(Received for publication, August 1, 1994)
From the
Previous studies have shown that proteins are transported across
the chloroplast thylakoid membrane by two very different mechanisms,
one of which requires stromal factors and ATP, whereas the other
mechanism is ATP independent but completely reliant on the thylakoidal
pH. We have examined the role of the
pH in the latter
mechanism by simultaneously monitoring the magnitude of
pH (by
9-aminoacridine fluorescence quenching) and the rate of import of the
23-kDa photosystem II protein into isolated pea thylakoids. We show
that protein import can take place, at low but significant rates, at
very low values of
pH (in the region of 1.2-1.4), and that
plots of the rate of protein import against proton concentration
gradient are probably hyperbolic in nature. There is no evidence for a
threshold level of
pH which is required to drive translocation of
the 23-kDa protein. Addition of uncouplers midway during import
incubations results in a rapid and complete inhibition of
translocation, showing that the continuous presence of the
pH is
required for translocation to take place. During import into intact
chloroplasts, the intermediate-size 23-kDa protein substrate for the
thylakoidal protein transport machinery is found only in the stromal
fraction at all values of
pH, suggesting that the initial
interaction with the machinery is relatively weak, reversible and
pH-independent. We therefore propose that the
pH is required
for both the initiation and completion of translocation; these roles
are in marked contrast to the roles of protonmotive force in
mitochondrial and sec-dependent bacterial protein transport.
Extensive efforts have been made to understand the mechanisms by
which proteins are translocated across biological membranes, and in
vitro translocation assays have been developed for the analysis of
most of the known protein-translocating membranes. Several unifying
features have emerged from comparisons of the different mechanisms,
among which is the observation that some form of energy input is always
required to drive the translocation process. Studies on the transport
of proteins across the endoplasmic reticulum, chloroplast and
mitochondrial envelopes, and bacterial cytoplasmic membranes have shown
that ATP (or GTP) is critical for the operation of each of these
translocation mechanisms, other than in exceptional circumstances (Theg et al., 1989; Wickner, 1988; Meyer, 1988; Deshaies et
al., 1988). In addition, protein transport tends to be coupled to
a transmembrane protonmotive force (p) if one is
available; mitochondrial protein import is heavily dependent on the
electrical potential (
) across the inner membrane (Pfanner
and Neupert, 1985) and bacterial protein export by the Sec-dependent
pathway requires the
p across the cytoplasmic membrane
(reviewed by Driessen, 1992).
The thylakoid membrane has proved to
be an interesting, and surprisingly complex, system for the study of
protein translocation. Initially, it was thought that proteins were
transported across the chloroplast thylakoid membrane by a mechanism
akin to the Sec-dependent pathway because virtually all imported
lumenal proteins are synthesized with bipartite presequences containing
an ``envelope transit'' domain which targets the precursor
into the stroma, and a ``thylakoid transfer'' signal which
resembles bacterial export (or signal) peptides in several respects
(von Heijne et al., 1989; Bassham et al., 1991). More
recent studies using assays for the import of proteins by isolated
thylakoids suggest that a subset of proteins are indeed
probably transported by a sec-type mechanism: the import of
plastocyanin and the extrinsic 33-kDa photosystem II protein (33K) ()into isolated thylakoids requires a stromal protein factor
and ATP (Hulford et al., 1994; Robinson et al., 1994), just as bacterial protein export requires a soluble factor
(SecB) and ATP for the function of the translocation ATPase, SecA
(reviewed in Wickner et al., 1991). Furthermore, the SecA
inhibitor azide has been shown to block the translocation of
plastocyanin and 33K across the thylakoid membrane (Knott and Robinson,
1994). Definitive evidence for the operation of a sec-related
pathway in chloroplasts is still lacking, but the circumstantial
evidence is fairly convincing at the present time.
The major
surprise to emerge from recent studies is that another mainstream
thylakoidal protein transport mechanism operates in chloroplasts, which
is fundamentally different in several respects to the putative sec-type mechanism. The extrinsic 23- and 16-kDa photosystem
II subunits (23K and 16K), photosystem II subunit T (PSII-T), and
photosystem I subunit N (PSI-N) are transported by a mechanism in which
stromal factors and ATP are completely unnecessary, and which appears
instead to be completely dependent on the thylakoidal pH (Mould
and Robinson, 1991; Mould et al., 1991; Cline et al.,
1992; Klösgen et al., 1992; Henry et
al., 1994; Nielsen et al., 1994). The
pH is not a
prerequisite for the transport of plastocyanin or 33K by the putative sec-type mechanism, although it may stimulate translocation
(Theg et al., 1989; Cline et al., 1992). Competition
studies (Cline et al., 1993) and studies on the translocation
properties of fusion proteins (Robinson et al., 1994) have
provided further compelling evidence for the existence of distinct
translocation systems for thylakoid proteins.
It is far from clear
why proteins are transported across the thylakoid membrane by two such
different mechanisms, but there are some clues as to the possible
origins of the two mechanisms. It appears likely that the putative sec-related mechanism was inherited from the
cyanobacterial-type progenitor of the chloroplasts, since both 33K and
plastocyanin are present in cyanobacteria, where they are probably
transported across the thylakoid membrane by such a mechanism
(Kuwabara et al., 1987; Briggs et al., 1990). In
contrast, 23K, 16K, and PSI-N are all absent from cyanobacteria (the
situation regarding PSII-T is unclear) suggesting that the development
of these proteins has occurred relatively recently in phylogenetic
terms. The implications for the evolution of the chloroplast
thylakoidal protein transport mechanisms are fascinating: there is a
good chance that the emergence of these proteins in chloroplasts has
been accompanied by the emergence of a novel system for their transport
across the thylakoid membrane. Alternatively, the pH-driven system
may in fact be present in cyanobacteria, in which case the above
proteins may have been preferentially transported by this system, for
unknown reasons.
The mechanism of action of the pH-driven
system is of particular interest because it is unique among known
translocation mechanisms in being ATP independent, and hence represents
an excellent (and relatively simple) system for studying the energetics
of protein translocation. In this report we describe kinetic data on
the role of the
pH as a driving force for the translocation of 23K
across the thylakoid membrane.
Thylakoids were isolated from 8 to 9-day-old pea seedlings as
described (Brock et al., 1993). The thylakoids were washed
twice in 10 mM HEPES-KOH, pH 8.0, 5 mM MgCl and resuspended in the same buffer to a concentration of 50
µg/ml chlorophyll. Wheat pre-23K was synthesized by in vitro transcription of a full-length cDNA clone followed by translation
in a wheat germ cell-free system (Promega) in the presence of
[
S]methionine. Thylakoid import assays were
basically as described in Brock et al.(1993) except that
incubation mixtures contained 940 µl of thylakoid suspension and 60
µl of translation product. Under these conditions, the
concentration of K
is approximately 10 mM and
that of dithiothreitol is 0.6 mM. Mixtures were incubated at
25 °C for periods indicated in the figure legends, after which
samples were analyzed directly or after incubation of the thylakoids
with 0.2 mg/ml thermolysin for 30 min on ice.
pH was estimated by
9-aminoacridine fluorescence quenching as described by Mills(1986)
except that the 9-aminoacridine concentration was 5 µM.
The intensity of the measuring beam was 0.1-0.2
watts/m
, and the
pH was driven by a quartz halogen
lamp filtered through a Corning 2-62 glass filter.
pH was
calculated from the fractional quenching, q, according to
pH = log [q/(1 - q)]
+ log (V/v) where V is total reaction
volume and v is the internal volume of the thylakoids (assumed
to be 10 µl/mg chlorophyll). The magnitude of
pH was altered
by changing the intensity of the incident light, and import reactions
were carried out in a random order to obviate any effects due to
changes in the import competence of the thylakoids during storage on
ice.
Chloroplast import assays and fractionation procedures were as
described in Nielsen et al.(1994). Nigericin was added from a
100 stock in ethanol, together with 10 mM KCl; all
incubation mixtures contained identical concentrations of ethanol and
KCl. ATP synthesis was measured by the luciferin/luciferase method as
described by Mills(1986). Fluorograms were quantitated by laser
densitometry using a Molecular Dynamics Imagequant v. 3.0.
As with all
techniques for monitoring the thylakoidal pH, this technique in
practice gives more of an estimate, than an accurate measure, of the
pH. However, the advantage of this method is that it allows
real-time monitoring of the
pH over the entire import incubation
period, during which samples were taken at given time points in order
to determine the rate of import. The magnitude of the
pH was
altered by changing the intensity of the incident light. A number of
tests (not shown) indicated that the import competence of the isolated
pea thylakoids did not change during a period of 60 min after
isolation, that import time course analyses over 8-min periods were
suitable for determining the rate of import, and that the magnitude of
pH did not change during these time courses to a significant
extent. In practice, up to six import reactions were possible with each
preparation of thylakoids.
The electrical potential, ,
was not measured during these experiments, and it is possible that it
may have contributed to the overall protonmotive force. However, under
these steady-state conditions the thylakoidal
is very low
indeed (in the order of 20-30 mV), and it is most unlikely that
this level could influence the rate of protein import to a significant
extent. The
can be effectively collapsed by the addition of
valinomycin, and we have shown previously that this compound does not
significantly inhibit the import of 23K by isolated thylakoids (Mould
and Robinson, 1991).
Figure 1:
Effects of varying pH on the
import of pre-23K into isolated thylakoids. Isolated pea thylakoids
were incubated with wheat pre-23K under varying light intensities as
described under ``Materials and Methods.'' The light
intensities and estimated
pH values are given on the left of the panels. In the bottom panel, the import assay was
carried out at a light intensity of 160 watts/m
in the
presence of 2 µM nigericin. Samples were removed at the
indicated time points and analyzed directly (- protease)
or after protease treatment of the thylakoids (+ protease). Lane T, pre-23K translation product; 23K, mature-size 23K.
Figure 2:
Import of 23K at low pH values.
Import assays were carried out as detailed in the legend to Fig. 1under the indicated light intensities. Bottom
panel, import was carried out at a light intensity of 200
watts/m
in the presence of 2 µM nigericin. Symbols are as in Fig. 1.
The
mature size 23K bands in the fluorograms were quantified by laser
densitometry, and Fig. 3shows the results of a typical
experiment. The data show that the rate of import is essentially
constant at each light intensity over the 8-min time period and that
initial rates of import can therefore be derived from this type of
experiment. Fig. 4shows the combined data from four different
experiments, with import rates this time plotted against the proton
concentration gradient across the thylakoid membrane (rather than the
pH which is a logarithmic function). The graphs show the same
general trend in each case, with import rates measurable at low values
of
pH but tending to saturate (with respect to proton gradient) at
higher values. The overall shape of the curve is hyperbolic in every
case, but the variation between experiments (and the inaccuracies
inherent in this technique) are such that it is not possible to
interpret the shape of the curve in great detail. Nevertheless, it is
worth pointing out that the shapes of the curves are consistent with
the involvement of a component which saturates at about
pH
= 2, although the gradual increases in import rate at higher
values of proton gradient are also consistent with the involvement of
another component which does not saturate at all.
Figure 3:
Initial rates of 23K import at varying
values of pH. Import time courses were carried out under varying
light intensities as detailed in Fig. 1, and the mature size,
protease-protected 23K bands in the fluorograms were quantitated by
laser densitometry. The graphs correspond to light intensities of 160,
95, 40, 2.2, and 0.05 watts/m
(filled squares, open diamonds, open squares, circles, and filled diamonds, respectively).
Figure 4:
Rate of import of 23K into thylakoids as a
function of proton concentration gradient. Four separate import time
course experiments were carried out as detailed in Fig. 1and Fig. 2, and the rates of import were calculated as in Fig. 3. The graph shows the import rates from the four
experiments plotted against the proton concentration gradient (antilog
of pH) across the thylakoid membrane (acid,
inside).
Although the
measurements of pH are only approximate for values under about
pH = 2 (proton gradient of 100:1, lumen/stroma), the data
clearly show that high rates of import take place at or around this
value and that import proceeds at significant rates at proton gradients
well below this figure. To put these figures into perspective, the
combined data in Fig. 4are shown as a ``best fit''
plot in Fig. 5, together with the values of proton gradient
required to drive the synthesis of ATP under these light intensities.
The results show that ATP synthesis only occurs at significant rates at
values of
pH of about 3 and greater, in good agreement with
previous work (Rumberg and Becher, 1984; Junesche and
Gräber, 1987) which showed ATP synthesis having a
threshold of
pH = 2.7 and saturating at
pH = 4,
depending on
Gp. In fact, in our experimental system ATP
synthesis was barely detectable even at the highest light intensities
(not shown) because no electron acceptors are present, and the maximum
values of
pH obtained (about 2.6) are thus in fact relatively low;
values of over 3.0 can be routinely measured if electron acceptors are
present to exploit the full potential of the thylakoidal electron
transport machinery. The results shown in Fig. 5Bwere obtained in the presence of the electron acceptor
methyl viologen, but this compound was found to inhibit thylakoidal
protein import (not shown), and hence the maximum values of
pH in Fig. 5A are significantly lower.
Figure 5:
Comparison of the dependence of 23K import
and ATP synthesis on magnitude of pH. A, the data from
the four experiments shown in Fig. 4are shown plotted as a
best-fit curve. B, the rate of ATP synthesis was determined
under the same light intensities and experimental conditions, except
that the electron acceptor methyl viologen was included at 10
µM.
The results shown
in Fig. 5are useful in two respects. First, the ATP synthesis
data serve as an internal control for the pH measurements because
the plot shown in Fig. 5B is similar to those obtained
in other studies using a variety of techniques (including 9-AA
fluorescence quenching) for the
pH estimations (for example, Quick
and Mills, 1987; Bizouarn et al., 1987). The calculated values
for
pH are not, therefore, gross under- or over-estimates. Second,
the data show that the levels of
pH required to drive protein
translocation are much lower than those required for ATP synthesis. A
proton concentration gradient of 100 can drive 23K translocation at
near maximal rates whereas a gradient of over 1000 is required for
efficient ATP synthesis.
Figure 6:
The intermediate form of 23K is soluble in
the stroma at all values of pH. Pre-23K was imported into intact
pea chloroplast in the absence of nigericin or in the presence of
nigericin at varying concentrations indicated above the lanes. After
import, the chloroplasts were reisolated, lysed in 10 mM HEPES-KOH, pH 8.0, 5 mM MgCl
for 10 min on
ice, and centrifuged for 5 min at 10,000
g. Samples of
stromal and thylakoidal fractions were analyzed. i23K and m23K, intermediate and mature forms of 23K,
respectively.
The combined data from Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6clearly
indicate that the magnitude of pH is important in determining the
rate of translocation of 23K across the thylakoid membrane, but a final
test was carried out to address the question: is the
pH the
driving force for translocation, or does it somehow activate the translocation machinery? This is an important question because
studies on the thylakoidal ATP synthase have clearly shown that the
pH activates this enzyme, as well as driving ATP synthesis per
se. We therefore carried out parallel time course analyses of the
import of 23K by isolated thylakoids and added nigericin midway through
one of the import reactions (Fig. 7). The results show that
nigericin rapidly and completely inhibits protein import, and we
conclude that the thylakoidal
pH is continuously required for
protein translocation to occur.
Figure 7:
The pH is continuously required for
import of 23K into thylakoids. Parallel thylakoid import assays were
conducted as detailed under ``Materials and Methods,'' and
samples analyzed at time points indicated; nigericin (2
µM) was added to one of the reactions after 4 min (filled diamonds). Mature size 23K was quantitated as detailed
under ``Materials and Methods.''
The unprecedented lack of requirement for ATP or soluble
factors in the 23K-type thylakoidal protein translocation mechanism
makes this process an interesting area of study but also complicates an
experimental analysis of the mechanism, since soluble factors and
ATP-requiring reactions are generally much easier to study than the
actual processes of membrane transfer (the soluble factors are more
amenable to study, and the time scales of the reactions in solution are
more protracted).Indeed, the only evidence to date for the presence of
proteinaceous translocation apparatus in the thylakoid membrane is the
observation that the transport of pre-23K into isolated thylakoids is
saturable (Cline et al., 1993). The pH plays an
all-important role in the 23K-type translocation mechanism, and it is
therefore crucial to understand the bioenergetics of protein transport
in this case. In this study we have examined the magnitude of
pH
required to drive translocation, and we have provided the first kinetic
data concerning the role of the
pH. Although many details remain
to be clarified, several important features of the translocation system
have become apparent.
1) There is no evidence for a threshold level of pH which is required to drive 23K transport across
the thylakoid membrane. Numerous plots of proton concentration gradient versus rate of 23K import have been made, and in every case
the graph shows the same basic characteristics: the rate of import
increases steeply at low values of proton gradient and levels off at
higher values. In no case have we found evidence for a threshold level
of
pH at which translocation is suddenly triggered. If such a
threshold exists, it must correspond to an extremely low
pH and
would be of questionable physiological significance.
2)
Translocation of 23K can be driven by extremely low values of pH.
The simple observation is that 23K transport can be detected, albeit at
low rates, if any
pH is apparent. The quenching of the
9-AA fluorescence signal at the lowest light intensities used in this
study is such that the estimated values of
pH are subject to an
enormous amount of error, but there is no doubt that a
pH of well
below 2.0 can support the translocation of 23K across the thylakoid
membrane. This may be physiologically important because several of the
known lumenal proteins (including 33K, 23K, 16K, and plastocyanin) are
present as mature size proteins in dark grown tissue in which the
etioplasts are photosynthetically incompetent (Sutton et al.,
1987). In these tissues it is unclear whether there is any
pH at
all across the thylakoid membrane, but we favor the possibility that
reverse action of the thylakoidal ATP synthase may generate a small but
sufficient proton gradient.
3) The data support a model in which the
pH provides the driving force for the translocation reaction.
There is little doubt that the continuous presence of the
pH is
required for translocation to proceed, and we thus conclude that the
pH probably functions in providing the driving force for
translocation across the membrane. There is no evidence that the
pH promotes binding to the translocation apparatus, although we
cannot formally rule out this possibility. Given the soluble nature of
the i23K polypeptide during chloroplast import reactions, we believe
that the initial interactions with the thylakoidal translocation
apparatus are weak, reversible, and probably
pH-independent. We
therefore propose that the 23K presequence is unlikely to protrude
across the thylakoid membrane and ``sense'' the acidic
lumenal pH, and we suggest instead that the
pH is required at an
early stage to initiate, and probably also to maintain, the
translocation process once binding has taken place. This situation
would contrast markedly with the Sec-dependent translocation mechanism,
where the ATP-dependent SecA reaction is proposed to provide the
impetus for the early stages of translocation, with the
p
providing the driving force for the later stages (reviewed in
Driessen, 1992). It is also likely that the thylakoidal mechanism
differs considerably from that of mitochondria, where the electrical
potential,
, is required for the transport of proteins
across the inner membrane (Pfanner and Neupert, 1985). It has been
proposed that the
drives the movement of presequences
(Martin et al., 1991), probably by an electrophoretic effect,
but the main driving force for translocation appears to be ATP
hydrolysis. The relative orientation of the
p is in any
case different from that of the thylakoid, since protons are pumped out
of the mitochondrial matrix but into the thylakoid lumen.
If the
pH is indeed required for the early stages of thylakoidal protein
transport, the above model also implies that the
pH plays a
dynamic role in the translocation process rather than translocation
relying on a pH difference across the membrane (acid, inside). We have
no information on how this is achieved, but obvious possibilities are
that protein translocation is coupled to the efflux of protons or the
influx of hydroxide ions (although many other mechanisms can be
envisaged). The graphs of proton gradient versus translocation
rate are consistent with this type of model, although further analyses
are required in order to obtain more accurate information on the
kinetics of translocation at low
pH values. If protein
translocation is coupled to proton movement, accurate data at low
values of
pH are absolutely essential for an estimate of the
protein-proton stoichiometry because a hyperbolic curve would be
indicative of 1 H
binding event per transport event,
whereas sigmoidal kinetics would suggest a higher ratio. Our data are
most consistent with a low proton-protein stoichiometry, but the
limited accuracy at the critical low values of
pH precludes any
firm conclusions.
Two key outstanding issues must be settled before
this mechanism can be understood in genuine detail. First, it will be
important to determine whether proton/ion movement is in fact coupled
to protein translocation; an answer to this question is obviously
central to an understanding of any pH-driven mechanism. Second,
further work is required to determine the type(s) of
protonmotive force which is (are) capable of driving protein transport
by this mechanism. We have consistently referred to this mechanism as
``
pH-driven'' but it should be emphasized that we do not
yet know whether translocation can be driven by the electrical
potential
. This is because the
across the
thylakoid membrane is almost non-existent under steady-state
conditions, and it would not therefore be surprising if, in the
presence of nigericin, such a small
were unable to support
translocation. It will be important to resolve this point because in
bacteria, both components of the total protonmotive force can support
protein translocation (Bakker and Randall, 1984; Shiosuka et
al., 1990). Further studies in which the
is
artificially enhanced will hopefully provide important clues concerning
the mechanism by which the thylakoidal
pH/
p drives protein
translocation.