From the Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom
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ABSTRACT |
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The nuclear psbY gene (formerly
ycf32) encodes two distinct single-spanning chloroplast
thylakoid membrane proteins in Arabidopsis thaliana. After
import into the chloroplast, the precursor protein is processed to a
polyprotein in which each "mature" protein is preceded by an
additional hydrophobic region; we show that these regions function as
signal peptides that are cleaved after insertion into the thylakoid
membrane. Inhibition of the first or second signal cleavage reaction by
enlargement of the The biogenesis of integral membrane proteins has attracted a great
deal of experimental attention in recent years, in an effort to
understand the mechanisms used to transfer hydrophobic regions from an
aqueous milieu into the membrane bilayer and to achieve the correct
final topology. Many of these studies have used bacterial proteins as
model systems, and the emerging evidence points to the operation of two
broad types of insertion mechanism, which can be classified as
"assisted" and "spontaneous." Some proteins clearly rely on the
membrane-bound elements of the Sec apparatus used for the export of
proteins into the periplasm, although the extrinsic SecA ATPase appears
to be used to a lesser extent unless large hydrophilic loops require
translocation across the membrane (1-3). Signal recognition particle
(SRP)1 has also emerged as an
important cytoplasmic factor that is required for the insertion of a
range of membrane proteins (4-7). Cross-linking studies suggest that
this ribonucleoprotein particle binds preferentially to highly
hydrophobic regions, consistent with a role in membrane protein
targeting (8). One such SRP-dependent protein has been shown to be inserted via the membrane-bound SecYEG complex, and it
appears likely that most, if not all, SRP substrates will follow this
route (9).
Other proteins are inserted by different means. The coat proteins of
the M13 and pf3 phages have been shown to insert into the
Escherichia coli plasma membrane by mechanisms that do not involve either SRP or the Sec machinery, and it has been proposed that
these proteins insert spontaneously into the membrane (reviewed in Ref.
10). It should be noted though that other, as yet undefined membrane
proteins could possibly assist in the insertion of this class of
proteins (none of these proteins have been shown to insert with the
correct topology into protein-free liposomes from an aqueous phase). A
small number of other membrane proteins have similarly been proposed to
follow either Sec- or SRP-independent insertion pathways (see
e.g. Ref. 11), but in general, these studies have been
carried out in vivo using Sec- or SRP-depleted cells, and
the precise insertion requirements are difficult to monitor under these
conditions. For example, one membrane protein previously designated
"Sec-independent" using a SecY-deficient strain has recently been
shown to be absolutely reliant on the Sec machinery in a SecE depletion
strain that exhibits a stronger phenotype (9). Partly as a result of
these problems, relatively few membrane proteins have been definitively
shown to insert by Sec/SRP-independent mechanisms, and it has remained
unclear whether this type of mechanism is widely used.
The chloroplast thylakoid membrane has emerged as an useful alternative
system for this type of study because, although genetic analysis is
more difficult, in vitro insertion assays have proved to be
relatively facile (reviewed in Ref. 12). Again, two basic types of
insertion mechanism have been characterized for the biogenesis of
membrane proteins. The multispanning light-harvesting
chlorophyll-binding protein (LHCP) of photosystem II is imported into
the chloroplast by means of an envelope transit signal, after which it
integrates into the thylakoid membrane by means of information
contained in the mature protein (13, 14). This process requires stromal SRP (cpSRP54) and nucleoside triphosphates (NTPs), and hence the overall insertion process may well resemble bacterial
SRP-dependent insertion events (15, 16). However, it should
be noted that an RNA molecule has yet to be identified in chloroplast
SRP, and certain aspects of the insertion process may therefore differ. Proteolysis of thylakoids destroys their ability to integrate LHCP
(17), indicating that membrane-bound protein transport apparatus is
required (probably the thylakoidal Sec machinery, although this remains
to be confirmed).
A very different insertion process has been demonstrated for a series
of abundant single-spanning thylakoid membrane proteins: subunit II of
the CFo assembly of the ATP synthase (CFoII)
and the X and W subunits of photosystem II (PsbX and PsbW). In contrast to LHCP (and most other multispanning proteins), these proteins are
synthesized with bipartite presequences in which the usual envelope
transit peptide is followed by a cleavable signal peptide. Signal
peptides usually specify an interaction with protein translocation systems, and most thylakoid lumen proteins are synthesized with this
type of peptide and translocated by either a Sec- or
To date, only simple, single-span proteins have been definitively shown
to insert into bacterial or thylakoid membranes by SRP/Sec-independent
mechanisms, raising the possibility that these factors tend to be
recruited for more complex proteins and that the biogenesis of
multispanning proteins may thus be rather more involved in general
terms. In this report, we describe the insertion pathway for a
thylakoid membrane polyprotein. Mant and Robinson (23) characterized an
unusual Arabidopsis thaliana cDNA encoding a protein
that contains two separate regions bearing high homology to
ycf32 open reading frames encoded by several algal plastid genomes. Whereas ycf32 genes encode small single-span
proteins, the Arabidopsis protein was predicted to contain
four hydrophobic regions, and it was proposed that this was in effect a
polyprotein of two separate Ycf32-related proteins, each of which was
preceded by a signal-type peptide. It has now been shown that the two
proteins are indeed found in thylakoids, associated with photosystem
II, and the gene has been designated psbY (24). The
individual polypeptides are designated PsbY-A1 and PsbY-A2. Here we
describe a complex insertion/maturation pathway for PsbY that involves
the use of two separate cleavable signal peptides, and we show that the
entire polyprotein can insert into the thylakoid membrane by an
SRP/Sec-independent mechanism.
Constructs--
Oligonucleotide-directed, site-specific
mutagenesis was carried out on the Arabidopsis cDNA
encoding pYcf32/pPsbY used in the study of Mant and Robinson (23),
using the inverse polymerase chain reaction method (25). 35-Mer primers
were used to alter the following Ala codons to Thr as follows: mutant
PsbY/1 (see under "Results"), codon 66 from GCC Import Assays--
Precursor proteins were synthesized in
vitro by transcription of cDNA clones followed by translation
in a wheat germ lysate in the presence of [35S]methionine
or [3H]leucine as detailed by Mant and Robinson (23) and
Robinson et al. (17). Assays for the import of proteins by
intact chloroplasts and isolated pea thylakoids were as in the same
references; a more complete description of the thylakoid import assay
is given in Brock et al. (26). Apyrase (Sigma, type VI) was
used to deplete the import incubation of NTPs using a protocol adapted
from (27). Twice-washed thylakoids (30 µg of chlorophyll for the
experiment shown in Fig. 6; 20 µg of chlorophyll for the experiment
shown in Fig. 7) resuspended in stromal extract were incubated for 10 min on ice with either 4 units of apyrase or 4 units of inactivated enzyme (100 °C for 10 min). Next, 10 µl of puromycin-treated
translation mixture (see below) was added to the thylakoids on ice and
incubated for a further 10 min before being transferred to the light
bath for 30 min. The final volume of each incubation was 50 µl, and stromal extract was present at 1.3× the equivalent concentration of
chlorophyll. Import buffer was 10 mM Hepes-KOH, pH 8.0, 5 mM MgCl2 (HM buffer). Postincubation, the
thylakoids were washed and analyzed directly (by mixing with protein
sample buffer), after protease treatment with thermolysin at 0.2 mg
ml Puromycin Treatment of Translation Mixtures--
In order to
avoid nonspecific association of precursor proteins with thylakoid
membranes, in vitro translation mixtures were treated with
0.1 mg ml Urea Washing of Thylakoid Membranes--
These analyses were
carried out essentially as described by Breyton et al. (28).
Thylakoid membranes (10-20 µg of chlorophyll) were washed in
ice-cold 20 mM Tricine-NaOH, pH 8.0, resuspended in a
freshly prepared solution of 6.8 M urea/20 mM
Tricine-NaOH, pH 8.0, and incubated for 10 min at room temperature
(around 22 °C). The samples were then subjected to two cycles of
freeze-thawing (dry ice-room temperature) and centrifuged at
120,000 × g for 15 min at 4 °C in a Beckman TL100
ultracentrifuge using a TLA100.3 rotor. Care was taken to remove the
top 80 µl of supernatant without disturbing the membrane pellet. The
remaining 20 µl of supernatant was discarded. The pellet was then
resuspended once more in 100 µl of urea solution, and the whole
process was repeated. The second extraction rarely removed any extra
material from the thylakoid membranes. Samples of the final membrane
pellet and the first supernatant (equivalent volumes loaded), along
with the other assay samples, were analyzed by SDS-polyacrylamide gel
electrophoresis followed by staining with Coomassie Brilliant Blue and fluorography.
Proteolysis of Thylakoid Membranes before Insertion
Assays--
The protocol was as described by Robinson et
al. (17) with the following modifications. After treating the
thylakoids with 60 µg ml Rapid Stopping of Chloroplast Import--
A stock solution of
0.2 M HgCl2 was prepared as described by Reed
et al. (29). Aliquots of intact chloroplasts from an import assay (30 µl; 10 µg of chlorophyll) were mixed with 2 µl of
HgCl2 at each sampling time. Chloroplasts were pelleted by
centrifugation at 2000 × g for 2 min at 4 °C, as
soon as possible after mixing with HgCl2. They were then
gently resuspended in 1 ml of 50 mM Hepes-KOH, pH 8.0, 330 mM sorbitol, 10 mM EDTA, recentrifuged, and
finally resuspended in the latter buffer plus protein sample buffer.
The PsbY Translation Product Contains Two Signal Peptides--
Our
initial aim in this study was to determine whether the psbY
gene product is indeed processed to two small single-span proteins
through the use of dual signal peptides as proposed in Ref. 23. Fig.
1 shows the overall primary structure of
the full precursor protein (which we term pPsbY) encoded by the
Arabidopsis psbY cDNA protein, in which the structure is
divided into five domains. The predicted sequence reveals an apparently
typical stroma-targeting envelope transit that is basic, hydrophilic, and enriched in hydroxylated residues. This is followed by the initial
"mature" protein (PsbY), which contains four hydrophobic regions:
(i) a predicted signal peptide, (ii) a region that is closely
homologous to algal ycf32 open reading frames (protein A1),
(iii) a second possible signal peptide, and (iv) a second region
homologous to single-span Ycf32 proteins (protein A2). Thylakoid signal
peptides are cleaved by a membrane-bound, lumen-facing TPP activity
that cleaves after short-chain residues at the
We first sought to clarify whether the internal hydrophobic region iii
is in fact a cleavable signal peptide. The cDNA coding region for
hydrophobic regions iii and iv was amplified using polymerase chain
reaction and an ATG codon incorporated at the 5'-end in order to
synthesize and import the second section of the PsbY polypeptide. This
construct is termed pre-A2. The import of wild-type pPsbY into
chloroplasts is shown as a time course analysis in Fig.
2A. In order to identify
possible processing intermediates, the samples were rapidly treated
with HgCl2, which has been shown to inhibit
import/processing events in studies on other chloroplast proteins (29,
31). Fig. 2A shows that the 23-kDa precursor protein, which
migrates as 19 kDa in this gel system, is processed to two small
polypeptides with mobilities of 6 and 7 kDa, as found by Mant and
Robinson (23). However, other processing products are apparent,
including the initial processed, imported form (PsbY), which migrates
just below the precursor protein (see below).
The pre-A2 construct can not be imported into chloroplasts because it
lacks an envelope transit peptide, and we therefore used assays for the
import of proteins into isolated pea thylakoids. Fig. 2B
shows that this construct is inserted and processed to a smaller
product, providing strong evidence that the construct does indeed
contain a cleavable signal peptide. This product has precisely the same
mobility as the upper band from a chloroplast import reaction (Fig.
2B, mark), and we therefore assign this upper band to
protein A2. These data provide further compelling evidence that pPsbY
is processed to two individual single-span proteins.
PsbY Inserts into Thylakoids as a Double-loop Structure--
The
above data and sequence information strongly suggest that pPsbY
contains a total of four hydrophobic regions, including two cleavable
signal peptides. Because TPP is known to be active on the lumenal side
of the thylakoid membrane, this protein offers attractive possibilities
in terms of identifying the topology of the polypeptide chain during
membrane insertion.
In order to address this topic more directly, we took advantage of the
highly precise nature of the TPP reaction, by substituting Thr residues
at possible
Analyses of several of the PsbY mutants are shown in Fig.
3. All of the mutant proteins are
imported by isolated chloroplasts and found exclusively in the
thylakoid fraction. The import and processing profiles of mutants
PsbY/1* and PsbY2* were found to be exactly as those shown in Fig. 1
for the wild-type protein (data not shown), strongly indicating that
these mutations do not lie at TPP cleavage sites. However, the
remaining mutants exhibit severe defects in processing. PsbY/2 is
imported and converted to a 10.8-kDa product, as shown in Fig. 3,
top panel. Clearly, the Thr residue prevents cleavage at the
second site by TPP, and a larger polypeptide accumulates. There is,
however, good evidence that the first signal peptide has been cleaved
because the mobility of the protein is consistent with a three-span
protein rather than a protein containing all four hydrophobic regions
(see below)
PsbY/1 is imported and converted to three polypeptides, one of
apparently mature size (7 kDa) together with a processing intermediate (designated int), which migrates as 10.2 kDa. The 7-kDa
protein co-migrates with protein A2 from chloroplast import assays with the wild-type protein or from thylakoid import assays using pre-A2 (see
below), and we therefore conclude that the A2 protein is correctly
removed from the PsbY/1 polyprotein upon insertion. Almost no A1 is
formed, however, indicating that the presence of the Thr residue has a
drastic effect on the release of this protein and providing very strong
evidence that the first hydrophobic region is in fact a cleavable
signal peptide that is recognized by TPP. Identical data were obtained
for the PsbY/1/Leu mutant (not shown), indicating that the appearance
of the PsbY/1 intermediate reflects an inhibition of processing rather
than any difficulty of translocating the more hydrophilic Thr residue
across the membrane.
A larger polypeptide was also apparent in this experiment (denoted by
an arrow), but the identity of this band is unclear because
it is usually present in lower quantities and is sometimes virtually
absent (see, for example, the PsbY/1 import results in Fig.
4). In contrast, the 10.2- and 7-kDa
proteins were always observed in approximately equal quantities.
The import experiment using the PsbY/1,2 double mutant is shown in Fig.
3, bottom panel. This protein was imported and cleaved to a
larger product that migrates as a 17.5-kDa protein which is thus only
marginally smaller than the full precursor protein. This product almost
certainly results from removal of the envelope transit peptide (see
below). Overall, these data strongly support the proposal that PsbY
contains two signal peptides that are removed upon insertion by TPP.
The results are, furthermore, consistent with data from the purified
spinach A2 protein (24), the N-terminal sequence of which,
ASEEIARGSDNRG, resembles the sequence following the second proposed TPP
cleavage site motif that was mutated in our Arabidopsis
mutant PsbY/2 (AAEAAAASSDSRG). However, the first cleavage
site region within spinach PsbY is PAFAVQLADIAAEAGTSDNRG, and the N-terminal sequence of purified spinach A1 was deduced to be
the AEAGTSDNRG sequence underlined above. This finding suggested that
TPP cleaved after the sequence QLADIA, whereas we believe that TPP
cleaves slightly upstream of this region (after PAFA in the spinach
sequence, which corresponds to the PALA sequence targeted for mutation
in our Arabidopsis sequence, as shown in Fig. 1). In our
view, cleavage after DIA is highly unlikely because charged
Further analyses of the processing mutants are shown in Fig. 4.
Panel A shows a comparison of all of the intermediate forms together with appropriate marker proteins. The autoradiogram confirms that the imported PsbY/1,2 polypeptide is significantly larger than the
intermediates generated during import of either PsbY/1 or PsbY/2 and
that the latter intermediates are in turn significantly larger than the
pre-A2 translation product containing two hydrophobic regions. This
result strongly suggests that the two single mutants are imported and
processed to polypeptides containing three hydrophobic regions. The
imported PsbY/1,2 mutant is only marginally smaller than the full
precursor and clearly contains all four hydrophobic regions. Fig. 4
also confirms the point made above, namely that the A2 protein is
cleaved from mutant PsbY/1, because the smaller import product in
lane 1 co-migrates precisely with the A2 protein generated
in a thylakoid import assay (adjacent lane T). We therefore conclude that neither of the single mutations prevents cleavage at the
unmutated TPP cleavage site. The only difference in the import profiles
of PsbY/1 and PsbY/2 is that a lower molecular mass cleaved product is
visible in the former but not in the latter. This reflects the nature
of the smaller cleaved species. Protein A2 is released from PsbY/1, and
this protein is stable under these conditions, whereas cleavage of
PsbY/2 at the first processing site leads to release of the signal
peptide. We have in fact found that this signal peptide is undetectable
even after over-exposure of the fluorographs and
[3H]leucine labeling (data not shown). After cleavage,
these polypeptides are clearly turned over very rapidly indeed, and our
attempts to visualize them have failed to date. Similar findings have
been made with the single-span proteins PsbW and PsbX, which are
also synthesized with cleavable signal peptides. Both proteins insert into thylakoids, yet the cleaved signal peptides are completely undetectable, despite being almost as large as the mature proteins (21).
All of the mutant forms are stably inserted into the thylakoid membrane
because each is resistant to extraction by urea washing. There is now
good evidence that this procedure effectively removes extrinsic
membrane proteins from thylakoids (23, 28), and Fig. 4B
shows that each of the intermediates is almost completely resistant to
this extraction procedure. In all cases, the protein from the thylakoid
fraction of a chloroplast import experiment (T) was almost
totally recovered in the pellet fraction containing the urea-washed
membranes (Pel), and very little protein was recovered in
the supernatants (Sn). However, the precise topologies of
the intermediates are difficult to determine. Studies on PsbW have shown that the precursor protein inserts as a loop intermediate prior
to cleavage by TPP in the lumen (22), and the two signal peptides in
PsbY probably form similar loops with their cognate mature proteins. It
is, however, difficult to determine whether both loops have formed in
the inserted PsbY/1 or PsbY/2 intermediate forms. In the case of
PsbY/1, the second loop must have formed for protein A2 to be released,
but it is notable that the remaining intermediate is highly sensitive
to digestion by thermolysin (Fig. 3, lane T+). The lack of
any defined protease digestion product means that we cannot be certain
that the first loop has formed. With PsbY/2, a thermolysin degradation
product is apparent that migrates as a 9-kDa protein (Fig. 3,
DP), but further analyses are required before the topology
of this protein can be determined.
Finally, the import data shown in Figs. 2-4 have another important
implication for the overall insertion mechanism used by PsbY. Hydrophobic regions i and iii can now clearly be designated as cleavable signal peptides that are processed upon reaching the thylakoid lumen, and these peptides must therefore form loop structures with their partner A1 or A2 proteins. This means that an additional cleavage event must take place, between the A1 protein and the second
signal peptide, and this event must furthermore take place on the
stromal side of the membrane (see under "Discussion"). Our data
indicate that this occurs relatively late in the maturation process
because, given that each of the PsbY/1 and PsbY/2 processing intermediates shown in Fig. 3 contains three hydrophobic regions, neither can have undergone this cleavage step.
PsbY Inserts Primarily by an SRP/Sec-independent
Mechanism--
The single-span proteins CFoII, PsbW, and
PsbX are of interest because each is synthesized with a cleavable
signal peptide, yet their insertion into thylakoids does not require
nucleoside triphosphates or stromal factors, ruling out an involvement
of SRP or SecA. Furthermore, extensive trypsin treatment of thylakoids blocks import by the Sec- and SRP-dependent pathways yet
has no effect on the insertion of this group of proteins, strongly
suggesting that the membrane-bound Sec apparatus is not involved
(18-21). In this context, the insertion mechanism used by PsbY is of
significant interest because this protein is far more complex in
structural terms and is effectively a multispanning protein during the
initial stages of the integration process. Similar tests were used to assess its mode of insertion into isolated thylakoids, and we used two
criteria as evidence of correct insertion. Firstly, PsbY insertion
should be accompanied by the appearance of the A1 and A2 subunits. This
alone would indicate that insertion has taken place because TPP is
active on the trans side of the membrane. Protease
resistance is often also used as an alternative criterion for
insertion, but this is unsatisfactory in the case of PsbY because
proteases tend to cleave the precursor protein to a size similar to
those of the mature A1 and A2 subunits (not shown). Instead, we used
urea washing as a second criterion, as described above for the
localization of the processing intermediates. However, control tests
have shown that urea washing yields slightly less clear-cut results
with small single-span proteins than with multispanning proteins, as
illustrated in Fig. 5, which shows the
urea resistance of authentic A1 and A2 proteins generated during a
chloroplast import experiment. A significant proportion of A1 and A2 is
washed from the membrane and recovered in the supernatant fraction,
apparently because the urea washing process is relatively harsh for
single-span proteins (we have found that a similar proportion of mature
CFoII and PsbW is likewise extracted by urea; data not
shown).
Fig. 6 shows assays for the insertion of
PsbY into thylakoids, in which apyrase was used to deplete the system
of all NTPs present. As a control, we analyzed the insertion of LHCP,
which depends entirely on NTPs for SRP-dependent insertion
(16, 32). After incubation of petunia pre-LHCP with thylakoids, the
membranes were reisolated, and the top panel of Fig. 6 shows
that this fraction (lane T) contains a mixture of precursor
protein and processed, mature size LHCP (the result of cleavage by
stromal processing peptidase present in the incubation). Treatment of
these membranes with 0.2 mg/ml thermolysin (lane T+) yielded
a significant amount of mature size protein, which represents inserted
protein; previous studies (14, 17) have shown that this particular LHCP
protein is completely resistant to further digestion under these
conditions when correctly inserted. We also subjected the thylakoids to
urea extraction, and the data show that a similar proportion of protein is found in the pellet fraction (Fig. 6, Pel), again
indicative of correct insertion (it is notable that a proportion of
both precursor and mature size protein is urea-resistant, indicating that insertion can precede proteolytic processing by the stromal processing peptidase). The apyrase-pretreated samples show a very different profile: essentially no protease-resistant protein was apparent, and the vast majority of protein was urea-extractable. These
data agree with previous studies (16, 32) concerning the NTP dependence
of LHCP insertion and show that urea resistance is a useful alternative
criterion for insertion.
PsbY also inserts into isolated thylakoids and is efficiently processed
to the A1 and A2 subunits in lane T of the control (minus apyrase)
incubation. Urea washing of the membranes showed that about 50% of the
protein is recovered in the pellet fraction (Fig. 6, Pel).
Because this level of urea resistance is similar to that found for
authentic A1 and A2 in chloroplast imports (Fig. 5), we concluded that
the mature proteins were correctly inserted in the thylakoid import
reactions. Significantly, apyrase treatment does not block import,
because a high level of urea-resistant mature A1 and A2 was again
generated. This indicates that NTPs are not required for insertion and
hence that neither SecA nor SRP is required. Nevertheless, some
inhibition was observed, and quantitation of the insertion reaction
shows that apyrase treatment reduces insertion efficiency to 47% of
the control value. In this respect the insertion of PsbY differs from
that of the single-span proteins, such as PsbX or PsbW, where apyrase
was found to have no effect whatsoever on insertion efficiency (21).
This result suggests that some PsbY molecules may insert with the aid
of SRP or SecA.
The same conclusion is reached in tests for dependence on stromal
factors. LHCP insertion into thylakoids is almost totally dependent on
the presence stromal extract, which contains the bulk of SRP (15, 32),
and Fig. 7 shows the results of this type
of analysis for PsbY. The data show that the presence of stromal
extract enhances insertion efficiency (from 20 to 34% of available
precursor), and it is notable that apyrase treatment largely abolishes
this stimulatory effect, reducing insertion efficiency to 14% in the
presence of stromal extract. This result is highly reproducible and
points to a proportion of PsbY molecules being targeted by a pathway(s)
that depend on both stromal factors and NTPs. Similar results were
obtained for the pre-A2 construct, indicating that the partial
NTP/stroma dependence is not simply due to the relative complexity of
the PsbY polypeptide.
Finally, we tested the effects of pretreating the thylakoids with
trypsin because this treatment has been shown to destroy their ability
to import substrates on the Our data indicate that the insertion and maturation of the PsbY A1
and A2 proteins takes place by an unusual pathway involving multiple
proteolytic processing steps. After import, the removal of the first,
envelope transit peptide is presumably carried out by the stromal
processing peptidase, as with all other import stromal/thylakoid
proteins (12). Thereafter, we propose that two distinct cleavable
signal peptides are used to assist the insertion of the A1 and A2
proteins. The evidence is overwhelmingly in favor of this premise:
these sequences are certainly removed and then degraded, the sequences
bear the typical hallmarks of thylakoid signal peptides (a hydrophobic
region followed by a helix-breaking Pro or Gly residue and then an
Ala-X-Ala motif) and the substitution of the The precise topologies of the mature A1 and A2 proteins have yet to be
established, but because TPP is active in the thylakoid lumen, it
appears inevitable that the N termini of the mature proteins are
located in this compartment and that the signal peptides therefore
function in a manner analogous to those of PsbW and PsbX. With these
single-span proteins, the role of the signal peptide appears to be to
assist integration by providing an additional hydrophobic region,
and pre-PsbW has been shown to form such a loop intermediate prior to
cleavage by TPP (22). Interestingly, this type of insertion process
appears to be used only when the protein is first imported into the
chloroplast. Genes encoding PsbX and CFoII have been
identified in cyanobacteria and the plastid genomes of several
eukaryotic algae, and in no case is the protein preceded by a
signal-type peptide (21, 33). This raises the possibility that the
signal peptides have been acquired only after the transfer of the genes
to the nucleus because the initial endosymbiotic events involving a
cyanobacterial-type organism, and that the more complex import pathway
necessitates the presence of the second hydrophobic region, for unknown
reasons. A basically similar situation applies to the Arabidopsis
psbY gene: the homologous proteins encoded by open reading frames
in the cyanobacterium Synechocystis PCC6803 and in several
plastid genomes are devoid of signal peptides (23), suggesting that
both of the PsbY signal peptides were acquired after transfer of the
gene to the nucleus. All known cyanobacterial/plastid-encoded
ycf32 genes encode single-span proteins, indicating that the
Arabidopsis gene arose by gene duplication, together with
the acquisition of the two signal peptides. A working model for the
insertion of PsbY is shown in Fig. 9. In
this model, the A1 and A2 proteins each insert as loop structures
together with their associated signal peptide. Because inhibition of
cleavage at either site has no apparent effect on cleavage at the
nonmutated site, we believe that the entire PsbY protein first inserts
as a double-loop structure, after which cleavage takes place at the two
TPP cleavage sites. A different, as yet unidentified peptidase is
believed to cleave between the A1 protein and the second signal peptide, and this step must take place at the end the insertion process
because no smaller cleaved products are apparent when TPP cleavage is
inhibited in the PsbY/1,2 double mutant. According to the above model,
this step has to take place on the stromal face of the thylakoid
membrane; any other scenario would require transmembrane segments to
reverse orientation, and this would be unusual in the extreme. One
interesting possibility is that this cleavage site is tightly
constrained in the PsbY polyprotein (perhaps as a tight loop) but that
it becomes accessible when the two TPP cleavage events release a
smaller, more flexible structure.
1 residues leads in each case to the accumulation
of a thylakoid-integrated intermediate containing three hydrophobic
regions after import into chloroplasts; a double mutant is converted to
a protein containing all four hydrophobic regions. We propose that the
overall insertion process involves (i) insertion as a double-loop
structure, (ii) two cleavages by the thylakoidal processing peptidase
on the lumenal face of the membrane, and (iii) cleavage by an unknown
peptidase on the stromal face on the membrane between the first mature
protein and the second signal peptide. We also show that this
polyprotein can insert into the thylakoid membrane in the absence of
stromal factors, nucleoside triphosphates, or a functional Sec
apparatus; this effectively shows for the first time that a
multispanning protein can insert posttranslationally without the aid of
signal recognition particle, SecA, or the membrane-bound Sec machinery.
INTRODUCTION
Top
Abstract
Introduction
References
pH-dependent translocase in the thylakoid membrane
(reviewed in Ref. 12). However, CFoII, PsbW, and PsbX have
all been shown to insert into thylakoids in the absence of SecA, SRP,
or NTPs, and proteolysis of thylakoids has been shown to block
Sec-dependent transport but have no effect on the insertion
of the above proteins (17-21). These findings represent strong
evidence that neither SRP nor the Sec apparatus is required, and it has
been proposed that these proteins may insert spontaneously into the
thylakoid membrane. In this insertion mechanism, the proposed role of
the signal peptide is to provide an additional hydrophobic section,
which, together with the corresponding region in the mature protein, is
able to partition into the membrane and drive the transport of the
hydrophilic region (the N terminus of the mature protein) into the
lumen. Cleavage by the thylakoidal processing peptidase (TPP) on the trans side of the membrane then yields the mature protein.
Inhibition of the processing step has been shown to generate such a
loop intermediate (22) akin to that involved in the insertion of M13
procoat (10).
EXPERIMENTAL PROCEDURES
ACC; PsbY/1*,
codon 78 from GCC
ACC; PsbY/2, codon 143 from GCT
ACT; PsbY/2*,
codon 150 from GCT
ACT. In mutant PsbY/1/Leu, codon 66 was altered
to CUC (Leu). The construct encoding pre-A2 was synthesized by
polymerase chain reaction amplification of the Arabidopsis
cDNA region encoding signal 2 and protein A2. The forward primer
(5'-GAG AGT AAA CAT ATG GTT GTT GGT CTA GG-3') introduced an
NdeI restriction site at glycine 118, altering it to
methionine. The reverse primer (5'-CGT AAG CTT GGA TCC TCT AGA GCG
GC-3') took advantage of the preexisting NotI linker
restriction site in the cDNA template. The amplified region was
cloned 5' NdeI-NotI 3' under control of the SP6
promoter of pGEM®5Zf (Promega) and completely sequenced before being
used as a template for in vitro transcription and translation.
1 for 40 min on ice, or after washing the membranes
with urea (see below).
1 puromycin (to dissociate ribosomes) for 2 min
at room temperature, at the end of the translation incubation. The
translation mixture was then centrifuged at 100,000 × g for 20 min at 4 °C to pellet any aggregated proteins.
The resulting supernatant was used in thylakoid insertion assays.
1 trypsin (Sigma, type XIII)
for 10 min on ice, the digestion was stopped by the addition of 120 µg ml
1 trypsin inhibitor (Sigma, type I-S), and the
thylakoids reisolated by centrifugation at 100,000 × g
for 10 min at 4 °C. The thylakoids were washed twice in HM plus 60 µg ml
1 trypsin inhibitor (centrifuged at 100,000 × g for 5 min at 4 °C) and finally resuspended in
stromal extract (for pPsbY and pre-A2) or HM buffer (p23K). Each sample
contained thylakoids equivalent to 20 µg of chlorophyll, 0.5 mM MgATP, and 10 µl of translation mixture all buffered
by HM (final volume, 50 µl). Stromal extract, when present, was at a
concentration equivalent to 1.3× the chlorophyll concentration. The
import/insertion incubation was carried out under a green safelight,
for 30 min at 26 °C, and the prevailing
pH was measured by
9-aminoacridine fluorescence quenching as described (17); this was
found to be invariably over 2 units.
RESULTS
3 and
1 positions in
the substrate relative to the processing site; the presence of Ala at
1 is essential for efficient cleavage (30). Ala is also usually found
at the
3 position. Hydrophobic regions i and iii above were
considered to be possible signal peptides on the basis that these
regions are followed by potential Ala-X-Ala TPP cleavage sites.
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Fig. 1.
Primary structure and proposed domain
organization of pPsbY. The Fig. shows the full predicted sequence
of Arabidopsis pPsbY (previously designated Ycf32 (23)). The
N-terminal region contains an envelope transit peptide that is believed
to be removed after import by the stromal processing peptidase (note
that this processing site has not been identified). Proteins A1 and A2
are indicated (see text), each of which is preceded by an apparent
signal peptide. The exact position of the junction between protein A1
and signal peptide 2 has not been determined. Hydrophobic regions in
the signal peptides and mature proteins are underlined.
Candidate 1 Ala residues of TPP cleavage sites are shown
italicized and underlined; these were altered to
Thr residues in four single mutants, the designations of which are
shown. Construct pre-A2 was synthesized by amplification of the coding
region for the C-terminal region of PsbY and the introduction of a
start codon in place of the Gly indicated by an arrow (see
under "Experimental Procedures").
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Fig. 2.
Protein A2 is preceded by a cleavable signal
peptide. A, [35S]Methionine-labeled pPsbY
was synthesized in vitro and incubated with intact pea
chloroplasts. At time intervals indicated above the lanes (in min)
samples were mixed with HgCl2 and centrifuged briefly to
pellet the chloroplasts. The chloroplasts were then washed with import
buffer containing EDTA and analyzed by SDS-polyacrylamide gel
electrophoresis and fluorography (see under "Experimental
Procedures" for further details). B, the pre-A2
translation mixture (lane Tr) was incubated with isolated
pea thylakoids (lane T). After incubation, the sample was
analyzed together with a marker sample (mark.) from the
30-min import of pPsbY shown in panel A. Proteins A1 and A2
are indicated together with pPsbY that is bound to the chloroplast
envelopes in the marker lane.
1 positions. This markedly inhibits the TPP processing
reaction (22, 30), and we reasoned that this would lead to the
identification of defined intermediates on the PsbY biogenesis pathway.
Any observed inhibition would furthermore help to define these
cleavable peptides as substrates for TPP because only minor, single
substitutions were made at each site, and identical substitutions had
no effect on the insertion of loop structures in pre-PsbW or pre-PsbX
(22). Both terminal Ala residues were therefore altered to Thr by
site-specific mutagenesis of the cDNA clone (the relevant residues
are shown in Fig. 1, italicized and underlined).
In addition, a mutant was made in which both sites were disrupted
(PsbY/1,2). Two further potential TPP cleavage sites were also
identified, and the
1 Ala residues mutated to Thr; these mutants were
designated PsbY/1* and PsbY/2*, as shown in Fig. 1. Finally, we
addressed the possibility that the presence of a Thr residue might
affect the translocation of the intervening regions as well their
removal by TPP. This was, in our view, extremely unlikely because Thr
residues had no detectable effect on the rate of insertion insertion of
PsbX or PsbW into the the thylakoid membrane or on the translocation of
the hydrophilic section into the lumen (22). Nevertheless, we tested
this possibility directly by altering the
1 Ala at the first
predicted cleavage site to Leu on the basis that this would similarly
prevent cleavage by TPP, while having no predicted effect on insertion
(if anything, the more hydrophobic Leu residue should enhance
insertion). This mutation is designated PsbY/1/Leu.
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Fig. 3.
Accumulation of processing intermediates in
the Ala Thr mutants. Mutants PsbY/1, PsbY/2, and the PsbY/1,2
double mutant (lanes Tr) were imported into intact
chloroplasts and samples analyzed of the chloroplasts (lane
C) and thermolysin-treated chloroplasts (lane C+).
Other aliquots of chloroplasts were pelleted after protease treatment
and lysed, after which, centrifugation yielded samples of stroma
(S) and thylakoids (T). Lane T+,
thermolysin-treated thylakoids. Processing intermediates
(int) and degradation products (DP) are denoted.
Arrow denotes larger cleavage product of unknown
significance (see text).
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Fig. 4.
PsbY processing intermediates contain three
hydrophobic regions and are stably inserted into the thylakoid
membrane. A, size comparison of the imported proteins.
The figure shows the mobilities of the PsbY/1, PsbY/2, and PsbY/1,2
import products (lanes 1, 2, and 1,2, respectively) together with a sample from a thylakoid import of pre-A2
and sample of pre-A2 translation product (lanes T and
TrA2 at the right) and a sample of the thylakoids
from thylakoid and chloroplast imports of pPsbY (lanes T and
C, respectively, at the lefts). Mobilities of
molecular mass markers are indicated on the right.
B, the three mutants described in A were imported
into chloroplasts and the thylakoid fraction prepared after lysis
(T). Samples of the membranes were then washed with urea and
samples analyzed of the the supernatant fraction (Sn) and
the pellet fraction (Pel) containing the membranes.
TrY, pPsbY translation product.
3
residues are unknown in thylakoid-targeting signals, and the
corresponding region in the Arabidopsis PsbY sequence (QIAQLA) would contain Gln as the
3 residue. Side-chains of this length at the
3 position are not tolerated by TPP (for example, both
Leu and Glu drastically inhibit processing (30)), and we propose
instead that TPP cleaves after PALA in the Arabidopsis sequence (PAFA in the spinach sequence shown above), both of which are
perfect TPP recognition sites. This would imply that a further cleavage
by an unknown protease takes place to yield the mature A1 protein
sequenced from spinach (24).
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Fig. 5.
Urea resistance of thylakoid-integrated A1
and A2 subunits. pPsbY was imported into intact chloroplasts, and
samples fractionated and analyzed as described in Fig. 3. The
thylakoid fraction (lane T) was then subjected to urea
washing as detailed under "Experimental Procedures," and samples of
the pellet (Pel) and supernatant (Sn) fractions
were analyzed. Other symbols are as in Figs. 2-4.
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Fig. 6.
Insertion of pPsbY into thylakoids does not
require nucleoside triphosphates. Petunia pre-LHCP
(pLHCP) and pPsbY were incubated with pea thylakoids in the
presence of either 4 units of boiled apyrase (control conditions) or 4 units of active apyrase, on ice, as described under "Experimental
Procedures." After incubation with thylakoids, samples were analyzed
directly (T) or after treatment of the thylakoids with 0.2 mg/ml thermolysin for 40 min on ice (T+). Other samples of
non-protease-treated thylakoids were washed with urea, and samples of
the pellet (Pel) and supernatant (Sn) fractions
were analyzed. LHCP denotes mature LHCP polypeptide.
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Fig. 7.
Stromal factors and NTPs stimulate but are
not a prerequisite for the insertion of PsbY into thylakoids.
pPsbY and pre-A2 were incubated with pea thylakoids in the presence or
absence of stromal extract as indicated; other incubations with (+) or
without ( ) stromal extract were preincubated with 4 units of apyrase
as described in Fig. 6. Control incubations contained the same amount
of boiled apyrase. Samples were analyzed of the thylakoids after
incubation; symbols are as in Fig. 6.
pH-, Sec-, or SRP-dependent pathways (17). The data (Fig. 8) show
that both pPsbY and pre-A2 are imported and processed after this
treatment, although a slight inhibition was apparent in the case of
PsbY, which again points to an assisted pathway being used by a subset
of molecules. The import of pre-23K into the lumen is completely
blocked by the trypsin treatment, and similar effects were observed for
LHCP and 33K, a Sec substrate (not shown). We therefore conclude that PsbY can insert into thylakoids in the absence of NTPs, a
pH, SecA,
SRP, or a functional Sec complex in the thylakoid membrane.
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Fig. 8.
Protease treatment of thylakoid membranes
does not block the insertion of pPsbY. pPsbY, pre-A2 (upper
panel), and pre-23K (lower panel) were incubated with
thylakoids under control conditions or with thylakoids that had been
pretreated with 60 µg/ml trypsin under conditions that retain a high
thylakoidal pH (see under "Experimental Procedures"). After the
import incubations, samples were analyzed directly (T) or
after thermolysin treatment of the thylakoids (T+).
23K, mature 23K.
DISCUSSION
1 alanine
residues results in the almost complete inhibition of TPP activity as
found in other studies using thylakoid signal peptides (22, 30). The
psbY gene therefore encodes the first chloroplast-targeted
polyprotein to be characterized in higher plants.
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Fig. 9.
Model for the insertion of PsbY.
Stage 1, stromal PsbY contains four hydrophobic regions
(shown as helices), which include proteins A1 and A2 together with
associated signal peptides (SP1 and SP2). This
protein inserts as a double loop structure (stage 2) with
the N terminus of the mature proteins located in the lumen. Cleavage at
the TPP cleavage sites (stage 3) yields the mature A2
protein together with protein A2 and the attached signal peptide 2. Cleavage of the latter intermediate by an unknown peptidase
(pept.) on the stromal face of the membrane generates the
mature A1 protein (stage 4).
Although the maturation of PsbY is unique among known chloroplast
proteins, our studies on the insertion requirements have more general
implications because PsbY is clearly able to insert in the absence of
either SRP or a functional Sec apparatus. No NTP hydrolysis is
required, and it was shown (23) that the thylakoid protonmotive force
is likewise not required for efficient insertion. Thus, the absence of
any identifiable essential insertion factor suggests that this protein
may insert spontaneously into the thylakoid membrane, a possibility
also proposed for CFoII, PsbX, and PsbW (17-21). However,
as in these previous studies, we would caution that other, as yet
unidentified proteins could conceivably assist in the insertion process
and that further studies are therefore required to confirm or refute
this proposal. Nevertheless, the data are of general relevance because,
although PsbY may resemble the above single-span proteins in some
respects, it is far more complex in structural terms and is effectively
a multispanning protein at the point of insertion. SRP has been
implicated in the insertion mechanisms for a range of bacterial
proteins and one thylakoid membrane protein (4-7, 15), and one
bacterial SRP substrate has now been shown to use the Sec apparatus
(9). However, our data demonstrate quite clearly that a complex
multispanning membrane protein can insert with high efficiency in the
absence of either SRP or Sec machinery, and there is in our opinion a high probability that the insertion process is indeed spontaneous. These findings have important implications for the mechanism used by
SRP. Cross-linking studies on both the bacterial and chloroplast SRP
have revealed a marked preference for binding to highly hydrophobic regions (8, 34), and the chloroplast SRP was not observed to bind to
the less hydrophobic signal peptides of Sec- and
pH-dependent lumenal proteins (34). This observation is
certainly consistent with the known substrate specificity of SRP, and
it was suggested that the binding sites for SRP are the more highly
hydrophobic transmembrane spans of integral membrane proteins. However,
hydropathy analysis of PsbY and the LHCP that has been shown to insert
by the SRP-dependent pathway (15), using, for example, the
dense alignment surface (DAS) or TopPred II prediction methods (35, 36), suggests that the transmembrane segments of PsbY are at least as
hydrophobic as those of LHCP (not shown). This raises the possibility
that SRP may recognize a rather complex determinant that includes
features other than a particularly hydrophobic region. In the case of
PsbY, however, it is also of interest that insertion does not only
occur by the stromal factor/NTP-independent route. Insertion is clearly
stimulated by the presence of stroma and NTPs, and we speculate that a
proportion of the PsbY molecules may in fact be targeted by the SRP
pathway. Further studies should help to reveal why this highly
hydrophobic molecule can insert with high efficiency by either of two
very different mechanisms when LHCP is so completely dependent on both
SRP and NTPs.
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FOOTNOTES |
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* This work was supported by Biotechnology and Biological Sciences Research Council Grant C07900 (to C. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-1203-523557;
Fax: 44-1203-523701; E-mail: CG{at}dna.bio.warwick.ac.uk.
The abbreviations used are: SRP, signal recognition particle; LHCP, light-harvesting chlorophyll-binding protein; NTP, nucleoside triphosphate; TPP, thylakoidal processing peptidase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
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