(Received for publication, August 9, 1994; and in revised form, November 2, 1994)
From the
Two distinct mechanisms have been previously identified for the
transport of proteins across the chloroplast thylakoid membrane, one of
which is unusual in that neither soluble factors nor ATP are required;
the system requires only the transthylakoidal pH. We have examined
this mechanism by studying the properties of one of its substrates: the
extrinsic 23-kDa protein (23K) of photosystem II. Previous work has
shown that this protein can be transported into isolated thylakoids as
the full-length precursor protein; we show that the stromal import
intermediate form of this protein is similarly translocation-competent.
Gel filtration tests indicate that the stromal intermediate is probably
monomeric. Protease sensitivity tests on both the initial in vitro translation product and the stromal import intermediate show that
the presequence is highly susceptible to digestion whereas the mature
protein is resistant to high concentrations of trypsin. The mature
protein becomes very sensitive to digestion if unfolded in urea, or
after heating, and we therefore propose that the natural substrate for
this translocation system consists of a relatively unfolded presequence
together with a tightly folded passenger protein. The ability of
thylakoids to import pre-23K is destroyed by prior treatment of the
thylakoids with low concentrations of trypsin, demonstrating the
involvement of surface-exposed proteins in the import process. However,
we can find no evidence for the binding of pre-23K or i23K to the
thylakoid surface, and we therefore propose that the initial
interaction of these substrates with the thylakoidal translocase is
weak, reversible, and probably
pH-independent. In the second phase
of the translocation mechanism, the
pH drives either the
translocation and unfolding of proteins, or the translocation of a
fully folded protein.
Recent studies on the biogenesis of cytosolically synthesized thylakoidal proteins in chloroplasts have pointed to the operation of a remarkable variety of mechanisms for their transport into and across the thylakoid membrane. Most integral thylakoid membrane proteins are synthesized in the cytosol with stroma-targeting (or ``envelope transit'') signals, and thus the information specifying integration into the thylakoid membrane is localized in the mature proteins (Viitanen et al., 1988; Lamppa, 1988; Cai et al., 1993). In contrast, proteins destined for the thylakoid lumen are usually synthesized with bipartite presequences containing an envelope transit and a ``thylakoid transfer'' signal in tandem. The envelope transit signal functions to target the precursor protein into the stroma and is usually removed by a stromal processing peptidase; the transfer signal then directs translocation across the thylakoid membrane, after which complete maturation is carried out by a thylakoidal processing peptidase (Smeekens et al., 1986; Hageman et al., 1986; James et al., 1989; Ko and Cashmore, 1989).
The properties of thylakoid transfer signals have elicited a great deal of interest because they share key features with ``signal'' sequences which mediate protein transport across the bacterial plasma membrane, namely the presence of a hydrophobic core region and short chain residues at the -3 and -1 positions, relative to the terminal cleavage site (von Heijne et al., 1989; Bassham et al., 1991). These observations led to the proposal that the thylakoidal protein transport apparatus evolved from a translocation system in a cyanobacterial-type progenitor of the chloroplast, and functional similarities between bacterial and thylakoidal translocation systems have emerged from the demonstration that thylakoid transfer signals can direct protein export in Escherichia coli (Seidler and Michel, 1990) and from the finding that E. coli signal peptidase and pea thylakoidal processing peptidase have very similar reaction specificities (Halpin et al., 1989).
Because all thylakoid transfer
signals contain apparently similar hydrophobic domains and processing
sites, it was widely assumed for some time that they were transported
across the thylakoid membrane by a common mechanism. Remarkably,
several recent studies have shown that lumenal proteins are transported
by two completely different translocation mechanisms and almost
certainly by two distinct translocases. The advent of in vitro assays for the import of proteins by isolated thylakoids has shown
that the transport of a subset of lumenal proteins, including the
extrinsic 33-kDa photosystem II (PSII) ()protein (33K) and
plastocyanin (PC), requires nucleoside triphosphates (NTPs) and a
stromal protein factor (Hulford et al., 1994; Robinson et
al., 1994). The transthylakoidal
pH is not a prerequisite for
the translocation of these proteins, although it may stimulate the
translocation rate (Theg et al., 1989; Cline et al.,
1992). In contrast, other lumenal proteins, including the extrinsic 23-
and 16-kDa photosystem II proteins (23K, 16K), photosystem I (PSI)
subunit N, and photosystem II subunit T, do not require either NTPs or
stromal factors for their transport across the thylakoid membrane;
these proteins are transported by a mechanism which appears only to
require the thylakoidal
pH (Mould and Robinson, 1991;
Klösgen et al., 1992; Cline et
al., 1992; Nielsen et al., 1994; Henry et al.,
1994).
Several further approaches have been used to confirm the
operation of separate translocation pathways for lumenal proteins. In
studies on the biogenesis of four lumenal proteins, Cline et
al.(1993) have shown that 23K competes only with 16K for transport
across the thylakoid membrane, and 33K competes only with PC. This
study provided strong evidence for the presence of parallel
translocation pathways, and there are now clear indications that these
lumenal proteins are indeed synthesized with two distinct types of
thylakoid transfer signal; Robinson et al.(1994) have shown
that PC is quantitatively diverted onto the pH-dependent pathway
if its presequence is replaced with that of 23K or 16K. Finally, it has
been found that azide, a known inhibitor of Sec-dependent protein
export in bacteria, also inhibits the transport of 33K and PC (but not
23K or 16K) across the chloroplast thylakoid membrane (Knott and
Robinson, 1994; Henry et al., 1994). It therefore appears
increasingly likely that 33K and PC are translocated by a Sec-related
mechanism, whereas 23K, 16K, PSI-N, and PSII-T are transported by a
mechanism with very different operational requirements.
An
intriguing evolutionary twist to the story emerges from a simple
analysis of the substrates for the two translocation mechanisms. Of the
lumenal proteins listed above, only 33K and PC are present in
cyanobacteria (the likely progenitors of chloroplasts), and in these
prokaryotes they are synthesized with presequences which resemble both
classical signal sequences and the thylakoid transfer signals of their
higher plant counterparts (Kuwabara et al., 1987; Briggs et al., 1990). 23K, 16K, and PSI-N, on the other hand, are all
absent in cyanobacteria (the situation with respect to PSII-T is
currently unclear). Thus, the evolutionary development of these
proteins in chloroplasts may well have been accompanied by the
emergence of a novel, pH-driven system for their transport across
the thylakoid membrane, although it is also possible that this
mechanism is in fact present in both cyanobacteria and chloroplasts, in
which case the chloroplast-specific proteins may have
``chosen'' this system in preference to a Sec-related one.
Although the basic requirements for the 23K-type system have been
mapped out (i.e. a continuous pH is required but not
stromal factors or ATP), very little is known about the actual
translocation mechanism. In this study we have analyzed the
conformation of the 23K substrate for the thylakoidal protein
translocase, and we provide evidence that the stromal intermediate form
of this protein is translocation-competent, monomeric, and comprises a
relatively unfolded presequence and a tightly folded mature protein
moiety. The results imply that the thylakoidal
pH drives either
the translocation and concomitant unfolding of thylakoidal proteins or
the translocation of fully folded proteins.
Figure 6:
Import of pre-23K into isolated thylakoids
requires surface-exposed proteins. A, the ATPase activity of
the CF ATP synthase was activated using light and
dithiothreitol as detailed under ``Experimental Procedures.''
Immediately after the activation process, the thylakoids were washed
and incubated with trypsin at the indicated concentrations for 15 min
on ice. The thylakoids were then incubated in the dark with 0.5 mM MgATP and the resulting
pH estimated by 9-aminoacridine
fluorescence quenching. B, the thylakoidal ATPase activity was
activated as in A, and thylakoids were treated with the
indicated concentrations of trypsin for 15 min on ice. The thylakoids
were then incubated with pre-23K in the presence of 0.5 mM MgATP as detailed under ``Experimental Procedures.''
Import efficiency was calculated by laser densitometry of the precursor
and mature size bands on the fluorograms.
We addressed these points by analyzing the
properties of pre-23K (i.e. full-length) and the stromal
intermediate form of 23K (i23K) which accumulates in the presence of
uncouplers, but before undertaking these experiments we addressed the
question: is this accumulated stromal i23K form a bona fide intermediate on the translocation pathway? Wheat pre-23K was
imported into intact pea chloroplasts for 20 min in the presence of the
uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), and
the chloroplasts were then washed to remove unbound precursor
molecules. Fig. 1shows that the i23K polypeptide effectively
accumulates over a 20-min period under these conditions, with no
evidence for the presence of the mature size protein. Some pre-23K
remains bound to the chloroplasts under these conditions but the
quantities were insignificant relative to the amount of stromal i23K
(data not shown). The light-driven proton motive force was then
restored by the addition of bovine serum albumin (which has a high
affinity for CCCP), and the remaining lanes show that the i23K form is
rapidly and quantitatively chased into the mature size form. Control
tests (not shown) confirmed that the i23K and mature size 23K were
indeed in the stroma and thylakoid lumen, respectively, and we
therefore conclude that the accumulated i23K is on the correct import
pathway. Konishi and Watanabe(1993) have similarly shown that the
stromal i23K polypeptide is a substrate for the transport apparatus,
although in their study the pH was generated by reverse action of
the ATP synthase in the dark. These data indicate that both the pre-23K
translation product, and the stromal i23K polypeptide are competent for
translocation across the thylakoid membrane, and further tests were
aimed at analyzing the properties of each of these polypeptides.
Figure 1: The stromal i23K protein which accumulates in the presence of uncouplers is competent for translocation across the thylakoid membrane. Pre-23K was incubated with pea chloroplasts in the presence of 3 µM CCCP, and samples were analyzed after 10 and 20 min. The chloroplasts were then re-isolated and bovine serum albumin (BSA; 1 mM) was added to the incubation mixture (at the time indicated by the arrow) and further samples were analyzed at time points (in minutes) indicated above the lanes. Samples of the chloroplasts were analyzed after protease treatment. Lane T, translation product. i23K and 23K, intermediate and mature forms of 23K, respectively.
Figure 2:
The stromal i23K polypeptide is probably
monomeric. Stromal i23K was generated using CCCP in an import
incubation as described under ``Experimental Procedures.''
The stromal fraction was mixed with molecular mass markers (Pharmacia
low molecular weight) and subjected to gel filtration chromatography as
detailed under ``Experimental Procedures.'' Fractions were
collected and analyzed by SDS-polyacrylamide gel electrophoresis for
the elution of i23K and the marker proteins. Peak elution volume was
determined by laser densitometry of the fluorograms (for i23K) or
stained gels (for markers). The graph shows the K plotted against log of the molecular mass of each of the four
marker proteins (in kDa). K
= (V
- V
)/(V
- V
), where V
=
elution volume, V
= void volume, and V
= total bed volume. The
molecular mass of i23K is calculated to be 36
kDa.
Figure 3: In vitro synthesized pre-23K consists of a protease-sensitive presequence and a protease-resistant mature protein. A, wheat pre-23K was synthesized in a wheat germ cell-free system, and equal aliquots of translation product were incubated with trypsin (concentrations indicated above the lanes) for 40 min on ice. Mature size 23K was generated by incubation of pre-23K with partially purified thylakoidal processing peptidase (TPP); the mobility of mature size 23K is indicated. F denotes a 22-kDa digestion fragment. B, a fraction containing authentic mature wheat 23K was prepared as described under ``Experimental Procedures,'' and trypsin was incubated with the fraction (at concentrations indicated above the lanes) for 40 min on ice. Wheat germ translation mix components were added so that the incubation conditions were similar to those in A.
Figure 4: Urea converts the 22-kDa polypeptide to a protease-sensitive conformation. A, pre-23K (lanes T) was incubated with trypsin (concentrations in µg/ml are given above the lanes) for 40 min on ice in the absence of urea, or after incubating pre-23K with 8 M urea for 30 min on ice, followed by dilution of the mixture 4-fold with 20 mM Tris-HCl, pH 8.0 (panel denoted + urea). Incubation conditions in the two mixtures were identical apart from the presence/absence of urea. B, pre-23K was incubated with trypsin at the indicated concentrations for 40 min at 25 °C. Symbols are the same as in Fig. 3.
Most of our tests on the protease sensitivity of 23K were carried out on ice. However, similar tests were carried out to exclude the possibility that 23K is partially unfolded at the temperatures used for import assays; Fig. 4B shows that the 22-kDa fragment is resistant to digestion with 15 µg/ml trypsin at 25 °C for 40 min. In other tests (not shown) we have found that equally high concentrations of thermolysin also generate a fragment of 22 kDa, and we therefore conclude that the 22-kDa polypeptide is highly resistant to proteolysis, and hence tightly folded, at both 0 and 25 °C.
Similar studies were carried out to assess the folding of the stromal i23K form which accumulates in the presence of uncoupler. Fig. 5shows the outcome of an experiment in which stromal i23K was incubated with increasing concentrations of trypsin (Fig. 5A) and in which the same concentrations of trypsin were incubated with a mixture of in vitro synthesized pre-23K and stromal extract as a control (Fig. 5B). The data show that given concentrations of trypsin cleave the two forms of 23K to a very similar extent, and high concentrations again generate only the 22-kDa form. The patterns of digestion products are furthermore almost identical (bearing in mind that no pre-23K band is present in the fluorogram shown in Fig. 5A; only i23K is incubated with trypsin). In this experiment it was not possible to confirm that the i23K becomes susceptible to digestion after heating or urea treatment, because both treatments led to extensive aggregation of the bulk stromal protein (not shown). Nevertheless, the data provide strong evidence that the mature 23K protein is tightly folded in both the initial in vitro translation product and in the authentic stromal intermediate form. A second experiment shown in Fig. 5C shows that a substantial proportion of i23K is converted to the 22-kDa polypeptide during digestion with as much as 30 µg/ml trypsin.
Figure 5: The stromal i23K form is protease-resistant. A, pre-23K was imported into intact chloroplasts in the presence of CCCP; the chloroplasts were then protease-treated, lysed, and centrifuged to generate a stromal fraction. Aliquots of the stromal fraction were incubated with trypsin at the given concentrations for 40 min on ice. An aliquot of the membrane fraction from an import assay containing no uncoupler was loaded to provide a mature size marker (lane m). B, in vitro synthesized pre-23K was mixed with stromal extract at a protein concentration identical to that in A, and trypsin added at the indicated concentrations. C, pre-23K was imported into chloroplasts in the presence of CCCP and the stromal fraction containing i23K (lane i) was incubated with trypsin at concentrations indicated above the lanes (in µg/ml). Lane m, mature size marker as in A. Symbols are the same as in Fig. 3and Fig. 4.
In this work we have sought to examine in greater detail the
pH-driven mechanism by which proteins are transported across the
thylakoid membrane. This translocation system is an interesting
experimental system because the data from previous studies have
suggested that its mechanism of action may be unique among known
protein translocation systems. Neither soluble factors nor ATP are
required for translocation of proteins such as 23K into isolated
thylakoids (Mould et al., 1991; Cline et al., 1992;
Klösgen et al., 1992), and the thylakoidal
pH appears to be the only requirement. Surprisingly, even
relatively low magnitudes of
pH are sufficient to support high
rates of translocation (Brock et al., 1995). Clearly, a
detailed understanding of the biogenesis of this group of lumenal
proteins is totally dependent on understanding how the
pH drives
translocation, and within this context a knowledge of the conformation
of the pre-23K or i23K forms prior to transport across the thylakoid
membrane is essential. Our data strongly indicate that the soluble 23K
substrates for the thylakoidal protein transport machinery are highly
folded, and we therefore propose that the unfolding of these proteins
is likely to take place during translocation across the
thylakoid membrane.
Several lines of investigation support the above proposal. First, the lack of requirement for stromal factors suggests that the pre-23K presented to isolated thylakoids consists of a folded mature 23K moiety with a relatively unfolded presequence. There is abundant evidence that chloroplast protein presequences are substantially, if not completely unfolded (von Heijne and Nishikawa, 1991; Theg and Geske, 1992; Pilon et al., 1992), and it is therefore to be expected that they are very susceptible to proteolysis. On the other hand, there is no evidence for the presence of a factor in cell-free translation systems which maintains chloroplast protein precursors in an unfolded conformation. On the contrary, the precursor of 5-enolpyruvylshikimate-3-phosphate synthase was found to be catalytically active, and thus presumably correctly folded, after synthesis in a reticulocyte lysate (Della-Cioppa et al., 1986). In a similar vein, an artificial construct comprising a chloroplast protein presequence followed by dihydrofolate reductase was shown to bind substrate analogs after synthesis in a wheat germ lysate, again indicating the presence of a correctly folded passenger protein (America et al., 1994). In the case of 23K, there is no assay for biological activity, but the protease sensitivity data show that the mature 23K protein is far more sensitive to digestion by trypsin after incubation with urea or at high temperatures. In the absence of these treatments, mature size 23K is resistant to concentrations of trypsin which would normally digest a wide variety of precursor proteins to completion. Strictly speaking, this observation indicates that 23K adopts a different conformation after heat or urea treatment, but obviously the most likely interpretation of these results is that mature 23K folds tightly after synthesis in cell-free translation systems. In our experience, dihydrofolate reductase is the only other mature size protein that can withstand incubation with 30 µg/ml trypsin for 40 min; this protein is known to fold very tightly, and the evidence indicates that 23K is a protein which likewise folds particularly tightly.
The data obtained from studies
on the stromal i23K polypeptide are entirely consistent with this
hypothesis. Fractionation data indicate that the protein is almost
certainly monomeric, which would in itself suggest that the 23K has
probably refolded after transport across the envelope membranes.
Furthermore, the protease sensitivity patterns closely resemble those
obtained in studies on the in vitro synthesized precursor
protein; the mature 23K moiety is likewise particularly resistant to
digestion over a similar range of protease concentrations, strongly
suggesting that it has adopted a similar conformation. Since the i23K
polypeptide is competent for translocation across the thylakoid
membrane, we propose that the natural substrate for the pH-driven
thylakoidal protein transport machinery consists of a folded 23K mature
protein together with an extended thylakoid transfer signal. The
conformation of 23K during translocation across the thylakoid
membrane is not known, but studies on every other protein-translocating
membrane analyzed to date have shown that the proteins are transported
in a substantially unfolded state.
As yet, we do not know how
protein translocation is harnessed to the pH, but it is clear that
surface-exposed thylakoidal proteins are involved in the translocation
process. Perhaps surprisingly, however, there is no evidence for the
binding of i23K to receptors on the thylakoid surface; recent studies
using intact chloroplasts (Brock et al., 1995) have shown that
i23K is soluble in the stroma at all values of
pH tested.
Similarly, we have failed to detect any binding of pre-23K to receptors
in studies with isolated thylakoids. Following incubation of pre-23K
with thylakoids in the presence of nigericin, a small proportion of
precursor was in fact found to be associated with the thylakoids, but
an identical amount of binding was observed with protease-treated
thylakoids, and we therefore concluded that the binding was entirely
nonspecific (data not shown). In view of these results, we propose a
basic two-step model for the translocation mechanism. The first step
involves the binding of i23K or pre-23K to the translocase, and we
believe that this binding is weak, reversible, and probably
pH-independent. The second step involves the translocation of the
protein across the membrane, in which the
pH most likely provides
the driving force for both translocation and unfolding of the 23K
mature protein.
Whatever the conformation of 23K during transport
across the thylakoid membrane, the emerging evidence indicates that the
pH-driven protein transport system is increasingly unusual among
known protein transport systems. All other known mechanisms require the
presence of nucleotide triphosphates, except in certain unusual
circumstances (Deshaies et al., 1988; Meyer, 1988; Wickner et al., 1991; Theg et al., 1989; Hulford et
al., 1994) and a second recurring theme in other systems is that
proteins are generally maintained in a relatively unfolded (and hence
translocation-competent) state during passage through soluble phases of the cell or organism. In bacteria, precursor proteins on
the Sec-dependent pathway are usually bound by the cytosolic chaperone
protein SecB, which prevents folding of the precursor proteins before
they reach the export machinery in the plasma membrane (reviewed by
Wickner et al.(1991)). Signal recognition particle performs a
similar role in the cytosol of eukaryotic cells, by arresting the
translation of secretory proteins until co-translational translocation
across the endoplasmic reticulum can take place (reviewed by Luirink
and Dobberstein(1994)). Against this background, the occurrence of
stable, folded intermediates on the
pH-dependent thylakoidal
protein transport pathway is most unexpected and provides a further
indication that this mechanism operates in a different manner.
In
some respects the thylakoidal translocation mechanism may more closely
resemble that of the chloroplast envelope-localized protein
translocase, because the precursor proteins for both systems are likely
in some cases to comprise protease-sensitive presequences and folded
mature proteins. For example, pre-23K synthesized in a wheat germ
lysate can be efficiently imported by both chloroplasts and thylakoids.
However, there may be fundamental differences between the two
translocation mechanisms. There is evidence that at least some
precursor proteins may be unfolded on the chloroplast surface prior
to transport across the envelope membranes in an
unfolded/partially folded state (America et al., 1994; Schnell et al., 1990), although pre-23K has not been analyzed in this
respect. This binding/unfolding process is very stable and
ATP-dependent. In contrast, any unfolding process occurs during
translocation of 23K by the pH-dependent thylakoidal system (it
remains to be determined whether other substrates for this system
contain folded mature protein moieties). What is clear is that the
substrates for the thylakoidal system do not bind stably, and
translocation is ATP-independent. We therefore suggest that the
thylakoidal system represents a novel class of protein translocase, in
which the
pH is harnessed to drive either the translocation and unfolding of proteins or the translocation of fully folded
proteins.