binding site. Second,
binding ATP. Third, a limited translocation
of approximately 20 residues. Fourth, ATP hydrolysis and proOmpA
release from SecA. Fifth, ApH+ -driven translocation of
proOmpA while it is not bound to SecA. Multiple cycles of
these steps allow translocation of the entire preprotein. In
this model, there is no coupling between the rate of ATP
hydrolysis and the rate of translocation. In the absence of
either SecA or ApH+, even reverse translocation can occur. We present studies of translocation intermediates,
as well as model studies of SecA-proOmpA
interactions,
suggesting that fundamentally different mechanisms couple the energy from ATP and from ApH+ to precursor protein translocation.
Results
ATP Binding Permits Sufficient Translocation
to Allow ProOmpA Processing
Though proOmpA will bind to translocase in the absence
of further energy input (Hart1 et al., 1990), ATP is required
for translocation. We tested whether the binding of ATP to
SecA is sufficient to initiate translocation. These studies
employed nonhydrolyzable
analogs of ATP that are known
to inhibit the overall translocation reaction (Chen and Tai,
1986). [35S]proOmpA was bound to inverted Escherichia
coli inner membranevesicles
and incubated at 37% either
Cell
928
CYTOPLASM
Figure
1. Current
Working
Model of the Translocation
of ProOmpA
(Step 0) Translocase
consists of the peripheral membrane protein SecA bound with high affinity to acidic membrane
lipids (Lill et al., 1990; J. P.
Hendrick and W. W., unpublished
data) and the integral membrane
protein SecYlE (Hart1 et al., 1990). In the absence of preprotein,
little ATP is
hydrolyzed
(Lill et al., 1989).
(Step 1) The complex of proOmpA and SecS (Lecker et al., 1989, 1990) binds to the SecA subunit of translocase
by virtue of the affinities of this
subunit for the leader peptide (Lill et al., 1990; Cunningham
and Wickner,
1989) the mature domain (Lill et al., 1990) and Se& (Hart1 et al., 1990).
The association
with preprotein activates the ATP-reactive
site of SecA (Lill et al., 1989).
(Step 2) ATP binds to the SecA domain of translocase
(Lill et al., 1989). It is not yet known when Se& dissociates
from proOmpA during the
translocation
process.
(Step 3) The energy of nucleotide binding drives a limited translocation
(Figures 2, 4, and 6).
(Step 4) The energy of ATP hydrolysis
drives release of the proOmpA translocation
intermediate
from its association
with SecA (Figure 9).
(Step 5) Under normal physiological
conditions,
the membrane electrochemical
potential (Au”+) drives a rapid and efficient forward translocation
(Figure 6) that is limited to that part of the catalytic cycle when the proOmpA is not complexed with SecAlATP. Not shown is the reverse translocation
that can occur at step 5 in the absence of membrane
potential (Figure 5).
(Steps 1 ‘and 2’, etc.) Translocation
intermediates
that have dissociated
from SecA can rebind (Figures 5 and 6) and undergo further ATP-dependent
translocation.
The associations
of translocation
intermediates
with lipid and with SecYlE are not known.
with no energy source, with ATP, or with the nonhydrolyzable ATP analogs AMP-PNP or ATP+.
The membranes
were from an uric- strain and therefore do not generate an
electrochemical
potential by ATP hydrolysis. Proteolytic
processing of proOmpA to OmpA by the leader peptidase
of these membranes required either ATP (Figure 2A, lanes
3 and 4) AMP-PNP (lanes 5 and 6) or ATPr-S (data not
shown). This result indicates that at least a small loop of
proOmpA had translocated, sufficient to expose the junction between the leader and mature domains to the periplasmic active site of leader peptidase. Imposition of a
membrane electrochemical
potential in the absence of
ATP did not allow such an initiation of translocation (data
not shown). To assay whether larger domains of proOmpA
had crossed the membrane, samples of these same reactions were incubated with proteinase K at 0% to hydrolyze
polypeptide domains that had not translocated, then were
analyzed by SDS-PAGE and fluorography. The translocation of large domains of proOmpA, measured by this assay
of inaccessability to protease, required hydrolyzable ATP
(Figure 28, lanes 3 and 4). These data show that the energy of ATP binding (see Figure 1, step 2) is sufficient to
permit a small domain of proOmpA to translocate (step 3),
while ATP hydrolysis (step 4) is required for more extensive translocation. Similar results are presented below for
later intermediates in translocation.
Translocation
Intermediates
Accumulate
at Low Concentrations
of ATP
Translocation intermediates allow the systematic dissection of intermediate steps in the secretion process and
evaluation of the effects of ATP, SecA, and ApH+ on their
interconversions.
Tani et al. (1969, 1990) have reported
translocation intermediates of 26 and 29 kd that can, in
the presence of ATP and/or ApH+, complete their translocation. Without a membrane potential, the rate of overall
A
’
2
.---r.b.--..“~~<
3
4
5
6
proOmpA
OmpA
Tmrb
I26
., -
Nucleofide
: 1
Time(min):
Figure
Event
2. The Energy
none(jA~~I(~-p&I
I
10
1
of ATP Binding
10
Drives
1
10
an Early Translocation
[35S]proOmpA-SecB,
SecA, and membrane
vesicles were preincubated for 2 min at 37%. Samples were further incubated at 37OC for
1 min (lanes 1, 3, and 5) or IO min (lanes 2, 4, and 6) with 10 uM ATP
in the presence of an ATP regenerating
system (lanes 3 and 4) 5 mM
AMP-PNP (lanes 5 and 6) or without any addition (lanes 1 and 2). (A)
One-tenth of each sample was analyzed by SDS-PAGE
and fluorography. (6) The remaining sample was treated with proteinase K, concentrated by trichloroacetic
acid precipitation,
and analyzed
by SDSPAGE and fluorography.
SecA,
929
Auk+,
and ATP in Preprotein
Translocation
t2345678
29-
i
proOmpA
OmpA
I-
‘26
i-
‘16
1814_
ITi
-(ll~~I(lralram?~Time(min):
Figure
0
0.5
3. Intermediates
I
during
2.5
5
10
Translocation
15
25
with Low ATP
[Yj]proOmpA-SecB
was preincubated
with 100 uglml membrane vesicles from E. coli KM9, 40 us/ml SecA, 2 mM dithiothreitol,
5 mM
creatine phosphate,
and 10 uglml creatine kinase for 2 min at 37%.
Translocation
was started by the addition of 2 PM ATP (0 min); 5 uglml
proOmpA and 2 mM ATP were added after 1 and IO min, respectively.
Samples (100 ul) were withdrawn
after the indicated times, chilled,
treated with proteinase
K, and analyzed
(see Experimental
Procedures).
proOmpA translocation is half-maximal at an ATP concentration of 105 PM (Driessen and Wickner, 1991) while
SecA is the only membrane ATPase that is directly involved in protein secretion (Lill et al., 1989; Brundage et
al., 1990). SecA function should therefore become limiting
for translocation at ATP concentrations
below this apparent K, and translocation
intermediates
may be more
readily detected. [35S]proOmpA, SecA, SecB, and inverted
inner membrane vesicles were preincubated for 2 min at
37OC to allow binding of proOmpA to translocase (Hart1 et
al., 1990), then ATP was added to a low concentration (2
$vl) to permit a slow translocation reaction. Partial synchlrony of translocation was achieved by adding an excess
of Inonradioactive proOmpA after 1 min. At various times,
aliquots of the reaction were assayed for translocation intermediates by incubation with proteinase K at 0°C followed by SDS-PAGE and fluorography (Figure 3). Successive peaks of proOmpA domains that were protected
from proteinase K were seen at low molecular size, at 16
kd and at 26 kd. These latter two species are characterized
in some detail below. After 10 min, the ATP concentration
was raised to 2 mM. Concomitant with appearance of fulllength translocated proOmpA and OmpA (lanes 7 and 8)
the lower molecularweight
species chased, indicating that
they are true translocation intermediates (also see Figure
4D). These datashow that translocation proceeds through
an ordered series of intermediates, termed II6 and &, according to the mass that has translocated and become
inaccessible to added proteinase. Each intermediate is of
course a full-length proOmpA molecule, and the different
sized fragments are only seen after digestion with protease to assay the extent of translocation.
Each of these
intermediates appears as a cluster in a narrow molecular
weight range rather than a unique molecular species.
Characterization
of Intermediates
Is6 lis a major kinetic intermediate (Tani et al., 1989) that
accumulates at low ATP levels (Figure 3). It satisfies several criteria as an authentic translocation intermediate:
First, 26 kd of the polypeptide chain is protected from protease (Figure 4A, lanes 1 and 3, intact membranes; lanes
2 and 4, disrupted membranes). Second, completion of
translocation is supported by ATP, but not by nonhydrolyzable ATP analogs (Figure 48). The latter support a limited
translocation of approximately 2 kd (Figure 48, lanes 3
and 4 verus lane l), which may be comparable with that
seen with nonhydrolyzable
ATP analogs at the beginning
of the translocation
reaction (see Figure 2). Third, the
membrane electrochemical
potential, termed the AuH+ or
protonmotive force (pmf), accelerates the conversion of lZ6
to fully translocated proOmpA (Figure 4C; also Tani et
al., 1989), as seen for the overall translocation reaction.
Fourth, introduction of a stable tertiary structure in proOmpA blocks ATP-driven translocation, causing accumulation of the translocation-arrested
species. Such an intermediate was created by sedimenting membranes bearing
lZ6 (Figure 4D, lane 2) through a solution of the oxidant
sodium tetrathionate, forming a disulfide bridge between
the cysteines at positions 290 and 302. Upon incubation
at 37OC with ATP, translocation yields lZ9 (lane 4) which
cannot proceed further until the disulfide bond is reduced
(Figure 4D, lane 5) or a membrane potential is imposed
(Tani et al., 1990). This result suggests that the introduction of a stable tertiary structure into proOmpA, such as by
a disulfide bond, impedes translocation.
To establish rigorously that lp6and lp9 are true translocation intermediates, the amount of lZ6, lZ9, and full-length
translocated
proOmpA was quantified by densitometry
and corrected to reflect the methionine content of each
species. lp6 (Figure 4D, lane 2) was 54% of the total
membrane-bound
proOmpA (lane 1). The fully translocated proOmpA formed upon reduction of oxidized lZ6(lane
3) was 58% of the the total proOmpA (lane 1). lm (lane
4) was 620/o, and the full-length translocated proOmpA
derived from 1%(lane 5) was 58%. The agreement between
theamountof lZ6(lane2), la(lane4), and full-length translocated proOmpA (lanes 3 and 5) demonstrates that lZ6and
I- are true translocation intermediates that can chase to
the fully translocated proOmpA.
Reverse Translocation
Au+++ and the translocation ATPase activity are not only
necessary to permit forward translocation but to prevent
reversal of translocation. The internal disulfide form of lZ9
(Figure 4E, lane l), which was stable in the presence of
ATP (lane 5) reversed its translocation in the absence of
added ATP to yield lZ6(lanes 3 and 9). However, lp9 did not
reverse at 37OC in the absence of ATP when the C-terminal
domain of the intermediate was first digested by proteinase K, suggesting that either the folding of this domain or
the function of exposed regions of translocase are necessary for the reversal of translocation (data not shown).
Removal or inactivation of SecA allows more extensive
reversal of translocation, revealing the earlier intermediate, lq6,a major kinetic intermediate at low ATP concentrations (see Figure 3) that is not prominent at high ATP concentrations (Tani et al., 1989). Antibody to SecA will cause
the disulfide form of 129(Figure 5A, lane 2) or 126(data not
shown) to reverse translocate to II6 (lane 3). The reverse
Cell
930
A
1
2
3
translocation is not seen at 0% (lane 4) or with control
antibody (lane 6). 116,once formed by inactivating SecA, is
stable during incubation at 37%. It will complete translocation if both SecA and ATP are added to the incubation
(see Figure 6) indicating that SecA can interact with par-
4
]26
Figure 4. Characterization
Translocation
sonicotionJLalkaline
’
’
126
2
3
-
4
-
<
proOmpA
OmpA
_
Appearance of proOmpA
mme (mln)
D
12345
proOmpA
I29
I26
<
4
A
Z-7;
ProK : ATP:OTT:-
E
t
-
1
proOmpA
OmpA 1.
ttt
t
t
t
-
OmpA
t
t
23456769
am
129;
I26
---II
DTT:
-
no
addition
+ATP
t
t
-
+AMP-PNP
-
t
-
tHexokinase
glucose
t
-
of Defined
Intermediates
in ProOmpA
(A) The translocated
part of lZ8 is protected from proteinase
K unless
the membrane is disrupted. Isolation of membranes
bearing lZ6: Translocation into 100 pg/ml inner membrane
vesicles was performed
at
37OC for 5 min in the presence of 10 pM ATP and an ATP regenerating
system (see Experimental
Procedures).
Membranes with iZ5were separated from untranslocated
proOmpA by sedimentation
(250,000 x g,
45 min, 0%) through a sucrose
solution (0.2 M sucrose,
50 mM
HEPES-KOH
[pH 7.5],50 mM KCI, 1 mM dithiothreitol).
The sediment
was resuspended
in 112 vol of buffer A. The isolated membranes,
bearing lZ8, were bath sonicated (2 min, 0%; lanes 1 and 2) or treated
with 100 mM sodium carbonate (pH 11) (lanes 3 and 4) in the presence
of 1 mglml proteinase
K (lanes 2 and 4) or without protease (lanes 1
and 3). The alkaline sample was neutralized
with 1 M HEPES-KOH
(pH 6). Proteinase K was added to the remaining samples (lanes 1 and
3) and all samples were incubated for 15 min on ice and analyzed.
(B) Effect of ATP and ATP analogs on the chase of lp6. Membranes
bearing Is6 were isolated as described
in (A). The ATP concentration
in the resuspended
sediment was 4-6 nM, as determined by the addition of [@P]ATP
to the initial incubation in which IX was formed, then
assay of the fraction of =P recovered
in the resuspended
membrane
sediment. The membranes
with lZ8were incubated (30 min, 37°C) with
2mMATP,
IO mM AMP-PNP, IOmM ATP-1-S or noaddition. Samples
were treated with proteinase
K, trichloroacetic
acid precipitated,
and
analyzed by SDS-PAGE
and fluorography.
(C)The chase of 1%(see Experimental
Procedures)
to the fully translocated proOmpA at 37°C was assayed in the absence of ATP, in the
presence of 2 mM ATP, or with 2 mM ATP and 5 mM NADH (pmf, i.e.,
ApLHt). Samples were withdrawn at the indicated times, chilled in ice
water, treated with proteinase
K, and analyzed.
(D) Sequential interconversion
of translocation
intermediates.
Inner
membrane vesicles bearing lZ8were centrifuged
(250,000 x g, 45 min,
0%) through a sucrose solution (0.2 M sucrose, 50 mM HEPES-KOH
[pH 7.51, 50 mM KCI) containing 200 uM sodium tetrathionate.
The
sediment was resuspended
in l/2 vol of buffer B (50 mM HEPES-KOH
[pH 81, 50 mM KCI, 5 mM MgCI,, 0.2 mglml BSA, 100 pM Ellmann’s
reagent). The oxidized lZ6 (lanes 1 and 2) was incubated with 2 mM
ATP in the presence (lane 3) or absence (lane 4) of 2 mM dithiothreitol
for 15 min at 37%
A sample prepared as described
in lane 4 was
further incubated for 15 min at 37OC after the addition of 2 mM dithiothreitol (lane 5). Samples were incubated at O°C with proteinase
K
(lanes 2-5) or without further addition (lane 1) and analyzed.
(E) Chase and stability of Ia. Oxidized L8. prepared as described in(D),
was incubated with 4 mM ATP for 15 min at 37%. The membranes
were layered on a sucrose solution (0.2 M sucrose,
50 mM HEPESKOH [pH 7.5],50 mM KCI, 100 uM Ellmann’s reagent) and centrifuged
(45 min, 250,000 x g, 0°C). The sediment was suspended
in equal
volumeof buffer B. Residual ATP(l0 PM), leftfrom the initial incubation
with 4 mM ATP, was present in the resuspended
membranes.
Samples
were incubated
with either 2 mM ATP, 10 mM AMP-PNP,
10 mM
glucose and 180 U/ml hexokinase,
or no addition for 15 min on ice.
Dithiothreitol
(2 mM) was added where indicated,
and the samples
were incubated at O°C (lane 1) or 37% (lanes 2-9) for 15 min, then
treated with proteinase
K and analyzed. To control for the effect of
the oxidizing reagent sodium tetrathionate
on translocase,
the two
cysteines
of proOmpA
were reacted with N-ethylmaleimide
(NEM),
preventing
the subsequent
formation of a disulfide bond. The NEMmodified proOmpA was treated with the oxidizing reagent in parallel
to proOmpA. The NEM-proOmpA
translocated
under oxidizing conditions without the appearance
of an la intermediate,
and the addition
of the reducing agent dithiothreitol
did not influence the rate of translocation (data not shown).
SecA,
931
AuH+,
A
and ATP in Preprotein
1234
proOmpA.
OmpA =-rim
-IIt
Temp. ‘C : 37
13
A
56
1234567
proOmpA
>
OmpA
8
anti-SecA
ontrbodies
control
antibodies
-
-
-
f
-
37
37
0
37
37
-
.#Per.S:AdditickA
B
3
IO II
gl-
-
-
-
+
+
12
+
-
3
0 37
--++---
+
+
4
12 I3
I4
-
^.
Temp.*C:O 37 37 31 37 31 37
ATP:--++---
“0
addition
DTT:
Translocation
-
5
6
-
7
37 31 31 37
37
-
+
_
-
+
-tt-t
6
910
proOmpA
>
6
proOmpA
OmpA
7
8
9
OmpA
46 [
>
-'I6
Time(min)
-&A,-AIPuT-S
0
1 25 5
II
I-eader
peptide
12 I3
14 15 I6 17
10
19 20
wOrnpA
(,hl)
:
0
123
126
2.5
10
25
>
OmpA
proOmpA
+
OmpA
-
Time(min1
0
I
25
5
Figure 6. ATP Binding to SecA Inhibits
Fully Translocated
ProOmpA
Figure
-SecA.tATP-r-S
10 0
I 25
5
5. Reverse
Translocation
of l28 and I28
(A) Incubation of I28 with anti-SecA antibodies.
Inner membrane
vesicles bearing lr) (100 pl, prepared
as in Figure 4D) were incubated
without antibodies (lanes 1 and 2), with 0.25 mglml anti-SecA immunoglobulins (IgG) (lanes 3 and 4) or with 0.25 mg/ml control IgG fraction
(lanes 5 and 6) for 2 hr on ice. After an incubation of 20 min at 0°C
(lane 4) or 37% (lanes l-3,5,
and 6) in the absence or presence of
1 mM dithiothreitol,
the samples were treated with proteinase
K and
analyzed by SDS-PAGE
and fluorography.
(B) Chase of h6 to fully translocated
proOmpA is not inhibited by leader
peptide. Membranes
bearing llB (A) were sedimented
through a sucrose solution to remove antibodies.
The membranes
were resuspended in one-half the orignial volume in buffer A. ATP (2 mM), followed by leader peptide and 40 uglml SecA, was added to isolated
llij (lanes l-4) or [“SjproOmpA-SecB
and membrane
vesicles (see
Experimental
Procedures;
lanes 6-9). As a control, membrane
vesicles bearing IJo received no SecA, ATP, or leader peptide (lane 5). The
concentration
of leader peptide was 0 (lanes 1, 5, and 6) 2.5 (lanes 2
and 7) 10 (lanes 3 and 6) or 25 uhf (lanes 4 and 9). Samples were
incubated
for 20 min at 37V, then treated with proteinase
K and
analyzed.
(C) Incubation of IX with urea. Membranes
bearing la were incubated
with 50 mM Tris-Cl (pH 6) (lane l), or 6 M urea, 50 mM Tris-Cl (pH
6) for 10 min on ice (lanes 2 and 3). The membranes
were sedimented
(250,000 x g, O°C, 45 min) and resuspended
in buffer A. ATP (2 mM)
and 40 ug/ml SecA were added to the sample in lane 3, and samples
in lanes 2 and 3 were incubated for 15 min at 37%. The samples were
treated with proteinase
K and analyzed.
tially translocated proOmpA. This forward translocation
reaction is not sensitive to the addition of synthetic leader
peptide (Figure 58, lanes l-4) at concentrations that completely block the overall translocation of proOmpA (lanes
6-9) indicating that the ability of SecA to recognize leader
10
0
1
25
Au,++ - Driven
5'
IO
Chase
of IIs to
(A) A translocation
reaction was performed
as described
in Figure 3.
The translocation
reaction was stopped after 2.5 min at 37%. and the
membranes
were sedimented
through
a sucrose
solution as described. The sediment was resuspended
in 112 vol of buffer C (50 mM
HEPES-KOH
[pH 8],50 mM KCI, 1 mM dithiothreitol,
0.2 mg/ml BSA).
The membranes
were divided into equal parts. The aliquots were incubated with or without 0.25 mglml anti-SecA immunoglobulins
for 1.5
hr at 0% followed by an incubation for 15 min at 37%. The membrane
vesicles were sedimented
through a sucrose solution to remove antibodies and resuspended
in an equal volume of buffer C containing 2
mM dithiothreitol.
Membranes
bearing II8 that had been treated with
anti-SecA
antibodies
(lanes 8-14) or incubated
without antibodies
(lanes l-7) were mixed with SecA (60 uglml), 5 mM ATP, or 5 mM
ATP--r-S where indicated. The samples were incubated for 15 min at
0% (lanes 1 and 6) or 37% (lanes 2-7 and 9-l 4) treated with proteinase K, and analyzed.
(B) Membranes
were prepared bearing II6 and SecA (lanes 1 i-20) or
without functional SecA protein, following incubation with anti-SecA
antibodies as described above (lanes l-10). Samples were incubated
at 37% in the presence of 5 mM NADH (A) with 5 mM ATP-1-S (lanes
6-10 and 16-20) or without added nucleotide (lanes l-5 and 11-15).
Samples were withdrawn
at 0, 1, 2.5, 5, and 10 min and treated with
proteinase
K and analyzed.
peptides (Cunningham
and Wickner, 1969; Fikes and
Bassford, 1969; Lill et al., 1990) is not required for SecA
recognition of l16. Incubation of membranes bearing 126
(Figure 5C, lane 1) with urea also caused reversed translocation to IIs (lane 2). Urea has previously been shown to
remove and inactivate the peripheral membrane protein
SecA (Cunningham et al., 1989). II8 formed in this fashion
will also complete translocation upon addition of SecA and
ATP (lane 3).
Cell
932
ATP Binding to SecA Drives Limited
II6 Translocation
When the energy for IIs translocation is derived from ATP,
SecA is needed to mediate translocation. I16 was selectively accumulated
as a kinetic intermediate when the
translocation reaction was performed for 2.5 min at 37%
with 2 PM ATP and an ATP regenerating
system. The
membrane vesicles bearing II6 were sedimented through
a sucrose solution to remove nontranslocated
proOmpA
and ATP. The isolated llB (Figure 6A, lane 1) was stable if
incubated at 37% (lane 2) and chased to fully translocated
proOmpA upon the addition of ATP (lane 3). Since SecA
was already bound to the SecYIE, additional SecA did not
increase the efficiency of the ATP-driven chase (lane 4).
The ATP analogs ATP-y-S (lanes 6 and 7) or AMP-PNP
(data not shown) caused further translocation of 116by
about 20 amino acids. This species is not digested by a
wide range of proteinase K concentrations
(data not
shown), which degrade SecA. As little as 0.5 mM ATPr-S
was sufficient to induce this shift of the II6 form of proOmpA. Thus, in addition to the initial limited translocation
driven by the energy of ATP binding (see Figure 2 and
Figure 1, steps 2 and 3) later translocation intermediates
also show limited translocation driven by nucleotide binding to SecA (Figures 48 and 6A; see Figure 1, step 2’).
To establish that the limited translocation of II6 that is
caused by nonhydrolyzable
ATP analogs involves SecA
protein, anti-SecA antibodies were incubated with the
membranes bearing II6 to inactivate the SecA protein (Figure 6A, lanes 8-14). Excess antibodies were removed by
sedimentation of the membranes through a sucrose solution. The isolated IIs (lane 8) was stable at 37%, either
without ATP (lane 9) or in its presence (lane 10). An ATPdriven chase of Ils to fully translocated proOmpA was only
seen if both ATP and SecA were added (lane 1 l), showing
that the previously bound SecA had, as expected, been
inactivated by the antibody treatment. The addition of
SecA alone to the SecA-stripped
II6 caused a small increase in the proteinase K-protected part of the intermediate (lane 12). Limited translocation of II6 upon addition of
ATPy-S required SecA (Figure 6A, compare lanes 13
and 14).
ApH+ Drives SecA-Free IIs to Fully
Translocated
ProOmpA
The ability to selectively inactivate SecA allows assay of
the separate contributions of ATP and Au!,+ to translocation. Membranes bearing the proOmpA translocation intermediate IIs were prepared by a brief incubation of membranes, proOmpA, and SecA with a low concentration of
ATP. These membranes were incubated with antibodies
to inactivate the SecA, and reisolated to remove ATP and
unbound antibodies. A ApH+ could be induced in these
uric- inner membrane vesicles by the NADH oxido-reductase. Upon addition of NADH, II6 chased to fully translocated proOmpA within 1 min in the absence of ATP or
functional SecA (Figure 6B, lanes l-5). In the absence of
SecA, this translocation was unaffected by ATP+S (lanes
6-10). However, interactions with bound SecA slowed
Au”+-driven translocation (lanes 11-l 5). The preincuba-
tion of these vesicles bearing he and SecA with apyrase
(to remove low levels of ATP) did not influence the chase
reaction (data not shown), confirming that a Au,++ can
efficiently drive the forward movement of II6 without ATP
hydrolysis. Strikingly, addition of ATP-y-S in the presence
of SecA prevented API++ - driven translocation
entirely
(lanes 16-20). These data clearly establish the importance
of ATP hydrolysis to release proOmpA from SecA (see
Figure 1, step 4) allowing But++-driven
translocation
(Figure 1, step 5) and further cycles of interaction with
SecA and ATP (Figure 1, steps l’, 2’, etc.). They also show
that AuH+ - driven translocation is most likely to occur during that part of the catalytic cycle when the proOmpA is
not bound to SecA-nucleotide
(step 5).
Synthetic Controlled
Translocation
Arrest
A careful examination of the possible coupling of ATP hydrolysis to net translocation was made possible by controlling the arrest of translocation at a defined stage. This was
achieved through covalent modifications of the cysteinyl
residues of proOmpA (Figure 7). Since even the disulfide
form of proOmpA can translocate if the translocation reaction is provided with both ATP and a A~IH+ (Tani et al., 1990)
we cross-linked bovine pancreas trypsin inhibitor (BPTI) to
the cysteinyl residues of proOmpA using the reversible
cross-linker N-succinimidyl-3(2-pyridyldithio)-priopionate
(SPDP) or the irreversible cross-linker N-maleimidobenzoylN-hydroxysuccinimide
ester (MBS). As expected, crosslinked derivatives were of higher apparent molecular
weight (Figure 7A, lanes l-6). Translocation of oxidized
proOmpA, proOmpA-SPDP-BPTI,
or proOmpA-MBS-BPTI
into proteoliposomes
bearing SecYlE protein yielded a
transmembrane
species with a translocated domain of 29
kd that was inaccessible to protease K (Figure 7A, lanes
8,10, and 12). Reduction of thedisulfide in the cross-linker
or the internal disulfide bridge in proOmpA allowed completion of translocation (lanes 7 and 1 l), while reductant did
not affect translocation of proOmpA-MBS-BPTI
(lane 9).
I= formation from these derivatized proOmpA species
required SecA, ATP, and SecYlE complex and was sensitive to leader peptide (Figure 78) while leader peptide did
not prevent the chase of Ia to fully translocated proOmpA
(data not shown). Together, these criteria establish that
Ia can be seen both with native membrane vesicles (see
Figure 4) and proteoliposomes
(Figure 7) and that it is on
the authentic translocation pathway from lZ6to full-length
translocated proOmpA.
In these studies, using proteoliposomes
reconstituted
with SecY/E protein, the BPTI cross-linked form of Ia (Figure 7C, lane 1) was stable in the presence of ATP (lane 4).
If the incubation is performed in the absence of ATP and
without reductant, thereby preventing forward translocation, the I29 reverses its translocation to yield earlier intermediates, which are not detected by our antibodies to
OmpA (lane 2). This does not represent a loss of sample
or lysis of the proteoliposomes,
since the readdition of
ATP again allows forward translocation to Ia (lane 6). This
forward translocation is from an early intermediate rather
than from free proOmpA, since it could not be blocked by
excess proOmpA leader peptide (lane 7).
SecA,
933
Apn+,
and ATP in Preprotein
Translocation
A
123456
7
proOmpA-( BPTI )*
proOmpA-BPTIE
~
wmeyr-
d
9
-
LUU
C
1234
proOmpA
OmpAg
12
proOmpA
--II
proOmpA proOmpA proOmpA
-SPDP-MBSBPTI
BPTI
proOmpA proOmpA proOmpA
-SPDP- -MBSBPTI
BPTI
DTT : t - t - + 1234
I1
-
proOmpA
B
10
t
56
.
.- -e
._..
56
7
T OmpA
I29
-+-+7
6
9 IO 11 12
4
I29
ipiii
IC
I29
Figure
7. The Introduction
of a Stably
Folded
Structure
at the C-Terminus
of ProOmpA
Causes
the Accumulation
of an Intermediate
(A) Characterization
of purified proOmpA-SPDP-BPTI,
proOmpA-MBS-BPTI,
and oxidized proOmpA.
Each of the proOmpA species (5 pg) was
incubated for 10 min at 37W with 1 mM dithiothreitol
where indicated and applied to SDS-PAGE,
which was stained with Coomassie
blue (lanes
l-6). Translocation
into SecY/E proteoliposomes
(see Experimental
Procedures)
was performed
in the presence or absence of 2 mM dithiothreitol
for 15 min at 37OC. Samples were treated with proteinase K, precipitated with trichloroacetic
acid, applied to SDS-PAGE,
transferred
to nitrocellulose.
and analyzed by immunoblotting
with anti-OmpA
antibodies (lanes 7-12).
(B) lmmunoblot
of translocation
reactions
of proOmpA-SPDP-BPTI
into SecY/E proteoliposomes.
Translocation
reactions
were as in (A) with
proOmpA-SPDP-BPTI
(lanes l-12) but either without SecA (lane 1), ATP (lane 2) SecY/E (lane 3) or SecB (lane 4). The sample analyzed in lane
3 contained liposomes
instead of SecY/E proteoliposomes.
The concentrations
of proOmpA leader peptide in lanes 6-6 were 2.5, 10, and 25 nM,
respectively.
Leader peptide was added immediately
prior to proOmpA-SPDP-BPTI.
Membranes
containing Is were disrupted by sonication
(lane
10) as described
in Figure 4A, or by the addition of 1% Triton X-196 (lane 12) in the presence
of proteinase
K. Lanes 9 and 11 represent
controls
where proteinase K was added after sonication (lane 9) or no detergent was present (lane 11). Samples were treated with proteinase K and analyzed
as in (A).
(C) Instability of I=. IB (proOmpA-SPDP-BPTI)
was accumulated
as in (A). Proteoliposomes
with this intermediate
were sedimented
in an airfuge
(30 min, 30 psi, 4OC), suspended
in buffer 8, and again collected by sedimentation.
Proteoliposomes
were resuspended
in an equal volume of buffer
B and incubated without further addition for 15 min at O°C (lane 1) or 37OC (lane 2) or incubated at 37OC with 2 mM ATP (lanes 3 and 4). The sample
in lane 3 also had 1 mM dithiothreitol
present during the incubation.
Samples, prepared as in lane 2, were further incubated for 15 min at 37OC
without any addition (lane 5) with 2 mM ATP (lane 6) or with 2 mM ATP and 25 M leader peptide (lane 7). Samples were treated with proteinase
K and analyzed.
ATP Hydrolysis Is Not Coupled
to Net Polypeptide
Movement
The availability of covalently modified species of proOmpA
that arrest in translocation as Ia allowed us to examine the
relationship between ATP hydrolysis and translocation.
While the progress of polypeptide chains through the different steps of translocation depends on ATP hydrolysis,
these two processes may only be said to be “coupled” if
ATP hydrolysis is equally dependent on the movement of
the preprotein polypeptide chain. Translocation ATPase
(Lill et al., 1989) has been defined as the hydrolysis of
ATP by SecA in response to the association of SecA with
preprotein, SecYIE, and acidic lipid.
To look for coupling, we assayed the ability of proOmpA,
Cell
934
Figure 8 Hydrolysis
of ATP
Translocation
of ProOmpA
SPDP or ProOmpA-MBS-BPTI
Time
(min)
proOmpA-SPDP-BPTI,
and proOmpA-MBS-BPTI
to undergo translocation
and to stimulate ATP hydrolysis by
SecA in the presence and absence of dithiothreitol. ATP
hydrolysis in response to proOmpA lasts for approximately
20 min (Figure 8A, open squares), after which the proOmpA is either translocated
or denatured
(data not
shown). If fresh proOmpA is added at this time, there is a
new burst of translocation
ATPase activity (open triangles). However, the substrate proOmpA-SPDP-BPTI,
which arrests in translocation at I=, supports a linear translocation ATPase reaction for at least 45 min (closed
squares). Addition of reductant, which frees the Ia to complete translocation, causes a rapid cessation of ATP hydrolysis (closed triangles) at a time that corresponds to the
completion of translocation (data not shown). Reductant
is without effect on translocation ATPase supported by
the noncleavable proOmpA-MBS-BPTI
(Figure 8B). Thus,
while ATP hydrolysis is essential for translocation, ATP
hydrolysis continues unabated when translocation
is
blocked by acovalent modification of the protein. The rates
of ATP hydrolysis measured under these conditions are
equivalent to the initial SecA ATPase activity observed
during translocation
of underivatized
proOmpA. These
data suggest that any forward translocation of the BPTI
form of I=, caused by ATP binding to SecA, is immediately
lost to reverse translocation after ATP hydrolysis drives its
release from SecA (see Figure 1, step 4).
We conclude that there is no strict mechanistic coupling
in vitro between ATP hydrolysis by SecA and net chain
translocation as exists, for example, between proton translocation and the hydrolysis or formation of ATP by the
FoF,-ATPase (Hoppe and Sebald, 1984).
The Role of ATP Hydrolysis
ATP hydrolysis by SecA is clearly necessary to release
proOmpA and allow AuH+-driven translocation (see Figure
6). This energy might directly alter the interactions be-
by SecA during
and ProOmpA-
Translocation
of proOmpA
and proOmpASPDP is shown in (A) and of proOmpA-MBSBPTI is shown in (B). SecYlE proteoliposomes
were incubated with 50 mM HEPES-KOH
(pH
8) 50 mM KCI, 5 mM MgCI,, 0.5 mg/ml BSA,
10 uglml SecA, 2 mM ATP, 100 uM Ellmann’s
reagent, and 4 mg/ml either proOmpA,
proOmpA-SPDP-BPTI,
proOmpA-MBS-BPTI,
or
urea. Dilhiothreitol
(2 mM) was added to the
proOmpA and one of the samples containing
proOmpA-MBS-BPTI.
Reactions were started
by the addition of one of the proOmpA species
or urea. The reaction mixtures were incubated
at 37OC and samples (50 pl) were withdrawn
after the indicated times. After 20 min, 2 mM
dithiothreitol
or 4 uglml proOmpA was added
to the indicated samples. ATP hydrolysis
was
quantified using malachite green (Lanzetta et
al., 1978). The assays in the presence of a proOmpA species were corrected for background
ATP hydrolysis
(in the presence
of urea).
tween SecA and proOmpA. Alternatively, it might be transduced from SecA to the SecYlE protein and modify interactionsof SecYlE with proOmpA. We therefore sought model
subreactions to explore the role of ATP hydrolysis.
As proposed for hsc70 proteins (Pelham, 1986; Beckmann et al., 1990) whose ATPase activity is stimulated
by interaction with protein substrates and which are then
released from these substrates (Flynn et al., 1989) SecA
may release the precursor protein upon ATP hydrolysis.
Such a reaction would be required at the membrane to
permit the translocation of successive domains of the preprotein. Liposomes bearing adsorbed SecA provide a suitable model system for the assay of ATP-dependent dissociation of proOmpA from SecA. In the absence of lipid,
proOmpA alone, upon dilution from urea, could be completely digested by concentrations of proteinase K as low
as 5-10 uglml at O°C (Brundage et al., 1990). ProOmpA
that adsorbs to liposomes remains protease accessible
(Brundage et al., 1990), but assumes an inherently protease-resistant conformation at the liposome surface (as
illustrated in Figure 9A, left panel). Association of proOmpA with liposome-bound
SecA preserves its protease
sensitivity (Figure 9A, center panel), while ATP hydrolysis
releases the proOmpA from SecA and allows its adsorption to the lipid in a protease-resistant
form (Figure 9A,
right panel). At a concentration of proteinase K of 300 Kg/
ml, the digestion of liposome-bound
proOmpA was incomplete, generating a reproducible pattern of proteolytic fragments that account for approximately
40% of the total
proOmpA added (Figure 9B, lane 1). If the proOmpA was
added to liposomes bearing adsorbed SecA protein and
incubated for 5 or 15 min at 37%, it was more readily
digested by protease (Figure 9B, lanes 5 and 6, respectively), owing to its association with the liposome-bound
SecA (Lill et al., 1990). Addition of ATP caused the release
of a substantial amount of this proOmpA for membrane
adsorption and gave rise to the same proteolytic fragments
SecA.
935
An”+,
and ATP in Preprotein
Translocation
Figure 9. ATP Hydrolysis
of ProOmpA from SecA
SecA
ATP
AMP-PNP
2
3
4
5
6
7
8 Std.
-
+
-
+
-
+
+
-
+
-
+
+
-
+
+
(Figure 9B, lane 7) observed in the absence of SecA. Incubation with AMP-PNP did not confer protease resistance
on SecA-bound proOmpA (Figure 96, lane 8).
We conclude that ATP hydrolysis by SecA promotes
the release of most of the bound proOmpA in this model
reaction. While proOmpA is trapped by adsorption to the
lipid bilayer upon dissociation from SecA in the liposome
reaction, during translocation the proOmpA may directly
interact with SecYIE. SecA may bind sequentially to separate domains of proOmpA after each ATP-dependent
release as the chain proceeds through the membrane (see
Figure 1).
Late Stages of Translocation
In agreement with the studies of Tani et al. (1990), we
find that the late stage of translocation, from the internal
disulfide form of Ia to full-length proOmpA, apparently
does not require SecA and ATP to the same extent as the
earlier intermediates
(see Figure 4E). After reduction of
the disulfide bond, almost the same extent of conversion
of Ia to full-length translocated proOmpA occurred in the
presence of AMP-PNP (lane 8) as with ATP at low concen,trations (lane 2) or high (lane 4). A more detailed kinetic
#analysis showed that the rate of the conversion of Ia to
,full-length translocated proOmpA was only increased 2-to
.Bfold by ATP compared with AMP-PNP (data not shown).
‘The binding of AMP-PNP seemed to provide enough energy for the chase of I= to full-length translocated protOmpA, while removal of residual ATP by hexokinase plus
glucose caused most of the I= to reverse translocate to lZ6
((see Figure 4E, lanes 8 and 9). In the absence of reductant
the Release
(A) A model of the binding of proOmpA
to
liposome-associated
SecA. Left panel: In the
absence of SecA, proOmpA binds to the lipid
and assumes a conformation
with high protease resistance.
Middle panel: In the presence
of SecA, proOmpA associates
with the SecA
and remains protease sensitive.
Right panel:
Upon ATP hydrolysis,
proOmpA
is released
from SecA and can adsorb to the liposome
surface.
(B) [%]proOmpA
(30,000 cpm per reaction)
was added from 6 M urea to eight reactions
containing 200 pglml liposomes prepared from
E. coli phospholipids
(see Experimental
Procedures) in buffer D. The final concentration
of
urea was below 150 mM. The indicated reactions contained 50 nglml SecA, 2 mM ATP, or
2 mM AMP-PNP.
Incubation at 37OC was for
15 min (reactions
1, 3, 4, and 6) or for 5 min
(reactions
2 and 5). In reactions 7 and 8, ATP
and AMP-PNP, respectively,
were added after
incubation for 5 min at 37%, and the incubation was continued
for 10 min at 37%. After
cooling on ice, protease
treatment
was performed for 15 min at 0% (300 uglml proteinase
K). Protease action was stopped by the addition
of trichloroacetic
acid, and precipitates
were
analyzed. The lane to the right (Std.) is a standard of proOmpA corresponding
to 10% of the
amount used in each reaction.
B
1
Allows
or ATP, where I= reverse translocates to lZ6(lanes 3 or 9)
AMP-PNP allows translocation to a species that is approximately 2 kd larger than lZ6(lane 7), as seen for the lZ6that
arises as a kinetic intermediate (see Figure 48, lanes 3
and 4).
Discussion
We have presented a working model (Hart1 et al., 1990) of
translocase-dependent
preprotein translocation. Our current studies now provide considerable detail to the membrane transit steps of this model (Figure 1). Some preproteins, such as ribose-binding protein, do not need SecB in
vivo (Kumamoto and Beckwith, 1985) and presumably
bind directly to translocase without stabilization or targeting assistance. Others, such as proOmpA and preMBP, are stabilized by interactions with SecB and then
bind to the SecA domain of translocase by virtue of the
ability of SecA to specifically recognize SecB (Hart1 et al.,
1990) and the leader and mature domains of precursor
proteins (Lill et al., 1990) activating the ATPase site of
SecA.
Each preprotein would be expected to have its own
unique set of stable translocation
intermediates, based
on its sequence and folding properties. While our studies
have been limited to proOmpA, this precursor is of average
size, has a typical leader sequence, and has been shown
to require AFT+ (Zimmermann and Wickner, 1983) SecA
(Wolfeet al., 1985) and SecY (Shibaet al., 1984)functions
for its in vivo export. The experiments described here, and
summarized in Figure 1, explore the roles of SecA, ATP,
Cell
936
and Ap,,+ in the establishment, maintenance, and further
movement of intermediates
in proOmpA translocation
(Figure 3) across the plasma membrane of E. coli. Several
of these intermediates were characterized in some detail
(Figures 4,5, and 7) establishing their authenticity. Based
on this characterization,
experiments were performed that
define a stepwise cycle promoting limited translocation of
SecA-bound intermediates. The proOmpA-SecB
complex
initially binds with high affinity to translocase (Hart1 et al.,
1990; Figure 1, step 1). The energy of ATP binding to SecA
(step 2) drives an initial, limited translocation event (Figure
2; Figure 1, step 3). Limited translocation events, driven
by binding nonhydrolyzable
ATP analogs, can also be
seen with llB (Figure 6) and with 1%(Figure 4). ATP hydrolysis then allows release of the bound segment of proOmpA
from SecA (Figures 6 and 9; Figure 1, step 4) permitting
further Ap,++ - dependent translocation (Figure 6; Figure
1, step 5).
The ability of SecA and ATP to drive the total translocation reaction suggests that hydrolysis may permit multiple
cyclesof SecA-proOmpA
binding, nucleotide binding, limited translocation, nucleotide hydrolysis, and release of
preprotein and nucleotide from SecA. In the absence of
SecA association, Af.rH+ alone promotes a rapid and efficient transit of a translocation intermediate, though SecA
association inhibits this transit and transit is blocked by
SecA plus a nonhydrolyzable
ATP analog (Figure 6). The
initiation of translocation, in which the first N-terminal loop
of proOmpA crosses the membrane, requires ATP and
SecA (Figure 1) and apparently cannot be driven by AftH+
alone, conferring a greater dependence
of the overall
translocation process on ATP during the early phases and
on AnH+ later on (Geller and Green, 1969). Our current
data strongly suggest that the potential-driven
translocation occurs after ATP hydrolysis has released each translocation intermediate of proOmpA from its association with
SecA (Figure 9). ATP hydrolysis is not coupled to net polypeptide chain movement (Figure8), suggesting that a‘slipping” reaction can occur. Indeed, in the absence of SecA,
there is reverse translocation (Figures 4E, 7C, and 5) that
is physiologically
prevented by the ApH+. While several
thousand ATP molecules are hydrolyzed for each proOmpA that translocates in the absence of ApH+, this ratio
falls to under 200 in the presence of a potential (E. S.,
unpublished data). Thus, translocation and ATP hydrolysis
are not “coupled” in the classical biochemical sense. Our
experiments do not address the question of whether part
of the energy of ATP hydrolysis may also be transferred
to the SecYlE protein to do work during translocation.
The forward or reverse movement of the polypeptide
chain is presumably driven by the net energy balance at
each stage of translocation. This energy balance may be
comprised of the relative energies for the transfer of residues into, or out of, the membrane (von Heijne and Blomberg, 1979); the energies of unfolding the precursor protein on the cytoplasmic membrane surface and refolding
it in the periplasm; and the energy contribution of ApH+,
which might include AY-driven electrophoresis
of acidic
residues and ApH-driven deprotonation of basic residues
in the cytoplasm and reprotonation in the periplasm. It is
striking that most of the translocation
reaction can be
driven by AB,++ (Figure 6). The inhibition of this potentialdriven translocation by SecA association suggests that
potentialdriven
translocation occurs after ATP hydrolysis
releases the proOmpA from its SecA association and before reassociation occurs. Energy imputsfrom either AILS+
or the binding and hydrolysis of ATP by SecA may contribute to a favorable energy balance for forward translocation, though byverydifferent
mechanisms(Figure
1). Similarly, pause or arrest of translocation may be due to factors
that affect any of the above elements of the energy equation. Thus, when covalent modification of the cysteine residues with BPTI blocks translocation, the remaining untranslocated chain may still bind to SecA (step 1’) facilitate
binding of ATP (step 21, and (without net translocation)
allow ATP hydrolysis.
Interesting mechanistic parallels may be drawn between
the translocation
of Set-independent
proteins, such as
Ml3 procoat (Wolfe et al., 1984), and Set-dependent
proteins, such as proOmpA. Procoat has a typical leader sequence (Kuhn et al., 1987) and requires ApH+ for its membrane insertion (Date et al., 1980) yet does not use either
SecA or SecY (Wolfe et al., 1984; Ohno-lwashita and Wickner, 1983). As with procoat, ApH+-driven proOmpA translocation occurs while the proOmpA is not associated with
SecA (Figure 6B). Further work will be needed to determine whether or not this potential-driven proOmpA translocation occurs while the protein is associated with SecYIE.
Each of the major themes of bacterial translocation-a
required ATPase activity, proteinaceous receptors, membrane transit that is sensitive to protein folding, and required chaperone function-is
also characteristic of protein transfer across eukaryotic membranes. Mitochondrial
import is thought to use the energy of ATP hydrolysis to
release some precursor proteins from cytosolic chaperones prior to translocation and to catalyze their correct
folding once import has occurred (Eilers et al., 1988; Ostermann et al., 1989; Hart1 and Neupert, 1990). Preprotein
transfer into the yeast or canine endoplasmic reticulum
requires ATP or GTP, respectively (Hansen et al., 1986;
Rothblatt and Meyer, 1986; Wilson et al., 1988). Each
translocation
event is mediated by protein receptors.
While the receptors for mitochondrial import are only now
being defined (Sollner et al., 1989; Pain et al., 1990; Hines
et al., 1990), mammalian endoplasmic reticulum import
relies on the combined functions of signal recognition particle, a peripheral membrane protein, and docking protein,
its integral membrane receptor (Walter et al., 1984).
Another shared theme is that preproteins cross membranes in an unfolded state (Wolfe and Wickner, 1984).
Stabilization of the folded state of an artificial mitochondrial preprotein was shown to prevent its import (Eilers and
Schatz, 1986) and protein folding can prevent transfer
across the plasma membrane of E. coli (Liu et al., 1989;
Phillips and Silhavy, 1990). In yeast, cytosolic heat shock
proteins of the hsp70 family have been shown to affect
translocation into both the endoplasmic reticulum and mitochondria (Deshaies et al., 1988; Chirico et al., 1988).
These proteins are believed to function as ATP-dependent
“molecular chaperones.” It has been shown that model
SecA,
937
AwH+, and ATP in Preprotein
Translocation
peptide substrates can activate the ATPase activity of two
:such chaperone components (Flynn et al., 1989), while
,ATP hydrolysis may promote chaperone/substrate
dissociation. There are, however, striking distinctions between
ithese several translocation
reactions. Whereas ATP is
llargely consumed during translocation on the noncyto(plasmic face of the mitochondrial or endoplasmic reticulum membrane by HspGOor BiP, respectively, SecA hydrolyzes ATP during bacterial export on the cytoplasmic face
of the membrane. The simplest unifying view would be
that, in each case, the ATPase (BiP, Hsp60, or SecA) is
acting through direct physical interaction with the preprol:ein to energetically drive membrane transit.
Recent advances may now allow an approach to remaining fundamental problems in bacterial translocation.
These include the precise roles of ApH and AY in translocation, the basis of recognition of exported proteins by
SecA and SecB, and the pathway by which the protein
traverses the membrane. The identification of transmembrane intermediates
may especially facilitate the latter
study.
Experimental
Procedures
Hlochemicais
Inverted E. coli inner membrane vesicles were prepared from the E.
coli strain KM9 (uric-::TnlO,
re/Al , spoTI, m&l;
Klionskyet
al., 1984)
as described (Chang et al., 1978). The following proteins were purified
according
to published methods:
SecA protein (Cunningham
et al.,
1989), [35S]proOmpA
(Crooke
and Wickner,
1987), unlabeled
proOmpA (Crooke et al., 1988), SecB (Weiss et al., 1988, as modified by
Lecker et al., 1989), and SecYlE complex (Brundage
et al., 1990).
ProOmpA leader peptide was synthesized
chemically
and purified by
high pressure
liquid chromatography
(Cunningham
and Wickner,
1989). SecY/E proteoliposomes
were reconstituted
from purified
SecY/E and E. coli phospholipids
(Avanti Polar Lipids, Pelham, AL)
as described
(Brundage
et al., 1990). Anti-SecA
immunoglobulin
G
fractions were purified according
to Lill et al. (1989). Liposomes were
prepared from E. coli phospholipids
(Avanti Polar Lipids, Pelham, AL)
at a concentration
of 20 mg of phospholipid
per ml in 50 mM HEPESKOH (pH 7.2), 30 mM KCI, 30 mM NH&I, 2.5 mM Mg(OAc)2. 1 mM
dithiothreitol
(buffer D) by sonication
(0.5 s pulses) at O°C for 2 min
with a microtip (Heat Systems-Ultrasonics,
NY). Liposome preparations were flushed with nitrogen and stored in the dark on ice. Prior to
use, bovine serum albumin (BSA) (fatty acid free, Sigma) was added
at 0.5 mglml to appropriate
dilutions of liposome stocks.
Synthesis
and Purltlcatlon
of Oxidized
ProOmpA,
ProOmpA-SPDP-BPTI,
and ProOmpA-MBS-BPTI
The disulfide bond of proOmpA was oxidized by incubating proOmpA
in 6 M urea, 50 mM Tris-Cl (pH 8), 200 NM sodium tetrathionate
(Sigma)
for 60 min at 0%. BPTI was coupled to the cross-linkers
SPDP (Carls.son et al., 1978) and MBS (Vestweber
and Schatz, 1988) as described.
The activated BPTI molecule was then cross-linked
to one of the two
cysteines
of proOmpA (Carlsson
et al., 1978; Vestweber
and Schatz,
1988). The unreacted
proOmpA and BPTI were separated
from the
adduct by chromatography
on a MonoQ column (FPLC, Pharmacia)
in
6 M urea, 50 mM Tris-Cl (pH 8.5), 1 mM EDTA at room temperature.
in Vitro Transiocation
of ProOmpA
Translocation
into inverted inner membrane
vesicles was performed
in buffer A (50 mM HEPES-KOH
[pH 8],50 mM KCI, 5 mM MgCI?, 0.5
mg/ml BSA [fatty acid free, Sigma], 2 mM dithiothreitol)
containing 40
pg/ml SecA, 5 to 15 @ml SecB, and 4 mM ATP. For ATP concentrations of 1 to 250 pM, the triphosphate
was regenerated
by the presence
of 10 mM creatine phosphate and IO @ml creatine kinase. Translocation reactions (50 or 100 ~1) were initiated with [35S]proOmpA (50,000
to 70,000 cpm; 10,000 cpmlng proOmpA).
P”S]proOmpA
was diluted
50-fold from solution in 6 M urea, 50 mM Tris-Cl (pH 7.6) into buffer
A containing SecB. Samples were incubated for the indicated times at
37OC and rapidly chilled. Translocation
into proteoliposomes
containing the purified SecYlE protein was as described
above, except
that 50 ug/ml SecB, 2 mM ATP, and 4 wg/ml purified proOmpA or
its derivatives
were used. Translocation
reactions were treated with
proteinase K (500 pglml) for 20 min at 0%. Digestion was stopped by
the addition of trichloroacetic
acid. Proteins were collected by sedimentation and resuspended
in 30 ~1 of SDS sample buffer, heated for
3 min at 95%, and layered on a polyacrylamide
slab gel with SDS.
The gels were analyzed by immunoblotting
or fluorography.
Miscellaneous
Protein was determined
using the Bradford reagent (Bio-Rad). Acid
precipitation:
Sample was mixed with an equal volume of ice-cold 25%
trichloroacetic
acid, incubated
for 15 min on ice, and collected
by
centrifugation
(Eppendorf centrifuge,
10 min, 4%). The sediment was
suspended
in 1 ml of acetone and centrifuged
as above. The acetone
was removed by aspiration followed by an incubation at 37% for 5 min.
Protein molecular weight standards
were purchased
from Bethesda
Research
Laboratories.
SDS-PAGE
and immunostaining
of proteins
after electrotransfer
to nitrocellulose
followed the methods of Ito et al.
(1980) and Towbin et al. (1979), respectively.
The fluorographs
were
quantified by a Zenith Soft laser scanner Model SLR-IDIPD.
Acknowledgments
We thank Marilyn Rice Leonard and Douglas Geissert for expert technical assistance.
This work was supported
by a grant to W. W. from the
National Institute of General Medical Sciences and gifts from Biogen,
Inc. E. S. and F.-U. H. are fellows of the Deutsche
Forschungsgemeinschaft
(DFG). A. D. is supported
by a grant from the Netherlands
Organization
for Scientific Research
(N. W.0).
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 USC Section 1734
solely to indicate this fact.
Received
October
22, 1990;
revised
December
14. 1990.
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