Plant Physiol. (1998) 116: 805-814
Identification of a Role for an Azide-Sensitive Factor in the
Thylakoid Transport of the 17-Kilodalton Subunit of the Photosynthetic
Oxygen-Evolving Complex1
Ellen A. Leheny,
Sarah A. Teter, and
Steven M. Theg*
Division of Biological Sciences, Section of Plant Biology, One
Shields Avenue, University of California, Davis, California
95616
 |
ABSTRACT |
We have examined the transport of the
precursor of the 17-kD subunit of the photosynthetic
O2-evolving complex (OE17) in intact chloroplasts in the
presence of inhibitors that block two protein-translocation pathways in
the thylakoid membrane. This precursor uses the transmembrane pH
gradient-dependent pathway into the thylakoid lumen, and its transport
across the thylakoid membrane is thought to be independent of ATP and
the chloroplast SecA homolog, cpSecA. We unexpectedly found that azide,
widely considered to be an inhibitor of cpSecA, had a profound effect
on the targeting of the photosynthetic OE17 to the thylakoid lumen. By
itself, azide caused a significant fraction of mature OE17 to
accumulate in the stroma of intact chloroplasts. When added in
conjunction with the protonophore nigericin, azide caused the
maturation of a fraction of the stromal intermediate form of OE17, and
this mature protein was found only in the stroma. Our data suggest that
OE17 may use the sec-dependent pathway, especially when
the transmembrane pH gradient-dependent pathway is inhibited. Under
certain conditions, OE17 may be inserted across the thylakoid membrane
far enough to allow removal of the transit peptide, but then may slip
back out of the translocation machinery into the stromal compartment.
| |
INTRODUCTION |
Although the chloroplast possesses its own genome, the majority of
proteins found in this organelle are nuclear encoded, synthesized on
cytoplasmic ribosomes, and posttranslationally imported. The process by
which these proteins are directed to their final destination is
complicated by the fact that chloroplasts possess three membranes that
define six distinct destinations for the newly imported protein.
Nuclear-encoded chloroplast proteins are generally synthesized as
higher-molecular-mass precursors that possess a cleavable, amino-terminal extension called a transit peptide. This topogenic sequence acts to direct the polypeptide from the cytoplasm to its final
location within the chloroplast. Proteins residing in the thylakoid
lumen are required to cross the envelope and thylakoid membranes, and
generally possess a bipartite transit peptide. The first region of the
targeting sequence directs the protein across the envelope membranes
into the stroma, where it is cleaved by the stromal-processing
protease, resulting in an intermediate-sized protein species. The
remaining region of the transit peptide then targets this stromal
intermediate to the thylakoid lumen, where it is removed by the
membrane-bound lumenal processing protease, generating the mature-sized
protein (for review, see Theg and Scott, 1993
; Cline and Henry, 1996;
Haucke and Schatz, 1997).
There have been extensive efforts to determine the mechanism by which
proteins are transported into or across the thylakoid membrane.
Currently, four pathways have been defined by their energy
requirements, and some of their components have been identified. The
first pathway appears to be spontaneous; no energy sources or
protease-sensitive membrane factors seem to be required (Michl et al.,
1994; Lorkovic et al., 1995). The second pathway shares some features
with protein translocation across the ER membrane. A chloroplast
homolog of the GTP-requiring signal-recognition particle 54-kD subunit
has been cloned and sequenced from Arabidopsis thaliana
(Franklin and Hoffman, 1993), and its involvement in the thylakoid
targeting of the light-harvesting, chlorophyll-binding protein has been
demonstrated (Li et al., 1995). The third pathway is homologous to the
sec pathway in Escherichia coli. Recently, chloroplast homologs of SecA (Nakai et al., 1994; Yuan et al., 1994;
Berghofer et al., 1995) and SecY (Laidler et al., 1995) have been
identified, and the involvement of cpSecA in thylakoid protein
translocation has been confirmed (Yuan et al., 1994). Transport of thylakoid proteins by the sec-dependent pathway
is inhibited by the action of azide on SecA (Yuan et al., 1994). As a
result, azide is used diagnostically in chloroplast import studies to
identify proteins that are translocated by this pathway (Knott and
Robinson, 1994). Proteins known to be transported in an azide-sensitive
manner include OE33, plastocyanin, and PSI-3 (Cline et al., 1992;
Karnauchov et al., 1994; Knott and Robinson, 1994; Yuan and Cline,
1994). As in E. coli, translocation of these proteins has
been demonstrated to require ATP. In addition, a transmembrane pH
gradient has been shown to stimulate their transport (Cline et al.,
1992).
The fourth pathway for thylakoid protein transport appears to be truly
unique in that it is dependent only on a transthylakoidal 
pH, making
it the only known energy-requiring protein transport process that can
drive translocation in the absence of nucleotide triphosphates. Cline
et al. (1992) demonstrated that ATP-depleting enzymes, as well as
compounds such as valinomycin, which dissipate the membrane potential,
had no discernible effect on protein transport by this pathway.
However, if the protonophore nigericin was used to collapse the pH
across the thylakoid membrane, protein transport into the thylakoids
was abolished, even when ATP was included in the assay medium. Because
these experiments were conducted in isolated thylakoids, an essential
role for stromal factors in translocation could also be eliminated.
Additional experiments have also demonstrated a requirement for the
pH throughout the entire import process, thus excluding the
possibility that it is used only to unfold proteins or to initiate
transport (Brock et al., 1995). Four lumenal proteins that use this
pathway have been identified: OE17, OE23, PSI-N, and PSII-T (Mould and
Robinson, 1991; Cline et al., 1992; Ikeuchi, 1992; Nielsen et al.,
1994).
Competition experiments using overexpressed precursor proteins from the
pH- and sec-dependent pathways revealed that proteins from one pathway do not compete with those from the other pathway during transport across the thylakoid membrane (Cline et al., 1993).
Thus, it has been suggested that each pathway displays substrate
specificity and possesses distinct components. A number of our own
experiments have led us to consider the possibility that there might be
shared components used by both pathways that had gone undetected in
previous studies. To address this question more completely, we examined
the effect of azide and nigericin on the thylakoid import of OE17, a
protein transported by the pH-dependent pathway. In these
experiments we have detected the involvement of an azide-sensitive
factor that results in the maturation, but not the compete transport,
of this protein in the presence of nigericin.
| |
MATERIALS AND METHODS |
Chloroplast Isolation and Preparation
Intact chloroplasts were isolated from 12- to 14-d-old pea
(Pisum sativum cv Progress 9) seedlings, as described by
Theg and Geske (1992). Chloroplasts were resuspended in IB that
contained 330 mm sorbitol and 50 mm K-Hepes, pH
8.0 (1× IB). Thylakoids were prepared from intact chloroplasts by
osmotic lysis. Chloroplasts were pelleted, resuspended in 10 mm K-Hepes, pH 8.0, with 5 mm MgCl2 (lysis buffer), and incubated on ice for 5 min. Membranes were collected by pelleting at full speed in a microfuge
for 5 min at 4°C. These membranes were washed twice in lysis buffer and their final concentration was adjusted to 1 mg/mL chlorophyll. Stroma was prepared by lysing chloroplasts at a high chlorophyll concentration with lysis buffer, incubating on ice for 5 min, pelleting
the membranes at full speed in a microfuge, and collecting the
supernatant (stroma). The stroma was then further centrifuged for 15 min at 4°C in a microfuge and transferred to a new tube for use.
Where indicated, thylakoids were resuspended at 1 mg/mL chlorophyll in
this stromal extract.
Protein Import Reaction Conditions
Cloned genes for OE17 and OE23 were transcribed and translated
with [3H]Leu (NEN-DuPont) in vitro, as
described previously (Cline et al., 1992). A typical import reaction
was performed in a volume of 60 µL and contained approximately
500,000 dpm of 3H-labeled precursor protein, 5 mm MgCl2, 5 mm ATP, and
0.33 mg/mL chlorophyll in 1× IB. Import reactions were conducted at
room temperature in the light (150 µE m
2
s1) for 30 min. Protease treatment of
chloroplast membranes was achieved by the addition of 200 µg
thermolysin/mL with 5 mm CaCl2. After
incubating for 15 min on ice, protease digestion was terminated by
washing the membranes twice in IB with 25 mm EDTA. Samples not digested with protease were treated similarly without the addition
of thermolysin. Unless otherwise noted, chloroplast import assays were
terminated by repurifying the chloroplasts through silicon oil into 1.5 m PCA (Leheny and Theg, 1994). With the exception of the
experiment presented in Figure 9, experiments performed with isolated
thylakoids were terminated by the addition of 1 mL of cold IB, followed
by two washes. The samples in Figure 1 were terminated by repurifying
the thylakoid membranes through silicon oil (a 50:50 [v/v] mixture of
Wacker AR20 and AR200 [Stauffer-Wacker Silicones Corp., Adrian, MI])
into PCA. Reactions that contained thylakoids with stroma were
terminated by the addition of an equal volume of 2× sample buffer
(0.125 m Tris, pH 6.8, 0.4% SDS, 20% glycerol, and 10%
-mercaptoethanol). Samples were separated on a 15% acrylamide
gel and visualized by fluorography. Quantitation of fluorograms for
Figure 8 was performed by densitometry (Bio Image, Ann Arbor, MI), as
described by Ettinger and Theg (1991), with individual calibrations
performed for each fluorogram.
View larger version (43K):
[in this window]
[in a new window]
| Figure 9.
A, Reactions run in the absence of inhibitor; B,
reactions run in the presence of azide; C, reactions run in the
presence of nigericin; and D, reactions run in the presence of azide
and nigericin. Lanes 1, Intact chloroplasts; lanes 2, isolated
thylakoids; lanes 3, stromal fraction; lanes 4, mock protease-treated
thylakoids; and lanes 5, protease-treated thylakoids. Intact
chloroplasts were preincubated with nigericin (6 µm) and
azide (30 mm) (or equivalent volumes of ethanol and water)
for 10 min in the dark on ice. Import reactions with precursor OE23 and
subsequent fractionations were performed as described in Figure 4,
except that an aliquot of thylakoids was removed before the protease
treatment. The standard (std) represents 20% of the precursor added to
each reaction; the positions of the precursor (p), intermediate (i),
and mature (m) proteins are noted.
|
|
View larger version (27K):
[in this window]
[in a new window]
| Figure 1.
Azide restores OE17 processing in the presence of
uncouplers. Where indicated, isolated intact chloroplasts were
preincubated with 30 mm azide, 15 µm CCCP,
and 6 µm nigericin (or equivalent volumes of water or
ethanol) for 10 min in the dark on ice. To start the reactions, the
chloroplasts were added to the import reaction mixture (final
concentrations of inhibitors were 10 mm azide, 5 µm CCCP, and 2 µm nigericin) and placed in
light for 30 min. Reactions were terminated by centrifuging into PCA as described in ``Materials and Methods''. The standard (std) represents
20% of the prOE17 added to each reaction; the positions of the
precursor (p), intermediate (i), and mature (m) proteins are noted.
|
|
View larger version (29K):
[in this window]
[in a new window]
| Figure 8.
Intact chloroplasts (lanes 5-8) and washed
thylakoids isolated from intact chloroplasts (lanes 1-4) were
preincubated for 10 min in the dark with 6 µm nigericin,
30 mm azide, or equivalent volumes of ethanol and water.
Reactions were initiated by the 3-fold dilution of chloroplasts or
thylakoids into the import mixture and proceeded for 30 min in the
light. Chloroplasts and thylakoids were reisolated by centrifugation
into PCA through Wacker AR200 and a 50:50 (v/v) ratio of Wacker AR20
and AR200 silicon oils, respectively. The standard (std) represents
20% of the precursor added to each reaction; the positions of the precursor (p), intermediate (i), and mature (m) proteins are noted.
|
|
Preincubation of Chloroplasts with Inhibitors and
ATP-Regenerating Compounds
Where indicated, chloroplasts or thylakoids were preincubated at 1 mg/mL chlorophyll for 10 min on ice with 6 µm nigericin (ethanolic stock), 30 mm sodium azide (aqueous stock), 15 µm CCCP (ethanolic stock), or an equivalent volume of
water or ethanol. In the experiment presented in Figure 8, 1 mm PMSF was included in the chloroplast preincubation
reactions. In the experiment presented in Figure 7, chloroplasts were
preincubated with 3 mm OAA and 3 mm DHAP or an
equivalent volume of IB for 10 min on ice in the dark.
View larger version (39K):
[in this window]
[in a new window]
| Figure 7.
A, Stromal extract does not process prOE17 to its
mature form. B, Thylakoid membranes are required for maturation. Lysis
buffer or concentrated stromal extract collected from the lysis of
chloroplasts at 6 mg chlorophyll/mL were preincubated with 6 µm nigericin (or ethanol) and 30 mm azide (or
water) for 10 min on ice in the dark. These solutions were added to 1×
IB containing radiolabeled prOE17, 5 mm MgCl2,
and 5 mm ATP, with no chloroplast membranes (A) or in the
presence of thylakoid membranes (B). The samples were incubated in the
light for 30 min and terminated by the addition of 2× sample buffer
without further purification. The standard (std) represents 20% of the
precursor added to each reaction; the positions of the precursor (p),
intermediate (i), and mature (m) proteins are noted.
|
|
9-Aminoacridine Measurements
Measurements of the thylakoid pH presented in Figure 3 were
made using the method described by Schuldiner et al. (1972). 9-Aminoacridine was purchased from Sigma, and its fluorescence and the
quenching thereof, in response to pH-generating illumination of
isolated thylakoids, was measured in an AB-2 fluorometer (SLM-Aminco, Rochester, NY).
View larger version (49K):
[in this window]
[in a new window]
| Figure 3.
Import reactions were initiated by the addition of
chloroplasts to 1× IB (final concentration in the reaction was 0.33 mg/mL chlorophyll) containing radiolabeled prOE17, 5 mm
MgCl2 and 5 mm ATP. A, Reactions incubated in
the absence of both inhibitors; B, reactions incubated with azide
alone; C, reactions incubated with nigericin alone; and D, reactions
incubated with both inhibitors. Lanes 1, Intact chloroplasts; lanes 2, washed thylakoids (containing contaminating envelope membranes); lanes
3, supernatant fraction after envelope lysis of sample in lanes 2 containing stromal components and envelope membranes; and lanes 4, protease-treated thylakoids. The reactions were treated as in Figure 1.
After this incubation, the samples were placed back in the light at
25°C for an additional 15 min. The chloroplast envelopes in lanes 2 and 4 were osmotically lysed in lysis buffer (see ``Materials and Methods'') containing 2 µm nigericin, whereas the
samples in lanes 1 were maintained in isotonic buffer (1× IB with 2 µm nigericin). Reactions were incubated on ice in the dark for 5 min. The osmoticum was then restored to samples in lanes 2 and 4 with the addition of 2× IB containing 2 µm
nigericin (an equivalent volume of 1× IB with 2 µm
nigericin was added to the samples in lanes 1). Samples in lanes 4 were
protease treated with thermolysin (200 µg/mL with 5 mm
CaCl2); samples in lanes 1 and 2 received 5 mm
CaCl2 only. After inactivating thermolysin with the
addition of 25 mm EDTA, membranes were pelleted and the supernatant collected from the samples in lanes 2. This supernatant was
precipitated with 1.5 m PCA and loaded in lane 3. The
standard (std) represents 20% of the precursor added to each reaction; the positions of the precursor (p) and mature (m) proteins are noted.
|
|
| |
RESULTS |
The import of OE17 into thylakoids has been shown to occur via the
pH-dependent pathway, thus making its translocation sensitive to
protonophores that collapse transmembrane pH gradients (Cline et al.,
1992; Klosgen et al., 1992). Figure 1
shows the import of OE17 into intact chloroplasts in the presence and
absence of azide and the ionophores nigericin and CCCP. As expected,
OE17 was transported into the thylakoid lumen and processed to its final, mature size in the presence or absence of azide, and the inclusion of nigericin or CCCP resulted in the inhibition of transport across the thylakoid membrane, as demonstrated by the accumulation of
the stromal intermediate. We were surprised to discover, however, that
when azide was included with these uncouplers in the preincubation, the
amount of stromal intermediate appeared to decrease with a corresponding increase in the abundance of mature-sized protein. Inclusion of the electrogenic ionophore valinomycin had no effect on
these experiments (data not shown). To our knowledge, this is the first
demonstration of a protein transported by the pH-dependent pathway
that is processed in the apparent absence of a transmembrane pH
gradient. Because the combined effect of azide occurred equally well
with nigericin and CCCP, we used only nigericin in the subsequent experiments.
There are two simple explanations for the effect of azide on OE17
maturation in the absence of a thylakoid pH gradient. The first is that
azide somehow inactivated, or at least decreased, the efficacy of
nigericin in collapsing the pH across the membrane. The second
possibility is that the combination of the two inhibitors lysed the
thylakoids and released the thylakoid-processing protease into the
stroma. These possibilities are addressed by the experiments reported
below.
Nigericin Is Still Active in the Presence of Azide
In the experiment presented in Figure 1, 2 µm
nigericin was used to dissipate the pH across the thylakoid
membrane, a concentration in excess of that required to inhibit OE17
transport (Mould and Robinson, 1991; Cline et al., 1992). We considered
the possibility that azide might necessitate the inclusion of a higher
concentration of nigericin to achieve the same result. Figure
2A demonstrates that concentrations as
high as 10 µm nigericin still resulted in the maturation
of OE17 when azide was present. To directly measure the membrane pH
gradient in the presence of these inhibitors, we used 9-aminoacridine,
a self-quenching indicator of the pH, with the acidification of the
lumen manifested as a decrease in fluorescence (Schuldiner et al.,
1972). As shown by the trace of relative fluorescence presented in
Figure 2B, no pH gradient was formed when the samples contained
nigericin, regardless of the presence of azide. Similar results were
observed with CCCP. This demonstrates that the OE17 maturation that was
observed in the presence of nigericin and azide (Fig. 1) was not the
result of the reestablishment of a pH across the thylakoid membrane. Measurements of the H+-pumping activities of
thylakoids (Dilley, 1970) independently confirmed that a pH was not
formed when nigericin and azide were present together (data not shown).
At this time, we cannot explain why a smaller pH gradient was
established in the presence of azide alone relative to the azide-free
control.
View larger version (28K):
[in this window]
[in a new window]
| Figure 2.
A, Increasing concentrations of nigericin do not
abolish OE17 processing in the presence of azide. Intact chloroplasts
were preincubated with 0, 6, 15, or 30 µm nigericin
(always with the same volume of ethanol) in the dark for 10 min on ice
in the presence or absence of 30 mm azide (or an equivalent
volume of water). Chloroplasts were diluted 3-fold into the import
mixture to start the assay, and reactions were allowed to continue for
30 min in the light. The import assay was terminated and the samples
analyzed as described for Figure 1. The standard (std) represents 20%
of the precursor added to each reaction; the positions of the precursor (p), intermediate (i), and mature (m) proteins are noted. B, Azide does
not inactivate the uncoupling activity of nigericin. Isolated thylakoids were preincubated with 6 µm nigericin (or an
equivalent volume of ethanol) and 30 mm azide (or an
equivalent volume of water) for 10 min on ice in the dark. Thylakoids
were then diluted 3-fold into import buffer containing 20 µm methyl viologen and 3 mm MgCl2
to a concentration of 20 µg/mL. 9-Aminoacridine (5 µm)
was added and the fluorescence was measured. Saturating red actinic
light was turned on and off at 20 and 100 s; the shutter in front
of the measuring beam was briefly shut at 75 s to determine if the
sample chamber was light tight.
|
|
The Combination of Nigericin and Azide Does Not Cause Chloroplast
Lysis
Although each inhibitor alone does not compromise thylakoid
membrane integrity, we investigated whether they might result in
vesicle lysis when present in combination, thereby exposing the stromal
intermediate to the thylakoid processing peptidase located on the
lumenal face of the membrane (Kirwin et al., 1989). To address this
question we allowed the mature protein to accumulate in the lumen
before the addition of nigericin and azide. If these compounds acted to
release the processing protease from the thylakoid vesicles, the mature
protein should also be released and become accessible to exogenous
protease. We saw no decrease in the amount of mature-sized protein in
the presence of nigericin and azide after protease treatment (Fig.
3D, lane 4) compared with the protected mature protein seen in the absence of this combination of inhibitors (Fig. 3, A-C, lanes 4). Furthermore, the mature protein was not precipitated from the supernatant after lysis of the envelope membranes
(Fig. 3D, lane 3) but, rather, pelleted with the thylakoids (Fig. 3D,
lane 2). These data indicate that the thylakoids were not lysed by the
combination of azide and nigericin.
mOE17 Formed in the Presence of Nigericin and Azide Is in
the Stroma
Having ruled out the more trivial explanations for the appearance
of mOE17 under our experimental conditions, we asked whether this
protein had been fully transported into the thylakoid lumen. To this
end, we conducted import experiments for 30 min, followed by
chloroplast fractionation and thermolysin treatment of the thylakoids
(Fig. 4). As expected, when inhibitors
were absent from the import assay, mOE17 fractionated with the
thylakoids and was protease resistant, confirming its location in the
thylakoid lumen (Fig. 4, A-C, lanes 4). Surprisingly, when the sample
that had been incubated with both nigericin and azide was fractionated, the mOE17 was found to be soluble in the stromal fraction (Fig. 4D,
lane 2), rather than in the thylakoid lumen (Fig. 4D, lane 4). A
similar appearance of mOE17 in the stroma was previously noted when the
targeting sequence had been replaced by that for the precursor of OE33,
and was attributed to inefficient and aberrant targeting of an
artificial chimeric protein (Clausmeyer et al., 1993; Henry et al.,
1994). However, our experiments were performed with authentic prOE17,
and so this cannot be the explanation for our observations.
View larger version (56K):
[in this window]
[in a new window]
| Figure 4.
A, Reactions incubated in the absence of both
inhibitors; B, reactions incubated with azide alone; C, reactions
incubated with nigericin alone; and D, reactions incubated with both
inhibitors. Lanes 1, Intact chloroplasts; lanes 2, stromal fractions;
lanes 3, washed thylakoids; and lanes 4, protease-treated thylakoids. A
4× import reaction was initiated by the addition of 80 µg of chlorophyll and a 30-min import reaction was conducted in the light. At
this time, one aliquot was removed and the sample was centrifuged
through silicon oil into PCA; these intact chloroplasts were run in
lanes 1. The remaining chloroplasts were pelleted for 30 s in a
microfuge, the supernatant was removed, and the chloroplasts were
resuspended in 0.5 volume of envelope lysis buffer containing 4 µg of
aprotinin, 4 µg of leupeptin, and 1 mm PMSF. To
distinguish between mOE17 bound to the lumenal face of the membrane and
proteins that were fully translocated, we protease treated one-half of
the thylakoids with thermolysin (lanes 4), whereas the other half was
mock-protease treated (lanes 3). The thylakoid fraction also contained
fragmented envelope membranes; thus, some prOE17 is seen in the samples
that are not protease treated (lanes 3). The standard (std) represents
20% of the precursor added to each reaction; the positions of the
precursor (p), intermediate (i), and mature (m) proteins are noted.
|
|
The results of the experiment shown in Figure 4 suggested two
possibilities for the mechanism by which OE17 is processed to the
mature size in the presence of nigericin and azide. First, it is
possible that iOE17 was cleaved by an uncharacterized stromal protease,
and did not engage the thylakoid-targeting apparatus. Alternatively,
some portion of the protein may have actually crossed the thylakoid
membrane far enough to be processed by the lumenal-processing protease,
but then slipped out of the transport pore back into the stroma. In
either case, this activity was manifested only in the presence of both
azide and uncoupler. In the reactions containing azide alone (no
uncoupler), a significant fraction of the mature polypeptide also
fractionated with the stroma (Fig. 4B, lane 2). These results suggest
that azide plays a previously undetected role in either OE17 processing
or transport.
Stromal Proteases Do Not Appear to Be Responsible for the
Generation of mOE17 That Accumulates in the Presence of Nigericin and
Azide
There are several proteases present in the stroma that have been
shown to digest imported proteins (Musgrove et al., 1989; Lindahl et
al., 1996). We explored the possibility that azide might favor a
nonnative conformation of iOE17, which would make it more susceptible
to these proteases, thereby leading to a mature-sized proteolytic
product. This postulate suggests that, although stromal factors are
thought not to be involved in OE17 transport (Cline et al., 1992),
iOE17 may in fact be complexed with another component, such as a
stromal chaperone.
Hsp70 chaperones have been identified as components of the chloroplast
envelope machinery (Marshall et al., 1990; Ko et al., 1992; Wu et al.,
1994) and as soluble factors in the stroma (Marshall and Keegstra,
1992). Presumably, these chaperones bind to polypeptides as they are
being translocated across the envelope membranes, perhaps to maintain
the proteins in a loosely folded conformation and to prevent their
premature folding. Upon complete translocation of the protein, ATP
would be expected to be hydrolyzed and the chaperones released. The
necessity for ATP hydrolysis by the chaperone suggests two
possibilities for a chaperone-mediated effect of azide on OE17
processing. First, through its effect on the chloroplast coupling
factor (Murataliev et al., 1991), azide might restrict ATP synthesis in
the plastid, which could inhibit the release of the chaperone from the
partially folded protein. Alternatively, azide might bind to the hsp70
ATP-binding site directly, again resulting in an extended interaction
between the chaperone and iOE17. In either case, maintenance of the
polypeptide in a loose conformation might result in the exposure of a
proteolytic site that is normally not accessible to stromal proteases
when the protein is tightly folded. We tested the first of these
postulates by including the Calvin-Bensen cycle intermediates OAA and
DHAP in our reactions. These intermediates allow high stromal ATP
concentrations to be maintained independently of photophosphorylation
(see Olsen and Keegstra, 1992). Figure 5
demonstrates that the inclusion of these compounds had no effect on the
appearance of the mature-sized protein when azide was included with
nigericin (Fig. 5, compare lane 7 with lane 8). This suggests that
azide does not act indirectly in our experiments through an effect on
stromal ATP concentrations. These data, however, do not exclude the
possibility that azide occupies a chaperone ATP-binding site.
View larger version (21K):
[in this window]
[in a new window]
| Figure 5.
Intact chloroplasts were preincubated with
OAA/DHAP (3 mm), azide (30 mm), nigericin (6 µm), or equivalent volumes of 1× IB, water, or ethanol,
respectively, for 10 min on ice in the dark. Reactions were initiated
by the 3-fold dilution of chloroplasts into the import mixture and
continued for 30 min in the light. The assay was terminated and samples
were analyzed as described in Figure 1. The standard (std) represents
20% of the precursor added to each reaction; the positions of the
precursor (p), intermediate (i), and mature (m) proteins are noted.
|
|
In an attempt to inhibit stromal proteases directly, import reactions
were performed with PMSF, a membrane-permeable Ser protease inhibitor.
Figure 6 shows that the inclusion of PMSF
in the import reactions did not result in a decrease in the maturation
of iOE17 in the presence of azide and nigericin, indicating that
this phenomenon is not caused by a stromal Ser protease.
View larger version (33K):
[in this window]
[in a new window]
| Figure 6.
Chloroplasts were preincubated with 1 mm PMSF (striped bars) or an equivalent volume of
isopropanol (open bars) in the presence of nigericin (6 µm) and azide (30 mm) (or equivalent volumes
of ethanol and water) for 10 min on ice in the dark. These chloroplasts were then diluted 3-fold into an import mixture containing 1 mm PMSF to initiate the protein-transport assay. Reactions
proceeded for 30 min in the light and were terminated by repurifying
the chloroplasts through silicon oil. Samples were separated by
SDS-PAGE, visualized by fluorography, and the fluorographs were
quantitated by densitometry. %IOD represents mOE17 as a fraction of
the total amount of radiolabeled protein found in the sample. The data
shown represent the average from three experiments.
|
|
To test directly for involvement of a stromal protease in OE17
maturation, we incubated the precursor in a concentrated stromal extract that had itself been preincubated with azide and nigericin, and
compared it with precursors that had been incubated in chloroplast lysis buffer alone (control). Figure 7A
reveals that stromal extract did not cause cleavage of the precursor to
the mature size, regardless of whether inhibitors were present. This
lack of processing was not attributable to a general decline in stromal
enzymatic activity because the precursor was processed to the
intermediate form by the soluble stromal processing protease. When
thylakoid membranes were included in the assay, however, maturation of
the protein in the presence of nigericin and azide was restored (Fig.
7B, lane 4). Furthermore, processing in the presence of membranes was
found to be inhibited by HgCl2 (data not shown),
a compound that modifies thiol groups and is often used to terminate
protein transport across the thylakoid membrane (Reed et al., 1990;
Yuan and Cline, 1994). These results, in conjunction with the fact that
PMSF had no effect on OE17 maturation in the presence of azide and
nigericin (Fig. 6), suggest that a stromal protease is not responsible
for the mature-sized product generated in our experiments, and that
iOE17 may interact with the thylakoid membrane during the cleavage
event.
Role of a Stromal Factor in OE17 Maturation
Because thylakoid membranes appeared to be necessary for
maturation of OE17 in the presence of nigericin and azide (Fig. 7B), we
asked whether a membrane-bound protease, exposed on the stromal surface, might be responsible for digesting the protein to a
mature-sized proteolytic fragment. To address this possibility, we
performed import experiments using isolated thylakoids that had been
washed several times to purify the membranes away from loosely bound stromal factors (Fig. 8). As expected,
OE17 import was prevented when only nigericin was present (Fig. 8, lane
3). However, in contrast to the situation found in whole chloroplasts,
the addition of azide to the nigericin-treated samples did not result
in maturation of the precursor (Fig. 8, lane 4). This was also true if
CCCP was used in place of nigericin (data not shown). Thus, it appears that a stromal-facing, membrane-bound protease is not solely
responsible for cleaving OE17 to a mature-sized protein. Furthermore,
this experiment suggests that a stromal factor may be required for this
process because maturation was seen only when thylakoids were incubated
with additional stroma (Fig. 7B). Unfortunately, reconstitution of
azide-/nigericin-sensitive maturation of OE17 in isolated thylakoids
was not reliably reproducible, and when achieved, occurred with low
efficiency. This precluded our investigation of the effects of a
broader range of exogenously added protease inhibitors on this process,
and of a potential requirement for nucleotide triphosphate
hydrolysis.
OE23 Processing Is Not Affected by Azide in the Absence of a
pH
To determine if the nigericin-/azide-dependent precursor
processing event is a general phenomenon, we carried out chloroplast fractionation after import of OE23, another polypeptide that uses the
pH-dependent pathway (Fig. 9). We
found that OE23 accumulated as a stromal intermediate in the absence of
a pH gradient (Fig. 9C, lane 3), even when azide was present (Fig. 9D,
lane 3). Thus, it appears that there is some difference between the
mechanism of OE17 and OE23 import that still allows the former, but not the latter, to be processed under conditions in which its complete translocation is inhibited.
| |
DISCUSSION |
We undertook the experiments presented here in an effort to
determine if there are common components used by the pH- and sec-dependent pathways that had previously gone undetected.
Using nigericin to inhibit transport by the pH-dependent pathway,
and azide to inhibit the sec-dependent pathway, we have
discovered what appears to be a role for an azide-sensitive factor in
the transport of a protein that follows the pH-dependent transport pathway into the thylakoid lumen. Our initial data demonstrated that
the addition of azide to chloroplasts that could not maintain a
thylakoid transmembrane pH resulted in restoration of prOE17 processing to the mature-sized protein (Fig. 1). Direct measurements of
the pH under these conditions indicated that the restoration of
processing was not simply the result of reduced efficacy of the
protonophore in the presence of azide (Fig. 2B). Furthermore, we showed
that processing was not simply a result of induced thylakoid lysis
(Fig. 3).
Although azide restored OE17 processing in the absence of a
transmembrane pH gradient, we found that protein targeting was still
aberrant because the mOE17 protein fractionated with the chloroplast
stoma instead of with the thylakoid lumen, its correct destination
(Fig. 4). This azide-induced mislocalization could also be observed to
a lesser extent when a pH was present across the thylakoid membrane,
indicating that azide plays an inhibitory role in OE17 import under
conditions in which the primary translocation pathway for this
substrate is still operative. In the experiment presented in Figure 2B,
we measured a smaller pH gradient in the presence of azide relative to
control thylakoids. However, because the pH generated under these
conditions was still well above levels required for efficient transport
via the pH-dependent pathway (Brock et al., 1995), it is unlikely
that this lower pH gradient accounts for the presence of mOE17 in the
stroma.
Further characterization of the effect of azide in restoring OE17
processing in the presence of protonophores suggested the involvement
of a stromal factor, since azide was ineffective at restoring OE17
maturation when isolated thylakoids had been purified away from stroma.
This was surprising because it has been amply demonstrated that stromal
factors are not required for efficient transport of proteins into the
thylakoid lumen via the pH-dependent pathway (Mould et al., 1991;
Cline et al., 1992). We explored the possibility that this newly
detected stromal factor might be the actual protease that is
responsible for OE17 maturation. The membrane-permeable protease
inhibitor PMSF had no effect on azide-induced restoration of processing
in nigericin-treated plastids (Fig. 6). Other protease inhibitors could
not be investigated because we were unable to consistently reconstitute
this assay using washed thylakoids and concentrated stroma. However,
the fact that we were unable to generate mOE17 by incubating it with stromal extract in the absence of thylakoid membranes (Fig. 7) makes it
unlikely that the processing event is mediated by a soluble stromal
protease.
We have also considered a model in which a stromal factor maintains
OE17 in a protease-sensitive conformation. In this view, a protease
that is associated with the stromal face of the thylakoid membrane
would cleave prOE17 polypeptides only in the presence of the
azide-sensitive stromal factor. A number of chaperones have been
identified in the stromal compartment (Hemmingsen et al., 1988;
Marshall and Keegstra, 1992; Moore and Keegstra, 1993). Although we are
unaware of any reports demonstrating an association of OE17 with any of
these stromal chaperones, it is possible that trapping this substrate
in the stromal compartment by inactivating its normal thylakoid
translocation pathway could allow it to form an association with
chaperones that had gone previously undetected. The azide effect
observed in our experiments could be explained by an azide-dependent
inhibition of ATP hydrolysis by the chaperone, which could result in a
prolonged chaperone-substrate interaction and an increased protease
susceptibility of the loosely folded substrate. It is noteworthy,
however, that we were unable to detect an association of OE17 with
other components present in the stroma (E.A. Leheny and S.M. Theg,
unpublished results).
Our data suggest that the proteolytic processing activity resides
either within the thylakoids or on the external thylakoid membrane
surface because processing does not occur during incubation of prOE17
with the stroma alone (Fig. 7). Recently, a homolog of bacterial FtsH,
an ATP-dependent metalloprotease, has been cloned and its protein
product has been localized to the thylakoid membrane (Lindahl et al.,
1996). Might this integral membrane protein be responsible for the
processing of OE17 in our experiments? In support of this notion, the
deduced amino acid sequence and topology of the plastid FtsH indicate
that the ATP- and Zn-binding sites required for proteolytic activity of
the related bacterial enzyme are both exposed to the stromal
compartment (Lindahl et al., 1996). However, azide does not affect
either proteolytic activity or in vitro ATP hydrolysis in the bacterial
FtsH homolog (Tomoyasu et al., 1995). Furthermore, FtsH cleaves target
proteins at many sites (Tomoyasu et al., 1995), whereas the cleavage of OE17 that we observed in our experiments was specific, resulting in
mOE17. If the chloroplast FtsH protease is responsible for this
processing, one would have to hypothesize that a stromal factor
stimulates its activity in the presence of azide, and that its
specificity is such that degradation products are the same size as
those created by the lumenal thylakoid processing protease.
Although we cannot rule out the possibility that a nonspecific protease
is responsible for the azide-/nigericin-dependent maturation of OE17,
we favor a third model, which posits that this protein is partially
translocated across the membrane in the presence of azide and
protonophores. Translocation of the amino-terminal portion would allow
processing by the thylakoid processing peptidase, but in the absence of
a pH to complete translocation, the transporting protein would then
be released from the translocation pore back into the stroma. A similar
retrograde transport event has been invoked by Roberts et al. (1991) to
explain the presence of mature-sized ricin in the stroma after its
import into chloroplasts behind lumen-directed transit peptides.
Retrograde movement of partially translocated precursors was also
observed in mitochondria in the absence of the transmembrane potential difference (Ungermann et al., 1996). This model is consistent with all
of our experiments, and is uniquely supported by our observation that
processing is sensitive to the translocation inhibitor
HgCl2. We recognize, however, that
HgCl2 may also inhibit stromal or
membrane-associated proteases in addition to the transport machinery.
The involvement of an azide-sensitive stromal factor in the maturation
of OE17 in the presence of ionophores suggests (although it does not
prove) a role for cpSecA in this process. Assuming that, as in
bacterial SecA, cpSecA undergoes repeated rounds of membrane insertion
and deinsertion (Economou and Wickner, 1994; Kim et al., 1994), and
that the deinsertion step is inhibited by azide (Nishiyama et al.,
1996), one can construct a reaction scheme in which azide-bound cpSecA
presents the amino terminus of OE17 in the lumen in such a way as to
allow cleavage of the transit peptide. In the absence of the pH
driving force, the protein would be unable to continue into the lumen
and would then slip back out into the stroma.
The postulate that SecA plays a role in the transport of certain
proteins that use the pH-dependent pathway has interesting implications. "Back-up" transport systems have been identified for
other translocation processes (Nunnari and Walter, 1996; Schatz and
Dobberstein, 1996), and our data suggest the possibility of pathway
redundancy in chloroplast protein transport as well. In this respect,
it is interesting to note that two nuclear mutations in maize that
cause defective targeting to the thylakoid lumen have been
characterized (Voelker and Barkan, 1995). The first, hcf106,
specifically affects proteins that are transported by the
pH-dependent pathway, whereas the second, tha1, affects
proteins on the sec-dependent pathway. The phenotype
associated with these mutations is that proteins accumulate as stromal
intermediates because their transport into the thylakoids is inhibited.
The mutation in tha1 has recently been identified as a null
mutation of the SecA gene (Voelker et al., 1997). In vivo data from
tha1 indicate that a certain fraction of the proteins
transported by the sec-dependent pathway are still correctly
transported to the lumen (Voelker and Barkan, 1995). This raises the
possibility that some components of the different translocation
machineries may be redundant and called into use if the pathway
normally used by a protein is inhibited.
This view is further supported by the recent study of Henry et al.
(1997) in which a "dual-targeting" transit peptide was constructed
that was capable of directing passenger proteins to either the
sec- or pH-dependent pathways. In these experiments, however, proteins normally on the pH-dependent pathway were found to
be incompatible with the sec pathway, such that
dual-targeting OE17 was transported only on the former pathway. The
experiments presented here suggest that under some conditions authentic
prOE17 might traverse the sec pathway, albeit inefficiently,
suggesting that our understanding of the structural requirements for
pathway-specific targeting remains incomplete.
At this time, we do not know the identity of the azide-sensitive factor
involved in the OE17 processing described here, and the model involving
cpSecA is speculative. It is consistent, however, with both our
observations and the known mode of action of azide on the SecA cycle.
Experiments are currently under way to test predictions generated by
this view. It will be interesting to determine if thylakoid protein
import will be another example of a cellular protein translocation
system that has developed redundancies as a fail-safe mechanism for
function.
| |
FOOTNOTES |
1
This work was supported in part by the U.S.
Department of Agriculture (grant no. 95-37304-2325 to S.M.T.) and by a
National Science Foundation training grant fellowship to S.A.T.
*
Corresponding author; e-mail smtheg{at}ucdavis.edu; fax
1-530-752-5410.
Received July 23, 1997;
accepted November 9, 1997.
| |
ABBREVIATIONS |
Abbreviations:
CCCP, carbonyl cyanide
m-chlorophenylhydrazone.
cpSecA, the chloroplast homolog
of bacterial SecA.
pH, transmembrane pH gradient.
DHAP, dihydroxyacetone phosphate.
IB, import buffer.
OAA, oxaloacetic acid.
OE33, OE23, and OE17, the 33-, 23-, and 17-kD subunits of the
O2-evolving complex, respectively.
PCA, perchloric acid.
prOE17, iOE17, and mOE17, the precursor, intermediate, and mature
forms of OE17, respectively.
| |
ACKNOWLEDGMENT |
We thank Marc Tormey for assistance with preparation of the
figures.
| |
LITERATURE CITED |
Berghofer J,
Karnauchov I,
Herrmann RG,
Klosgen RB
(1995)
Isolation and characterization of a cDNA encoding the SecA protein from spinach chloroplasts: evidence for azide resistance of sec-dependent protein translocation across thylakoid membranes in spinach.
J Biol Chem
270:
18341-18346
[Abstract/Free Full Text]
Brock IW,
Mills JD,
Robinson D,
Robinson C
(1995)
The pH-driven, ATP-independent protein translocation mechanism in the chloroplast thylakoid membrane.
J Biol Chem
270:
1657-1662
[Abstract/Free Full Text]
Clausmeyer S,
Klosgen RB,
Herrmann RG
(1993)
Protein import into chloroplasts: the hydrophilic lumenal proteins exhibit unexpected import and sorting specificities in spite of structurally conserved transit peptides.
J Biol Chem
268:
13869-13876
[Abstract/Free Full Text]
Cline K,
Ettinger WF,
Theg SM
(1992)
Protein-specific energy requirements for protein transport across or into thylakoid membranes: two lumenal proteins are transported in the absence of ATP.
J Biol Chem
267:
2688-2696
[Abstract/Free Full Text]
Cline K,
Henry R
(1996)
Import and routing of nucleus-encoded chloroplast proteins.
Annu Rev Cell Biol
12:
1-26
[CrossRef][Web of Science][Medline]
Cline K,
Henry R,
Li C,
Yuan J
(1993)
Multiple pathways for protein transport into or across the thylakoid membrane.
EMBO J
12:
4105-4114
[Web of Science][Medline]
Dilley RA
(1970)
The effect of various energy-conversion states of chloroplast on proton and electron transport.
Arch Biochem Biophys
137:
270-283
[Medline]
Economou A,
Wickner W
(1994)
SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion.
Cell
78:
835-843
[CrossRef][Web of Science][Medline]
Ettinger WF,
Theg SM
(1991)
Physiologically active chloroplasts contain pools of unassembled extrinsic proteins of the photosynthetic oxygen-evolving enzyme complex in the thylakoid lumen.
J Cell Biol
115:
321-328
[Abstract/Free Full Text]
Franklin AE,
Hoffman NE
(1993)
Characterization of a chloroplast homologue of the 54-kDa subunit of the signal recognition particle.
J Biol Chem
268:
22175-22180
[Abstract/Free Full Text]
Haucke V,
Schatz G
(1997)
Import of proteins into mitochondria and chloroplasts.
Trends Cell Biol
7:
103-106
Hemmingsen SM,
Woolford C,
van der Vies SM,
Tilly K,
Dennis DT,
Georgepolous C,
Hendrix RW,
Ellis RJ
(1988)
Homologous plant and bacterial proteins chaperone oligomeric protein assembly.
Nature
333:
330-334
[CrossRef][Medline]
Henry R,
Carrigan M,
McCaffery M,
Ma X,
Cline K
(1997)
Targeting determinants and proposed evolutionary basis for the Sec and delta pH protein import systems in chloroplast thylakoid membranes.
J Cell Biol
136:
823-832
[Abstract/Free Full Text]
Henry R,
Kapazoglou A,
McCaffery M,
Cline K
(1994)
Differences between lumen targeting domains of chloroplast transit peptides determine pathway specificity for thylakoid transport.
J Biol Chem
269:
10189-10192
[Abstract/Free Full Text]
Ikeuchi M
(1992)
Subunit proteins of photosystem II.
Bot Mag Tokyo
105:
327-373
[CrossRef]
Karnauchov I,
Cai DG,
Schmidt I,
Herrmann RG,
Klosgen RB
(1994)
The thylakoid translocation of subunit 3 of photosystem I, the psaf gene product, depends on a bipartite transit peptide and proceeds along an azide-sensitive pathway.
J Biol Chem
269:
32871-32878
[Abstract/Free Full Text]
Kim Y,
Rajapandi T,
Oliver D
(1994)
SecA protein is exposed to the periplasmic surface of the E. coli inner membrane in its active state.
Cell
78:
845-853
[CrossRef][Medline]
Kirwin PM,
Meadow JW,
Shackleton JB,
Musgrove JE,
Elderfield PD,
Mould R,
Hay NA,
Robinson C
(1989)
ATP-dependent import of a lumenal protein by isolated thylakoid vesicles.
EMBO J
8:
2251-2255
[Web of Science][Medline]
Klosgen RB,
Brock IW,
Herrmann RG,
Robinson C
(1992)
Proton gradient-driven import of the 16 kDa oxygen-evolving complex protein as the full precursor protein by isolated thylakoids.
Plant Mol Biol
18:
1031-1034
[CrossRef][Web of Science][Medline]
Knott TG,
Robinson C
(1994)
The SecA inhibitor, azide, reversibly blocks the translocation of a subset of proteins across the chloroplast thylakoid membrane.
J Biol Chem
269:
7843-7846
[Abstract/Free Full Text]
Ko K,
Bornemisza O,
Kourtz L,
Ko ZW,
Plaxton WC,
Cashmore AR
(1992)
Isolation and characterization of a cDNA clone encoding a cognate 70-kDa heat shock protein of the chloroplast envelope.
J Biol Chem
267:
2986-2993
[Abstract/Free Full Text]
Laidler V,
Chaddock AM,
Knott TG,
Walker D,
Robinson C
(1995)
A SecY homolog in Arabidopsis thaliana: sequence of a full-length cDNA clone and import of the precursor protein into chloroplasts.
J Biol Chem
270:
17664-17667
[Abstract/Free Full Text]
Leheny EA,
Theg SM
(1994)
Apparent inhibition of chloroplast protein import by cold temperatures is due to energetic considerations, not by membrane fluidity.
Plant Cell
6:
427-437
[Abstract]
Li X,
Henry R,
Yuan J,
Cline K,
Hoffman NE
(1995)
A chloroplast homologue of the signal recognition particle subunit SRP54 is involved in the posttranslational integration of a protein into thylakoid membranes.
Proc Natl Acad Sci USA
92:
3789-3793
[Abstract/Free Full Text]
Lindahl M,
Tabak S,
Cseke L,
Pichersky E,
Andersson B,
Adam Z
(1996)
Identification, characterization, and molecular cloning of a homologue of the bacterial FtsH protease in chloroplasts of higher plants.
J Biol Chem
271:
29329-29334
[Abstract/Free Full Text]
Lorkovic ZJ,
Schroder WP,
Pakrasi HB,
Irrgang KD,
Herrmann RG,
Oelmuller R
(1995)
Molecular characterization of psbw, a nuclear-encoded component of the photosystem II reaction center complex in spinach.
Proc Natl Acad Sci USA
92:
8930-8934
[Abstract/Free Full Text]
Marshall JS,
DeRocher AE,
Keegstra K,
Vierling E
(1990)
Identification of heat shock protein hsp70 homologues in chloroplasts.
Proc Natl Acad Sci USA
87:
374-378
[Abstract/Free Full Text]
Marshall JS,
Keegstra K
(1992)
Isolation and characterization of a cDNA clone encoding the major hsp70 of the pea chloroplastic stroma.
Plant Physiol
100:
1048-1054
[Abstract/Free Full Text]
Michl D,
Robinson C,
Shackleton JB,
Herrmann RG,
Klosgen RB
(1994)
Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway.
EMBO J
13:
1310-1317
[Web of Science][Medline]
Moore T,
Keegstra K
(1993)
Characterization of a cDNA clone encoding a chloroplast-targeted Clp homologue.
Plant Mol Biol
21:
525-537
[CrossRef][Web of Science][Medline]
Mould RM,
Robinson C
(1991)
A proton gradient is required for the transport of 2 lumenal oxygen-evolving proteins across the thylakoid membrane.
J Biol Chem
266:
12189-12193
[Abstract/Free Full Text]
Mould RM,
Shackleton JB,
Robinson C
(1991)
Transport of proteins into chloroplasts: requirements for the efficient import of two lumenal oxygen-evolving complex proteins into isolated thylakoids.
J Biol Chem
266:
17286-17289
[Abstract/Free Full Text]
Murataliev MB,
Milgrom YM,
Boyer PD
(1991)
Characteristics of the combination of inhibitory Mg2+ and azide with the F1 ATPase from chloroplasts.
Biochemistry
30:
8305-8310
[CrossRef][Medline]
Musgrove JE,
Elderfield PD,
Robinson C
(1989)
Endopeptidases in the stroma and thylakoids of pea chloroplasts.
Plant Physiol
90:
1616-1621
[Abstract/Free Full Text]
Nakai M,
Nohara T,
Sugita D,
Endo T
(1994)
Identification and characterization of the Sec-A protein homologue in the cyanobacterium Synechococcus PCC7942.
Biochem Biophys Res Commun
200:
844-851
[CrossRef][Medline]
Nielsen VS,
Mant A,
Knoetzel J,
Moller BL,
Robinson C
(1994)
Import of barley photosystem-I subunit-N into the thylakoid lumen is mediated by a bipartite presequence lacking an intermediate processing site: role of the pH in translocation across the thylakoid membrane.
J Biol Chem
269:
3762-3766
[Abstract/Free Full Text]
Nishiyama K-I,
Suzuki T,
Tokuda H
(1996)
Inversion of the membrane topology of SecG coupled with SecA-dependent preprotein translocation.
Cell
85:
71-81
[CrossRef][Web of Science][Medline]
Nunnari J,
Walter P
(1996)
Regulation of organelle biogenesis.
Cell
84:
389-394
[CrossRef][Web of Science][Medline]
Olsen LJ,
Keegstra K
(1992)
The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space.
J Biol Chem
267:
433-439
[Abstract/Free Full Text]
Reed JE,
Cline K,
Stephens LC,
Bacot KO,
Viitanen PV
(1990)
Early events in the import/assembly pathway of an integral thylakoid protein.
Eur J Biochem
194:
33-42
[Web of Science][Medline]
Roberts LM,
Meadows JW,
Robinson C
(1991)
Targeting of ricin A chain into pea chloroplasts.
FEBS Lett
293:
134-136
[CrossRef][Medline]
Schatz G,
Dobberstein B
(1996)
Common principles of protein translocation across membranes.
Science
271:
1519-1525
[Abstract]
Schuldiner S,
Rottenberg H,
Avron M
(1972)
Determination of pH in chloroplasts. 2. Fluorescent amines as a probe for the determination of pH in chloroplasts.
Eur J Biochem
25:
64-70
[Web of Science][Medline]
Theg SM,
Geske FJ
(1992)
Biophysical characterization of a transit peptide directing chloroplast protein import.
Biochemistry
31:
5053-5060
[CrossRef][Medline]
Theg SM,
Scott SV
(1993)
Protein import into chloroplasts.
Trends Cell Biol
3:
186-190
Tomoyasu T,
Gamer J,
Bukau B,
Kanemori M,
Mori H,
Rutman AJ,
Oppenheim AB,
Yura T,
Yamanaka K,
Niki H,
and others
(1995)
Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor 
32.
EMBO J
14:
2551-2560
[Web of Science][Medline]
Ungermann C,
Guiard B,
Neupert W,
Cyr DM
(1996)
The 
and hsp70/MIM44-dependent reaction cycle driving early steps of protein import into mitochondria.
EMBO J
15:
735-744
[Web of Science][Medline]
Voelker R,
Barkan A
(1995)
Two nuclear mutations disrupt distinct pathways for targeting proteins to the chloroplast thylakoid.
EMBO J
14:
3905-3914
[Web of Science][Medline]
Voelker R,
Mendel-Hartvig J,
Barkan A
(1997)
Transposon-disruption of a maize nuclear gene, tha1, encoding a chloroplast SecA homolog: in vivo role of SecA in thylakoid protein targeting.
Genetics
145:
467-478
[Abstract]
Wu C,
Seibert SS,
Ko K
(1994)
Identification of chloroplast envelope proteins in close physical proximity to a partially translocated chimeric precursor.
J Biol Chem
269:
32264-32271
[Abstract/Free Full Text]
Yuan J,
Cline K
(1994)
Plastocyanin and the 33-kDa subunit of the oxygen-evolving complex are transported into thylakoids with similar requirements as predicted from pathway specificity.
J Biol Chem
269:
18463-18467
[Abstract/Free Full Text]
Yuan J,
Henry R,
McCaffery M,
Cline K
(1994)
SecA homolog in protein transport within chloroplasts: evidence for endosymbiont derived sorting.
Science
266:
796-798
[Abstract/Free Full Text]
Top
Abstract
Introduction