(Received for publication, May 18, 1995)
From the
A Sec-type system is responsible for the translocation of a
subset of proteins across the thylakoid membrane in higher plant
chloroplasts. Previous studies have suggested that the thylakoidal
pH plays a minor role in this translocation mechanism, but we show
here that it can be essential for the translocation process, depending
on the identity of the passenger protein and the concentration of ATP.
Studies using chimeric proteins show that, whereas the presequence
dictates the translocation pathway, the
pH requirement is dictated
exclusively by the passenger protein; some passenger proteins are
virtually
pH-independent whereas others are absolutely dependent.
pH requirement is not related to charge characteristics of the
passenger proteins, ruling out an electrophoretic effect. Analysis of
the 33-kDa photosystem II protein reveals an inverse relationship
between
pH requirement and ATP concentration; import into isolated
thylakoids is inhibited 14-fold by nigericin at moderate ATP
concentrations, and totally inhibited when the ATP concentration is
reduced to 2 µM. The results indicate that the roles of
the
pH and ATP overlap and suggest that the
pH may be
obligatory when the passenger protein is abnormally difficult to
translocate, possibly due to the folding of the polypeptide chain. We
compare the energetics of this system with those of prokaryotic systems
from which the chloroplast system is believed to have evolved.
In plants and green algae, a number of photosynthetic proteins are synthesized in the cytosol and targeted across all three chloroplast membranes into the thylakoid lumen. This complex import pathway can be broadly divided into two phases, the first of which involves the transport of a cytosolically synthesized precursor protein into the stroma, after which the stromal form is transported across the thylakoid membrane into the lumenal space (Hageman et al., 1986; James et al., 1989; Ko and Cashmore, 1989; Hageman et al., 1990). The two translocation events are directed by distinct signals in the presequences of lumenal proteins. The first ``envelope transit'' signals resemble the presequences of imported stromal proteins in both structural and functional terms, whereas the second ``thylakoid transfer'' signals have differing properties which are more reminiscent of the signal peptides which direct transport across the bacterial plasma membrane (von Heijne et al., 1989; Bassham et al., 1991).
The available
evidence suggests that most proteins are transported across the
envelope membranes by a common mechanism, but recent studies have
pointed to the operation of at least two completely different
mechanisms for protein transport across the thylakoid membrane. The
development of assays for the import of proteins into isolated
thylakoids has shown that lumenal proteins fall into two clear groups
in terms of import requirements. A subset, including plastocyanin (PC) ()and the extrinsic 33-kDa photosystem II protein (33K)
require the presence of a stromal protein factor and nucleoside
triphosphates (NTPs) for their transport across the thylakoid membrane
(Hulford et al., 1994; Robinson et al., 1994),
whereas neither of these elements is required for transport of the
extrinsic 23- and 16-kDa photosystem II proteins (23K and 16K),
photosystem II subunit T, and photosystem I subunit N; these proteins
appear to be dependent only on the transthylakoidal
pH (Mould et al., 1991; Klösgen et al.,
1992; Cline et al., 1992; Nielsen et al., 1994; Henry et al., 1994). In contrast, both 33K and PC can be transported
across the thylakoid membrane of intact chloroplasts in the complete
absence of a
pH (Theg et al., 1989; Cline et
al., 1992; Nielsen et al., 1994).
Other studies have
shown that these differing requirements reflect the operation of
separate thylakoidal protein translocation systems. Robinson et
al.(1994) used chimeric proteins to show that the presequences of
23K and 16K are able to direct translocation of mature PC solely by the
23K/16K-type pathway, indicating that the presequences of 23K and PC
contain different types of targeting signal which almost certainly
specify translocation by distinct translocases. A similar conclusion
was reached by Cline et al.(1993), who showed that 33K and PC
compete for transport across the thylakoid membrane, and 23K competes
with 16K, but the two groups do not compete with each other. These
findings are remarkable in view of the similarities between thylakoid
transfer signals and typical signal sequences, all of which contain
hydrophobic core regions and apparently identical terminal cleavage
sites for a signal-type peptidase (von Heijne et al., 1989;
Halpin et al., 1989). However, recent studies have shown that
transfer signals for the pH-driven system contain a twin arginine
motif which is critical for translocation across the thylakoid membrane
(Chaddock et al., 1995).
The origins of the pH-driven
translocation mechanism are presently unclear, but there are
indications that the ATPdependent, azide-sensitive mechanism may have
been inherited from a sec-related mechanism in the
cyanobacterial-type progenitor of the chloroplast. 33K and PC are
present in cyanobacteria, where they are synthesized with presequences
resembling signal peptides and transfer signals and (probably)
transported across the thylakoid membrane by a Sec-dependent mechanism
(Kuwabara et al., 1987; Briggs et al., 1990).
Furthermore, the transport of these proteins across the chloroplast
thylakoid membrane is blocked by azide (Knott and Robinson, 1994; Henry et al., 1994), a known inhibitor of bacterial SecA proteins
(Oliver et al., 1990), and a SecA homolog has been shown to
participate in the translocation process (Nakai et al., 1994;
Yuan et al., 1994). Recent studies by Mant et
al.(1994) and Karnauchov et al.(1994) on the import of
PSI-F are consistent with this model: like 33K and PC, PSI-F is present
in cyanobacteria (Chitnis et al., 1991) and is targeted in
chloroplasts by the 33K/PC-type pathway. None of the proteins targeted
by the other, ATP-independent pathway have been found in cyanobacteria,
and it has therefore been suggested that their appearance in
chloroplasts may have been accompanied by the emergence of a novel
mechanism for their translocation across the thylakoid membrane.
Because PC, 33K, and PSI-F can all be transported across the
thylakoid membrane of intact chloroplasts in the absence of a pH,
this pathway has tended to be regarded as essentially
``
pH-independent.'' In this study we have used two
approaches to examine the energetics of the Sec-type mechanism more
closely. First, we show through the use of chimeric proteins that some
proteins are completely dependent on a
pH for transport across the
thylakoid membrane and that the
pH dependence is strictly linked
to the identity of the passenger protein rather than the presequence.
Second, we show that a protein which appears to be
pH-independent
in intact chloroplasts becomes wholly reliant on the
pH when the
ATP concentration is lowered to micromolar levels.
In ATP-depletion experiments, agarose-linked hexokinase (2U) was used to treat 50 µl of stromal extract, in the presence of 10 mM glucose for 10 min on ice. The mixture was agitated to ensure the agarose beads remained in suspension. After the incubation, the beads were pelleted in a microcentrifuge, and the stromal supernatant was removed to be used in a thylakoid import assay. The presence of 10 mM glucose had no adverse effects on control incubations.
Figure 1: Effects of nigericin on the translocation of lumenal proteins by the Sec-dependent pathway. Precursors of spinach plastocyanin (pre-PC), and pre-33K from spinach (spin) and wheat were incubated with intact pea chloroplasts in the presence or absence of nigericin (nig) as indicated. After incubation, the chloroplasts were protease-treated, and samples were analyzed of the stromal and thylakoid fractions (S and T) and the thylakoids after protease-treatment (T+). i33K denotes stromal intermediate form of 33K. Lanes Tr, translation products.
In order to examine the influences of the presequence and mature
protein on the energetics of transport by the Sec-type pathway, a
variety of chimeric proteins were tested in assays for the import of
proteins by intact chloroplasts. We analyzed the import characteristics
of two constructs (33/16 and PC/16) in which the presequences of
spinach 33K or PC are followed by mature size spinach 16K, a protein
which is usually transported by the pH-dependent pathway
(Klösgen et al., 1992). In the case of
33/16, the precursor form is imported into chloroplasts and converted
to two forms in the control incubations: a stromal intermediate form,
which presumably results from processing by SPP, and mature-size 16K
which is located in the thylakoid fraction (lane T). The
mature size 16K is resistant to proteolysis of the thylakoids (lane
T+) demonstrating that it has been correctly targeted into
the lumen. In the presence of nigericin, only the stromal intermediate
form is found, indicating that translocation across the thylakoid
membrane has been completely blocked.
Similar results are obtained with the PC/16 fusion protein (lower panel). In the control panel, the chimera is imported into chloroplasts and mature size 16K is again found in the thylakoid lumen in a protease-protected form. In addition, two prominent polypeptides are found in the stromal fraction, one of which (denoted iPC/16) probably results from the action of SPP. The second main stromal form (denoted i2) is only slightly larger than mature size 16K, and we believe that this polypeptide results from proteolysis or aberrant cleavage by SPP. In the presence of nigericin, translocation across the thylakoid membrane is again completely inhibited, and only the stromal polypeptides are detectable.
The
likely conclusion from the results shown in Fig. 2is that the
thylakoidal pH is essential for the translocation of 16K by the
Sec-dependent mechanism, but this conclusion is valid only if the 33K
and PC presequences do indeed transport 16K by this pathway. An
alternative interpretation is that in these chimeras the 16K mature
protein is dictating translocation by the ATP-independent pathway.
Additional tests were therefore carried out to determine the pathway
followed by these chimeras; our previous studies have shown that the
presequences of 23K and 16K are able to direct PC onto the
ATP-independent pathway, but reciprocal types of construct (in which
the presequences originate from Sec-dependent substrates) were not
analyzed. Previous studies have shown that the 33K/PC-type mechanism is
azide-sensitive (Knott and Robinson, 1994), and Fig. 3shows
that azide blocks translocation of 33/16 across the thylakoid membrane,
suggesting that the 33K presequence is directing translocation of 16K
by the Sec-dependent mechanism. We also carried out control tests to
verify that azide had no effect on the
pH-dependent pathway, and,
rather than using pre-23K or pre-16K in these tests, we analyzed the
transport of another fusion protein consisting of the presequence of
23K linked to mature size 16K. This was deemed useful in order to test
whether fusion proteins containing 16K are fortuitously sensitive to
azide irrespective of import pathway. The lower panel of Fig. 2shows that this fusion protein (23/16) is efficiently
targeted into the thylakoid lumen in both the absence and presence of
azide, indicating that translocation is taking place by the
pH-dependent pathway as expected. Finally, we have found that
PC/16 does not compete with overexpressed 23K for translocation across
the thylakoid membrane, demonstrating that translocation is not taking
place by the
pH-dependent pathway (not shown). In each of these
chimeras, therefore, the presequence dictates the import pathway
followed.
Figure 2:
33/16 and PC/16 fusion proteins require a
pH for translocation across the thylakoid membrane in intact
chloroplasts. 33/16 and PC/16 were synthesised in vitro and
imported into chloroplasts in the absence or presence of nigericin as
indicated above the lanes. After import, the chloroplasts were lysed
and samples analyzed of the stromal and thylakoidal fractions (lanes S and T) and the thylakoid fractions after
protease treatment (lanes T+). Lanes Tr,
translation products. iPC/16, probable intermediate form
resulting from cleavage by the stromal processing peptidase; i2, intermediate size form probably resulting from aberrant
processing or proteolysis.
Figure 3: The 33/16 fusion is transported by the azide-sensitive thylakoidal translocation mechanism. 33/16 was imported into chloroplasts in the absence or presence of 10 mM sodium azide as indicated above the lanes; after import, samples of stromal fraction, thylakoids, and protease-treated thylakoids were analyzed as in Fig. 1. Symbols are as in Fig. 2.
The overall conclusion from Fig. 2and Fig. 3is that the pH is critical for the translocation of
the 16K passenger protein by the Sec-type mechanism. This is the first
indication that translocation by this pathway can be absolutely
pH-dependent, and there is no doubt that it is the passenger
protein which dictates the translocation energetics in this case:
pre-PC and PC/16 contain the same presequence but exhibit diametrically
opposite
pH dependences. This phenomenon is further exemplified by
studies with chimeras in which the presequences and mature proteins of
spinach pre-PC and pre-33K are exchanged (33/PC and PC/33). The top
panel of Fig. 4shows that 33/PC is efficiently imported
into the thylakoid lumen (there is no evidence for a stromal
intermediate form), and that nigericin has no apparent effect on the
translocation process, in that a stromal intermediate does not appear.
Translocation of PC/33 across the thylakoid membrane, on the other
hand, is completely inhibited by nigericin. When these results are
compared with those obtained with the authentic precursor proteins (Fig. 1), it is again apparent that the passenger protein
dictates the
pH dependence of translocation. There is a slight
difference in the effects of nigericin on the translocation of pre-33K
and PC/33 across the thylakoid membrane, in that the latter is totally
inhibited whereas the former is almost completely inhibited.
However, this is a minor difference which may simply reflect the fact
that PC/33 is a poor substrate for the translocation machinery; this
construct is translocated with the lowest efficiency among those used
in this study. As with the 33/16 and PC/16 fusion proteins, azide
sensitivity and competition tests were used to confirm that the PC/33
and 33/PC are transported across the thylakoid membrane by the Sec-type
mechanism (data not shown).
Figure 4: Influences of presequence and mature protein on the translocation energetics of 33/PC and PC/33. 33/PC and PC/33 were imported into pea chloroplasts, and the organelles were subsequently fractionated, and the lanes labeled, as detailed in the legend to Fig. 1. A stromal intermediate size form of iPC/33 is indicated by an arrow; 33K and PC, mature-size proteins.
Fig. 5shows the results of
assays for the import of i33K by pea thylakoids. As shown by Hulford et al.(1994), stromal extract is required for efficient import
of i33K, although washed thylakoids are capable of a low level of
import. The presence of nigericin reduces import efficiency to 7%
of the control level, showing that the
pH does indeed stimulate
the translocation process to a significant extent under these
conditions. The ATP concentration in this assay is 120 µM,
and additional MgATP does not improve import efficiency in the absence
of nigericin; the presence of 1 mM MgATP does, however, rescue
import efficiency to some extent in the presence of nigericin (up to
15% of the control value).
Figure 5: Effects of uncouplers and increased ATP concentrations on the import of i33K by isolated pea thylakoids. i33K was incubated with isolated thylakoids in the absence of stromal extract (-S) or in the presence of stromal extract (+S) as detailed under ``Experimental Procedures.'' Incubation N contained stromal extract and 2 µM nigericin. All of the remaining incubations contained stromal extract, as well as MgATP at the indicated concentrations (in mM), in the presence or absence of nigericin. Import efficiencies were quantitated by laser densitometry and are given as a precentage of the control value (incubation +S). Maximum import efficiency in this experiment was 16% of available i33K. The inset shows the protease-treated lanes from the fluorogram; the order of the lanes corresponds to the graph.
Thylakoid import assays were also used to
address another important question: is the importance of the pH
related to the prevailing ATP concentration? The two sources of energy
overlap to an extent in bacteria, in that the K
for ATP is much lower in the presence of a proton motive force
(Shiozuka et al., 1990), and the requirement for a
µH
can be overcome by adding an
excess of SecA (Yamada et al., 1989b). A detailed
understanding of the thylakoidal Sec-type system similarly requires an
understanding of the precise contributions of ATP and
pH. Again,
the intact chloroplast assay is unsuitable because fairly high ATP
concentrations (above 100 µM) are required to first
translocate proteins across the envelope membranes (Theg et
al., 1989). For these studies we developed a low ATP variant of
the thylakoid import assay, in which the mixed stromal extract and i33K
translation mixture were passed through a NAP 10 desalting column in
order to remove the vast majority of free ATP. These columns are
designed to remove small molecules from DNA-protein samples, but we
found that the actual concentration of ATP in the eluate was 1-2
µM (possibly because some ATP is weakly bound to
macromolecules in the concentrated stromal extract). Fig. 6shows that the import efficiency remains high after
desalting of the stromal extract-translation mixture (lanes S and D), indicating that 2 µM ATP is
sufficient to drive translocation. Significantly, nigericin completely
inhibits import under these conditions (lanes N) showing that
the
pH is essential at low ATP concentrations.
Figure 6:
Import of i33K by thylakoids is totally
dependent on the pH at low ATP concentrations. Washed pea
thylakoids were mixed with stromal extract and i33K translation mixture (lanes S) or stromal extract-translation mix which had been
desalted by chromatography through a NAP-10 column (lanes D)
as detailed under ``Experimental Procedures.'' Lanes
H, washed thylakoids were incubated with desalted
stroma-translation which had been preincubated with hexokinase and
glucose as detailed under ``Experimental Procedures.'' A
further incubation containing desalted stroma-translation was carried
out in the presence of 2 µM nigericin (lanes N).
Samples were analyzed directly after incubation (left panel)
or after protease treatment (righthand
panel).
The efficient import evident in lane D raises the possibility that the required ATP is actually bound to the stromal translocation factor during passage through the NAP 10 column, and we have attempted to determine whether this is the case. The desalted stromal extract-translation mix was preincubated with a mixture of hexokinase and glucose in order to hydrolyze free ATP, and lane H shows that this treatment totally blocks import. We therefore believe that micromolar levels of free ATP are sufficient to drive the translocation process, although we cannot exclude the possibility that prebound ATP does in fact contribute to the translocation process in lane D (the presence of hexokinase/glucose may shift the equilibrium between bound and free ATP).
Fig. 7shows a more detailed
assessment of the role of NTPs in the translocation reaction, using the
same type of assay. Two points emerge from the data. First, the
inhibition by nigericin can be relieved to a substantial extent by the
inclusion of 1 mM MgATP in the assay mixture. The average
import efficiency in the presence of nigericin, 1 mM MgATP is
31% of the control value, and we conclude that the pH dependence
is linked to ATP concentration. Additional ATP does not improve import
efficiency in the absence of nigericin, suggesting that the available 2
µM ATP is sufficient to drive translocation at high rates
in the presence of a
pH. Second, we find that 100 µM 5`-adenylyl-imidodiphosphate (AMP-PNP, a non-hydrolyzable ATP
analog) completely inhibits 33K translocation in the presence of a
pH, and it therefore appears extremely likely that hydrolysis of
ATP is an essential step in the translocation of 33K. A second analog,
adenylyl-(
,
-methylene)-diphosphate (AMP-PCP) is less
effective at inhibiting import at this concentration, but is likewise
capable of inhibiting import completely at 1 mM concentrations
(not shown). It should be emphasized that the figures shown in Fig. 7were averaged from several experiments, for the following
reason. We find that the effects of nigericin and AMP-PNP under these
conditions are fully reproducible: import is invariably completely
blocked. However, the extent to which added ATP is able to restore
import in the presence of nigericin varies between experiments. The
cause of this variability is presently unknown.
Figure 7: Hydrolysis of ATP is required for import of i33K into thylakoids. Washed thylakoids were incubated with i33K and stromal extract which had been desalted as in Fig. 6(D). Other incubations contained desalted i33K/stroma and 100 µM MgATP, AMP-PNP, or AMP-PCP (samples ATP, PNP, PCP). Incubations N and N+ATP contained 2 µM nigericin, and nigericin + 1 mM MgATP, respectively.
Is the translocation
of PC likewise dependent on the pH at low ATP concentrations? Fig. 8shows the effects of nigericin in ``standard''
thylakoid import assays (ATP concentration, 120 µM) and
after desalting of the translation mix-stromal extract as described
above. In the standard import assay, PC translocation is affected to a
somewhat lesser extent by nigericin (import efficiency is reduced by a
factor of 6), and transport continues at an appreciable rate in the low
ATP assay (again, a 6-fold reduction of import efficiency) whereas
import of 33K was totally inhibited under these conditions. These data
are fully consistent with the data obtained with the chimeric proteins
and reinforce the conclusion that PC is least reliant on a
pH
among the proteins studied to date, although the
pH stimulates
translocation even in this case.
Figure 8:
Pre-PC can be imported into thylakoids in
the absence of a pH at low ATP concentrations. Graph A,
washed thylakoids were incubated with spinach pre-PC in the presence of
stromal extract in the absence of further additions (S) or in
the presence of 2 µM nigericin (S+N) or 2
µM nigericin + 1 mM MgATP (N+ATP). Graph B, as in A except that
the thylakoids were incubated with a mixture of pre-PC and stromal
extract which had been passed through a PD-10 desalting column as
described in Fig. 5and Fig. 7for i33K (D).
In this study we have sought to examine the precise
contributions of the pH and NTPs to the azide-sensitive protein
translocation mechanism, in order to build a more detailed picture of
the energetics of this translocation process. The role of the
pH,
in particular, has been a source of confusion in recent studies, and no
details have emerged concerning the type of NTP required (or whether
NTP hydrolysis is a prerequisite for translocation). In the present
study we have used chimeric proteins and more controlled in vitro translocation assays to examine these requirements. The overall
message from these studies is clear: the presequence dictates the
translocation pathway inside the chloroplast, whereas the passenger
protein dictates the
pH requirement for transport across the
thylakoid membrane when targeted by the Sec-dependent mechanism.
A
significant point to emerge from this study is that the pH
probably stimulates, to a large extent, the transport of most or all of
the substrates for the Sec-type mechanism. In previous studies using
intact chloroplasts, nigericin was found to have no effect at all on
the transport of pre-PC or wheat pre-33K into the lumen (Theg et
al., 1989; Robinson et al., 1994; Nielsen et
al., 1994), suggesting that the translocation of some proteins is
truly
pH-independent. The use of thylakoid import assays has shown
that this is clearly not the case; translocation of both spinach pre-PC
and wheat pre-33K is severely affected when the
pH is collapsed.
We now suggest that the translocation of these proteins is similarly
affected in intact chloroplasts, but that the efficiency of
intraorganellar sorting is such that stromal intermediates still fail
to accumulate in many cases. This idea is supported by observations on
the translocation efficiency of pre-33Ks in intact chloroplasts. Under
standard assay conditions, nigericin almost totally blocks
translocation of spinach 33K in intact chloroplasts, has a less marked
effect on pea 33K (about half of the imported protein is present as
stromal intermediate; Cline et al., 1992), and has little or
no apparent effect on wheat 33K. We suggest that the transport
of all three species of 33K is probably inhibited to a similar extent
but that the proteins are transported across the thylakoid membrane
with varying efficiencies. Support for this possibility comes from the
observation that, even in the absence of nigericin, a prominent stromal
form of spinach 33K can be detected (suggesting relatively slow
translocation across the thylakoid membrane) whereas no stromal form of
wheat 33K is usually apparent. Thus, it may well be the case that
translocation of the wheat protein is much more efficient, and under
these circumstances a
10-fold inhibition of transport by nigericin
(which is the extent to which translocation is inhibited in standard
thylakoid import assays) may not be detectable in intact chloroplasts
because translocation is still too efficient for the stromal form to
accumulate.
The second point is that the Sec-dependent passenger
proteins vary greatly in terms of their requirement for the pH.
The least responsive is PC, which can be transported across the
thylakoid membrane in the complete absence of a
pH, either in
intact chloroplasts or in isolated thylakoids at low ATP concentrations
as shown in this study. We have shown that the PC mature protein is
likewise least reliant on a
pH (in chloroplasts) when fused to the
spinach 33K presequence because no stromal intermediate can be detected
during import of 33/PC in the presence of nigericin. 33K, on the other
hand, demonstrates an intermediate level of sensitivity. In intact
chloroplasts nigericin does induce the appearance of a stromal
intermediate but only with precursor proteins from some plant species.
The effects of nigericin in standard thylakoid import assays are more
pronounced with 33K than with pre-PC and, critically, translocation is
totally blocked in low ATP assays whereas that of pre-PC is not. The
third natural substrate of the azide-sensitive mechanism is PSI-F (Mant et al., 1994; Karnauchov et al., 1994). We have not
been able to demonstrate import of this protein into isolated
thylakoids, but uncouplers slightly inhibit the transport of this
protein across the thylakoid membrane in intact chloroplasts,
suggesting that this protein also exhibits an intermediate level of
pH requirement. Other proteins, however, are totally dependent on
the
pH for translocation to occur, even in intact chloroplasts.
The example illustrated in this study is 16K, which is completely
reliant on a
pH for translocation across the thylakoid membrane
when directed onto the Sec-dependent pathway. The data therefore
indicate that passenger proteins are not simply passively translocated
across the thylakoid membrane, but instead influence to a major extent
the physiological conditions required during the translocation process.
The actual translocation route, on the other hand, is specified
entirely by the presequence.
Why do some proteins need a pH
more than others? An electrophoretic effect appears unlikely since the
electrical potential component (
) of the thylakoidal proton
motive force is small under steady state conditions, and dissipation of
the
does not block translocation of 33K or PC (Mould and
Robinson, 1991; Cline et al., 1992). One obvious possibility
is that proton flux is coupled to protein translocation, in which case
it might be expected that protons re-entering the thylakoids from the
stroma might help to protonate acidic residues before translocation.
Unfortunately, there is no obvious correlation between
pH
dependence and the charge characteristics of the various passenger
proteins studied to date. PC, which is least dependent on a
pH,
has a net negative charge (-9 in spinach), but so has spinach
33K(-5) which is almost completely dependent on a
pH.
Equally striking is the observation that barley PSI-F and spinach 16K
have very similar overall charges (+3 in each case) but notably
different
pH dependences: PSI-F can be efficiently translocated in
the absence of a
pH (Mant et al., 1994; Karnauchov et
al., 1994) whereas 16K is absolutely dependent on a
pH.
Finally, there is no correlation between
pH requirement and
polypeptide size, since PC, PSI-F, and 16K are not dissimilar in terms
of chain length. One possibility is that the
pH has to be
harnessed to drive the translocation of proteins which are more highly
folded, and previous work on bacterial systems would favor this idea
(see below).
Another intriguing aspect of the pH requirement
(at least in the case of 33K) is that it depends to some extent on the
concentration of ATP in the reaction mixture. At very low ATP
concentrations the
pH becomes essential for the translocation of
wheat 33K, and we therefore conclude that there is some overlap between
the roles of the two sources of energy, as in bacterial systems. It is
tempting to speculate that, in the absence of sufficient energy from
ATP hydrolysis, the
pH provides an input of free energy which
enables the translocase to overcome the activation energy for the
translocation reaction. Nevertheless, there is little doubt that the
predominant driving force in this mechanism is provided by ATP
hydrolysis because removal of ATP using hexokinase or apyrase
completely blocks translocation even at high
pH values, and ATP
analogs are potent inhibitors of translocation. The
pH is a
prerequisite only for the translocation of a few proteins at
physiological ATP concentrations.
How do the energetics of this
translocation reaction compare with those of prokaryotic Sec-dependent
translocation mechanisms, from which the chloroplast mechanism is
supposed to have evolved? It appears that there are numerous
similarities, suggesting that the basic mechanism has been highly
conserved. As with the chloroplast system, both an energized membrane
and ATP are involved in driving protein translocation in bacteria
(reviewed in Driessen, 1992), although both the pH and
contribute to the proton motive force,
p (Bakker and
Randall, 1984). Furthermore, the importance of the
p also
varies between different proteins. In a study of three precursors,
Ernst et al.(1994) found that the rates of import of three
proteins (precursors of LamB, OmpA, and Skp) fell to between
10-40% when the
p was collapsed, and Yamada et
al. (1989a) reported that transport of pro-OmpA was reduced to 25%
of the control value. It was not established in these studies whether
the presequences or the mature proteins dictate
p dependence, but other experiments using constructs strongly
suggest that the
p is required to drive the translocation
of folded domains in the mature proteins, since the introduction of a
disulfide bond into a passenger protein increases the
p dependence of translocation (Tani et al., 1990; Tani and
Mizushima, 1991). These similarities notwithstanding, it may be that
the two mechanisms rely on the
p to different extents in vivo. An energized membrane is essential for bacterial
protein export in vivo but merely stimulates translocation in vitro. The reasons for this discrepancy are not entirely
clear, but possible explanations suggested by Driessen(1994) are that
excess SecA in the in vitro assays may suppress the
p dependence of translocation, that the in vitro assays are
able to generate only low levels of
p, and that the
collapse of the
pin vivo has the effect of
lowering the internal pH, thereby inactivating the translocase.
Alternatively, it may be that those proteins which are absolutely
dependent on a
p rapidly jam the translocation machinery
and block translocation of all other substrates. The situation may be
somewhat different in chloroplasts. The
pH is essential for
efficient translocation into isolated thylakoids but is less important
(at least for PC and some species of 33K) within intact chloroplasts in
which the conditions are usually regarded as being near in vivo for thylakoid protein translocation. Nigericin has no detectable
effect on the translocation of PC or wheat 33K in the presence of ATP
at concentrations varying from 0.1 to 8 mM (not shown). These
observations suggest either that the thylakoidal system is more
efficient than the bacterial system or that the thylakoidal system has
evolved to become less reliant on a
p, perhaps because
the thylakoidal
pH fluctuates enormously during the lifetime of
the chloroplast. It will be of interest to study thylakoid protein
translocation in intact plants or algae to ascertain the true
importance of the
pH in vivo.