psak paper
TRANSCRIPT
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M1:02914, revised version. July 7, 2001
Insertion of PsaK into the thylakoid membrane in a 'horse-shoe' conformation
occurs in the absence of signal recognition particle, nucleoside triphosphates or
functional Albino3
Alexandra Mant1*, Cheryl A. Woolhead1,2, Misty Moore3, Ralph Henry3, and Colin Robinson1
1Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom
2Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom
3Department of Biological Sciences, University of Arkansas, Fayetteville AR 72701
Corresponding author: Colin Robinson
Telephone: +44 2476 523557
Fax: +44 2476 523568
Email: [email protected]
*Present address
Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and
Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.
Running title: Sec/SRP/Alb3-independent insertion of PsaK
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on July 12, 2001 as Manuscript M102914200 by guest on February 6, 2018
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SUMMARY
The photosystem I subunit PsaK spans the thylakoid membrane twice, with the N- and C-termini
both located in the lumen. The insertion mechanism of a thylakoid membrane protein adopting this
type of topology has not been studied before, and we have used in vitro assays to determine the
requirements for PsaK insertion into thylakoids. PsaK inserts with high efficiency and we show that
one transmembrane span (the C-terminal region) can insert independently of the other, indicating
that a 'hairpin'-type mechanism is not essential. Insertion of PsaK does not require stromal extract,
indicating that signal recognition particle (SRP) is not involved. Removal of nucleoside
triphosphates inhibits insertion only slightly, both in the presence and absence of stroma, suggesting
a mild stimulatory effect of a factor in the translation system and again ruling out an involvement of
SRP or its partner protein, FtsY. We furthermore find no evidence for the involvement of known
membrane-bound translocation apparatus; proteolysis of thylakoids destroys the Sec and Tat
translocons but does not block PsaK insertion, and antibodies against the Oxa1/YidC homolog,
Alb3, block the SRP-dependent insertion of Lhcb1 but again have no effect on PsaK insertion.
Because YidC is required for the efficient insertion of every membrane protein tested in
Escherichia coli (whether SRP-dependent or -independent), PsaK is the first protein identified as
being independent of YidC/Alb3-type factors in either thylakoids or bacteria. The data raise the
possibility of a wholly spontaneous insertion pathway.
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INTRODUCTION
Studies in bacteria and plant thylakoids have demonstrated the operation of a complex signal
recognition particle (SRP)-dependent pathway for the insertion of membrane proteins. This pathway
was first demonstrated in bacteria (reviewed in [1]) where the insertion of several plasma membrane
proteins was shown to require the activity of two soluble/extrinsic proteins, SRP and FtsY.
Escherichia coli SRP is a complex comprising a 4.5S RNA molecule together with a homolog of
the 54 kDa subunit of eukaryotic SRPs, which binds to particularly hydrophobic regions such as
nascent or newly-synthesized membrane proteins. The SRP-substrate complex interacts, at least in
some cases, with the SecYEG translocon in the plasma membrane and, in a poorly-understood
sequence of events, the substrate is transferred into the translocon with the assistance of an
additional factor, FtsY [2-8]. Both SRP and FtsY are GTPases and the insertion process depends
totally on GTP hydrolysis.
A broadly similar pathway has been characterised for the insertion of the major light-harvesting
chlorophyll-binding protein, Lhcb1, into the thylakoid membrane of plant chloroplasts. This
conservation of insertion pathway is perhaps not surprising, given that chloroplasts are widely
accepted to have evolved from endosymbiotic cyanobacterial-type organisms. After import from the
cytosol, Lhcb1 binds stromal SRP to form a soluble targeting complex [9]. Integration of the
polytopic Lhcb1 protein into the thylakoid membrane further requires FtsY [10,11], GTP and a
membrane-bound translocase. The translocase has yet to be fully characterized; antibodies to SecY
strongly inhibit the insertion of SecA-dependent lumenal substrates but do not block the insertion of
Lhcb1 [12,13]. This raises the possibility that the thylakoid SecYEG complex is not required,
although this work could not rule out the possibility that the SRP interacts with a different SecY
determinant that is unaffected by antibody binding. Interestingly, the chloroplast SRP particle
differs from that of E. coli in that (i) no RNA is present and (ii) this SRP possesses a novel 43 kDa
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subunit [14], which binds to a novel SRP recognition element in Lhcb1 that is found in other
members of the light-harvesting chlorophyll a/b –binding protein family (LHC proteins) [15,16].
Nevertheless, there are clear parallels with the E. coli insertion pathway and the similar natures of
the pathways are reinforced by recent findings concerning the essential involvement of Oxa1-type
proteins. Oxa1 is a mitochondrial inner membrane protein that is involved in the biogenesis of a
range of mitochondrial inner membrane proteins, specifically those that insert from the matrix side
of the membrane [17, 18]. An Oxa1 homologue, termed Alb3, is also essential for the insertion of
Lhcb1 in thylakoids [13] and recent work has shown an E. coli homologue, YidC, to be essential for
the insertion of at least some SRP-dependent membrane proteins in this organism [19].
Critically, YidC is also essential for the biogenesis of E. coli membrane proteins that do not require
SRP or the Sec apparatus. The insertion mechanisms of M13 procoat and Pf3 coat protein have
been characterised in some detail [20] and shown to insert efficiently into the plasma membrane in
the complete absence of functional SRP, SecA or the membrane-bound SecYEG translocon. On the
basis of these data it had been suggested that these proteins may insert spontaneously into the
membrane, but depletion of YidC led to a rapid block in their insertion indicating a central role in
their insertion. Thus, it appears that one pool of YidC may be associated with the Sec apparatus [21]
while another pool may in effect represent a novel form of translocase dedicated for the insertion of
some, if not all, SRP-independent membrane proteins. Like M13 procoat, a subset of thylakoid
membrane proteins are synthesized with cleavable signal peptides but inserted in the absence of
SRP [22,23], but the possible involvement of the YidC homolog, Alb3, remains to be clarified.
All of the previous studies on thylakoid protein insertion have focused either on relatives of the
well-studied Lhcb1 (LHC proteins), or on proteins that bear cleavable N-terminal signal peptides. In
this report we have sought to characterise the insertion of a different type of thylakoid membrane
protein, PsaK, which is not synthesized with a cleavable signal peptide and which is unrelated to
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LHC proteins. We show that this protein inserts with high efficiency into thylakoids by a
mechanism that does not involve SRP, NTP hydrolysis or any of the known translocation
machinery in the thylakoid membrane, including Alb3.
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EXPERIMENTAL PROCEDURES
DNA Constructs
A full-length cDNA clone encoding the precursor of barley PsaK (pPsaK) in the plasmid
pBluescriptSK(-) was a gift from B. L. Møller [24]. A construct encoding the mature PsaK
protein was prepared by polymerase chain reaction amplification of the aforementioned barley
cDNA. The forward primer (5’-CAG GGG ATC CGC ATG GAC TAC ATC GGC-3’) introduced a
BamH I site between bp 287 and 288, and altered cysteine 42, which is the last amino acid of the
transit peptide, to an initiating methionine residue. The reverse primer (5’-CCC GAA GCT TGC
AGA ACA GCT ATG-3’) introduced a Hind III restriction site between bp 603 and 604, after the
stop codon at bp 565. The amplified region was cloned 5’ BamH I-Hind III 3’ into pGEM4Z, and
sequenced completely before being used as a template for transcription and translation. A cDNA
clone encoding pea pLhcb1 was provided by N.E. Hoffman [25], while the cDNA clone encoding
wheat p23K has been described [26].
Transcription and Translation
The plasmid encoding pPsaK was linearized with Xho I, whereas linearization was found to be
unnecessary for the plasmid encoding PsaK. Transcription in vitro was performed according to
Promega protocols, using either T3 RNA polymerase (for pPsaK), or SP6 RNA polymerase (for
PsaK, pLhcb1 and p23K). Radiolabeled proteins were prepared using a wheat germ lysate system
(Promega) in the presence of [35S] methionine (Amersham Pharmacia), also according to the
manufacturers’ instructions. The translation mixtures were treated with puromycin and centrifuged
prior to use in insertion assays, as described in Thompson et al. [27].
Import assays
Assays for the import of precursor proteins by intact pea chloroplasts and isolated thylakoid
membranes were essentially as described in [28] except that the light intensity was 300 µmol
photons m-2 s-1. The proton ionophore nigericin (Sigma) was dissolved in ethanol, and used at a
final concentration of 2 µM in the presence of 10 mM KCl; control samples were identical, except
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they contained an equivalent volume of ethanol instead of nigericin. Assays to measure the effect of
apyrase (Sigma, type VI) were carried out as described in [27]. 10 mM stock solutions of the non-
hydrolyzable ATP and GTP analogues, AMP-PNP and GMP-PNP (Sigma) were prepared in 10
mM Hepes, 5 mM MgCl2 and the pH adjusted to 7. When used, the final concentrations of these
analogues were 0.5 mM. Proteolysis of thylakoid membranes, prior to insertion assays, was as
described in [27] and Alb3 antibody-inhibition tests were as described in [13], except that the
thylakoids were incubated with anti-Alb3 antibodies for 2 h instead of 1 h.
Treatment of Thylakoid Membranes, Post Assay
In order to test if PsaK was correctly inserted in the thylakoid membrane (either after import into
intact chloroplasts, or after insertion into isolated thylakoid membranes), the membranes (between
10 and 20 µg chlorophyll, depending on the experiment) were washed with 0.5 ml ice-cold10 mM
Hepes-KOH pH 8, 5 mM MgCl2 (HM) and then reisolated by centrifugation at 18,000 g and 4°C for
5 min in a microcentrifuge. Next, they were washed with 100 µl 20 mM Tricine-NaOH, pH 8 (TB)
and reisolated as above, then subjected to one round of extraction with 6.8 M urea, using a protocol
adapted from Breyton et al. [28] and described in detail in [27]. Next, the membranes were
resuspended in TB and digested with 0.2 mg ml-1 trypsin (Sigma, type XIII) in a final volume of
100 µl for 30 min on ice. Trypsin digestions were stopped by the addition of 0.5 mg ml-1 trypsin
inhibitor (Sigma, type I-S), followed by centrifugation at 18,000 g and 4°C for 10 min in a
microcentrifuge. Finally, the thylakoid membranes were resuspended in 15 µl TB containing 5 µg
trypsin inhibitor, and an equal volume of 2 X protein sample buffer, then immediately boiled for 5
min. Insertion efficiencies were measured by exposing dried SDS-PAGE gels in phosphorimager
cassettes, followed by quantitation in a Molecular Dynamics PhosphorImager.
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RESULTS
Structures of the precursor and mature forms of PsaK
PsaK is a nuclear-encoded subunit of photosystem I that is important for the stable interaction of
LHCI with the photosystem I core complex (29). Barley PsaK is synthesized in the cytosol as a 13.7
kDa precursor, imported into the chloroplast and subsequently inserted into the thylakoid membrane
[24]. The presequence is a typical stroma-targeting peptide that is predicted to be removed by the
stromal processing peptidase (SPP). From sequence analysis, the 9 kDa mature protein is predicted
to span the membrane twice with the N- and C-termini both located in the lumen and the positively
charged loop region remaining on the stromal (cis) side of the thylakoid membrane according to the
‘positive-inside rule’ [30]. Recent high-resolution crystallographic analyses of Synechococcus
elongatus photosystem I have confirmed these predictions for the cyanobacterial PsaK, which is
highly similar to eukaryotic PsaK (P. Jordan, P. Fromme and N. Krauss, personal communication).
This 'horse-shoe' configuration is illustrated in Figure 1, which also indicates the locations of the
methionine residues in the mature barley protein (two in the first transmembrane region, one in the
second). The stroma-exposed loop region contains numerous positively-charged residues and
trypsin is therefore predicted to cleave in this region and generate two degradation fragments
containing the indicated numbers of methionine residues. The amino acid sequence of pPsaK is also
given in Fig. 1 together with the start site of a mature-size construct described below.
Insertion of PsaK does not require SRP or nucleoside triphosphates, but is stimulated by the ∆µH+
Figure 2 shows a chloroplast import experiment conducted with [35S]-methionine-labeled barley
pPsaK. The precursor protein (pPsaK) is imported into chloroplasts (lane C) where it is processed to
the mature form and resistant to added protease (lane C+); the mature protein has an apparent mass
of 7 kDa (in agreement with [24]) and is found exclusively in the thylakoid fraction (lane T). The
protein is highly resistant to urea extraction (lane Turea), which has been shown to be an effective
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means of identifying many membrane proteins [28]. Trypsin-treatment of the thylakoids from lane
T (lane 1) leads to the generation of two fragments, one of which contains precisely twice as much
[35S] radioactivity as the other (data not shown, from Phosphorimager analysis). On the basis of the
data described above, the stronger, lower band most likely represents the first transmembrane
region (TM1) and the other (TM2) is the C-terminal transmembrane span. If the thylakoids are
washed with urea after trypsin-treatment, a proportion of the fragments are extracted (lane 2)
whereas the same fragments observed in lane 1 are obtained if the membranes are urea-treated prior
to proteolysis (lane 3). Clearly, mature PsaK is far more resistant to urea-extraction than either of
the single-span degradation fragments and we believe that this reflects the ability of urea to partially
extract small, single-span proteins, whereas proteins containing two or more spans are almost
totally resistant. We have observed a similar phenomenon with single-span proteins bearing signal
peptides: the precursor proteins adopt loop conformations in the membrane that are far more
resistant to urea extraction than the single-span mature proteins [27].
The primary aim in this work was to analyse the mechanism by which PsaK inserts into the
thylakoid membrane, and for these experiments we used in vitro assays for the insertion of
radiolabeled protein into purified pea thylakoids. Our criteria for correct insertion were (i) that the
protein should be resistant to urea extraction and (ii) that trypsin should generate the two
degradation products observed after import of pPsaK as shown in Fig. 2. Fig. 3A shows the results
of incubating the pPsaK translation product (lane Tr) with thylakoids in the presence of stromal
extract: the mature protein is efficiently generated by SPP in the extract, a large proportion of
mature PsaK becomes associated with the thylakoids (lane T) and urea-extraction followed by
trypsin digestion produces the two diagnostic degradation products (TM1 and TM2). These results
effectively confirm that insertion is taking place. However, the full precursor protein is unsuitable
for detailed tests on the insertion requirements because stromal extract (which contains essentially
all of the SRP) can not be omitted since it also contains almost all of the stromal peptidase required
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to generate the mature form of PsaK. The N-terminus of mature PsaK is located in the lumen, which
means that processing to the mature size must precede insertion. We have found that the full
precursor form does not insert correctly in the absence of stromal extract (see below), which is
unsurprising because the entire presequence would have to be translocated across the thylakoid
membrane. Further experiments were therefore conducted with a mature-size PsaK translation
product synthesized from a truncated cDNA as described in Experimental Procedures.
Fig. 3B shows assays for the insertion of this construct (denoted PsaK) into isolated thylakoids.
After each assay, samples of the thylakoids were either treated with trypsin (upper panel) or urea-
extracted (lower panel). PsaK again inserts into isolated thylakoids and the inserted protein is again
converted to the diagnostic degradation products TM1 and TM2 ('+ trypsin' panel). As a control, we
incubated the translation product with trypsin in the absence of membranes (lane Tr+) and this
procedure leads to an almost complete degradation of PsaK, with no evidence for either the TM1 or
TM2 bands (providing further evidence that TM1 and TM2 represent membrane-integrated
regions). In this assay, TM1 should contain three times as much [35S]-Met as TM2 because of the
initiation methionine introduced during the cloning procedure, which is located in the lumen after
insertion and hence protected from proteolysis. In repeat assays such as those shown in Fig. 3B, the
TM1:TM2 35S-Met ratio was always near 3 (within 15% in each case) confirming correct insertion,
and this is supported by the observation that the mature protein is equally resistant to urea extraction
('+ urea' panel). We have routinely found that PsaK inserts with very high efficiency in this assay
system; 34% of translation product was found to be inserted in this particular experiment (based on
the recovery of 35S-Met in TM1 and TM2) and insertion efficiencies of over 40% have been
observed in other experiments (data not shown).
The left hand panel of Fig. 3B shows assays carried out in the presence of stromal extract (SE) or
Hepes-magnesium buffer (HM), and in each of these cases insertion was assayed either with or
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without pretreatment of the assay mixture with apyrase (indicated as +/- above the lanes). This
enzyme hydrolyzes nucleoside triphosphates in the mixture and totally blocks insertion by the SRP
route [9,11]. These lanes show that insertion is most efficient in the HM incubations, i.e. in the
absence of stromal extract (and hence SRP). The presence of apyrase does, however, lead to a
reduction in insertion efficiency (to 71% of the control value) but this is not due to SRP
involvement because the presence of stromal extract also leads to a slight reduction in insertion
efficiency (down to 81% of the HM value). Stromal extract contains essentially all of the SRP (see
below) and this result rules out an SRP involvement.
In the same experiment we also assessed the effects of including GMP-PNP and AMP-PNP (non-
hydrolyzable analogs of GTP and ATP, respectively) in an attempt to identify the cause of the
reduction observed with apyrase. GMP-PNP, in particular, is an effective inhibitor of SRP [11].
However, neither analog inhibits insertion to any marked extent, either in the absence or presence of
stromal extract (right hand panel in Fig. 3B).
As a control for these experiments we used insertion assays with Lhcb1, a known substrate for the
SRP pathway. After insertion into thylakoids, Lhcb1 becomes highly resistant to trypsin digestion
and a characteristic stable degradation product is generated [9,11-13]. Fig. 3C shows that insertion
is completely dependent on stromal extract, as shown by the appearance of the degradation product
(DP) in the SE panel. Insertion is also completely dependent on the presence of NTPs in the mixture
as pretreatment with apyrase (lanes indicated by +) totally blocks insertion, as found previously
[9,11]. On the basis of these data we conclude that PsaK does not require SRP for its insertion and,
because insertion is not even stimulated by the presence of stroma, we propose that insertion is
indeed completely independent of SRP. Insertion is, however, slightly inhibited by apyrase
treatment even in the absence of stromal extract, which indicates a mild stimulatory influence of an
ATP or GTP-hydrolyzing factor in the wheatgerm translation system. We believe the most likely
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explanation to be an anti-aggregation effect of chaperones in the wheatgerm extract which may
prevent some PsaK molecules from adopting an insertion-incompetent conformation.
The above studies rule out a central role for NTP hydrolysis in the insertion of PsaK but to obtain a
more comprehensive picture of the energy requirements we tested whether the thylakoidal proton
motive force stimulates insertion. Figure 4A shows the effect of nigericin (a proton ionophore) on
the import and sorting of pPsaK in intact chloroplasts. In the control assay, the protein is found
exclusively in the thylakoid fraction, and only as the mature form. With nigericin present, however,
some imported protein is found in the stromal fraction and it is notable that the primary stromal
form is the full precursor protein. Some mature PsaK is also found in the stroma, together with a
further, intermediate-size protein (iPsaK) that may represent a processing intermediate
(alternatively, this may result from proteolysis of pPsaK). From these data it is evident that optimal
insertion efficiency is dependent on the thylakoidal proton electrochemical gradient, ∆µH+. The
appearance of the full precursor protein furthermore suggests that pPsaK is not necessarily
processed immediately upon entry into the stroma, and we suggest in addition that at least some
molecules may be processed only during the later stages of the insertion pathway, since the
appearance of pPsaK most likely stems from an inhibition of insertion.
To obtain further data on the ∆µH+-dependence of insertion we conducted thylakoid insertion
assays with PsaK in the presence of nigericin (Fig. 4B). The data show that the presence of
nigericin (lanes N) reduces insertion efficiency to a moderate extent when compared with the
control samples shown in lanes C (in this experiment by 20-25%) in both the presence and absence
of stromal extract. Taken together, these data indicate that the ∆µH+ stimulates PsaK insertion but is
not essential.
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PsaK insertion does not require the thylakoidal Sec machinery or Alb3
The data shown in Figs. 3 and 4 exclude the involvement of SRP or NTPs in PsaK insertion but it is
equally important to understand whether membrane-bound translocation machinery is involved. The
question of Alb3 involvement is critical because the homologous YidC protein is required for the
efficient insertion of every E. coli membrane protein tested to date [19], and the SecYEG complex
is also a prime candidate since this translocon is also used for some membrane proteins in bacteria
(reviewed in [1]). We addressed these possibilities in two ways. Previous studies [31] have shown
that the insertion of Lhcb1 is totally inhibited by pretreatment of the thylakoids with trypsin, and the
same study showed that translocation of Sec-dependent lumenal proteins is also completely
blocked. This technique provides a simple means of destroying both the membrane-bound Sec
apparatus and inhibiting integration by the SRP pathway. In previous studies using this approach we
have maintained a ∆µH+ by driving the ATP synthase in reverse in the dark (the synthase is highly
resistant to trypsin) and we used the same method in this study since insertion of PsaK is stimulated
by the ∆µH+.
Fig. 5 shows experiments in which thylakoids were treated with trypsin, washed with buffer
containing trypsin inhibitor to remove protease, and then assayed for their ability to import pre-23K
(a substrate for the twin-arginine translocation, or Tat pathway), Lhcb1 and PsaK. The pre-23K
imports serve as a test for the establishment of the ∆µH+ since transport of this protein into the
lumen is completely dependent on the proton gradient [1], and the data show that in the absence of
trypsin treatment this protein is indeed imported with high efficiency and processed to the mature
size, in total darkness. This observation confirms the presence of a ∆pH, and import is abolished by
trypsin treatment which has been shown previously to inactivate the Tat system [31]. Insertion of
Lhcb1 is also completely inhibited by this treatment; very little Lhcb1 is found associated with the
thylakoids after the incubation (lane T of the 'Trypsin' panel) and essentially no resistant
degradation product is found after protease-treatment (lane T+) and other experiments (not shown)
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confirmed that import of a Sec substrate was also blocked. However, the upper panel shows that
PsaK still inserts into trypsin-treated thylakoids, which indicates that the Sec system is not required.
The specific question of Alb3 involvement was approached by more direct means. Pre-incubation of
thylakoids with polyclonal anti-Alb3 antibodies almost blocks Lhcb1 insertion without affecting the
Tat- or Sec-dependent pathways [13] and the same technique was used to test for its involvement in
PsaK insertion. Fig. 6A shows a control assay using Lhcb1, in which insertion was monitored after
incubation of the thylakoids with buffer (HM; as a control) with pre-immune antibodies (PI) or anti-
Alb3 antibodies. The pre-immune serum causes a slight inhibition of insertion (to 89% of the
control value) but the Alb3 antibodies reduce insertion efficiency to 27% of the control value. A
similar level of inhibition was observed previously [13]. In contrast, Fig. 6B shows that neither the
pre-immune nor the Alb3 antibodies affect insertion of PsaK into thylakoids, and the levels of urea-
resistant protein or TM1 or TM2 degradation products remain undiminished. These data clearly
indicate that PsaK is not dependent on Alb3 for insertion.
The C-terminal transmembrane span of PsaK can insert independently
The above data show that PsaK inserts by a relatively simple mechanism that does not rely on any
known translocation apparatus, including Alb3. This type of mechanism is highly unusual and we
have sought to obtain further details on the overall insertion mechanism. One possibility, proposed
for many membrane proteins [1], is that the two transmembrane spanning regions may form a
'helical hairpin' that is able to insert with high efficiency due to the simultaneous partitioning of two
hydrophobic regions. Several membrane proteins are known to form loop intermediates in which
the loop region is on the trans side of the membrane, and similar principles may operate for those
proteins, such as PsaK, where the loop remains on the cis side. We tested whether a single span of
PsaK can insert independently into the thylakoid membrane, by simply using the full precursor
protein instead of the mature PsaK construct, under conditions where cleavage by SPP is prevented.
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As explained above, the N-terminus of mature PsaK lies in the lumen, hence cleavage of the large
and highly charged presequence would appear to be essential before this N-terminal region can
translocate across the membrane. We therefore carried out thylakoid import assays under two
conditions: in the complete absence of stromal extract (using thylakoids that had been thoroughly
washed to remove residual SPP), and after protease-treating the thylakoids (to destroy any SPP on
the membrane surface). Mature-size PsaK was used as a control since this protein inserts under both
sets of conditions as shown above.
The data (Fig. 7) show that the mature-size PsaK ('PsaK' panel) behaves as in experiments shown
above and the TM1 and TM2 fragments again appear with a labeling ratio which was calculated to
be close to 3:1. Insertion occurs with both the washed and protease-treated thylakoids. However,
very different results are obtained when the full precursor protein is used ('pPsaK' panel). The upper
degradation fragment (TM2) is again observed after insertion into either washed or protease-treated
thylakoids but the lower band (TM1) is now completely absent. A low-intensity smear of label is
present beneath the TM2 band, presumably due to degradation of non-inserted PsaK regions, but no
band is present in the TM1 region. We conclude from this result that the N-terminal transmembrane
span is indeed unable to insert when the presequence is present, as predicted above, but the clear
presence of TM2 is strong evidence that this region is able to insert independently under these
conditions. These data also serve to reinforce the efficacy of the in vitro assay because they provide
a third line of evidence that the bands denoted TM1 and TM2 do indeed represent inserted
transmembrane regions; the N-terminal hydrophobic region is clearly highly susceptible to
proteolysis (or is simply removed when the thylakoids are washed after the insertion reaction) when
not inserted in the thylakoid membrane.
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DISCUSSION
Several thylakoid membrane proteins have been previously analyzed in terms of insertion
mechanism, and in this respect they fall into two broad categories. Lhcb1 follows a complex
pathway involving the input of numerous factors, both in the stroma and at the membrane surface,
while several signal peptide-bearing proteins use an apparently simpler insertion mechanism that
does not rely on any of the known protein machinery, although the issue of Alb3 involvement has
yet to be addressed. PsaK is unlike any of the above proteins in that both the N- and C-termini are
transported to the lumen, the protein is not synthesized with a signal-type peptide and it is not a
member of the LHC super-family of proteins.
The data from this study all point to a strictly SRP-independent insertion mechanism. Stromal
extract contains essentially all of the SRP but is not required at any stage, and insertion does not
depend at all on NTP hydrolysis. Both of these factors are critical for Lhcb1 insertion. FtsY
involvement can also be ruled out since this factor hydrolyzes GTP during its operating mechanism.
We can not rule out the possibility that other, as yet unidentified soluble factors may assist PsaK
insertion, and the slight inhibitory effect of apyrase does raise the possibility that proteins in the
wheatgerm translation system (eg chaperones) may aid insertion, but the data nevertheless indicate
that the insertion of PsaK is fundamentally different from that of Lhcb1. It is as yet unclear why
Lhcb1 is so dependent on SRP activity whereas other thylakoid membrane proteins studied to date
are not.
We also find no evidence for the involvement of membrane-bound translocation machinery in PsaK
insertion. Several studies have shown that trypsin treatment blocks the translocation of lumenal Sec
substrates, very strongly suggesting that the Sec apparatus is inactivated. This treatment slightly
inhibits PsaK insertion (as indeed it does for PsbY, [24]) but the effect is not marked and we
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conclude that the Sec translocon does not play a major role in this pathway. We also find no
evidence for Alb3 involvement, since antibodies raised against Alb3 severely inhibit Lhcb1
insertion yet have no effect on the insertion of PsaK. On the basis of these data alone we can not
exclude the possibility that PsaK may interact with Alb3 in a manner which is not affected by the
antibodies used in this study. However, other ongoing studies in this lab (not shown) have
demonstrated that Alb3 is completely degraded when thylakoids are treated with trypsin under the
conditions used in this study (eg in Figs. 5 and 7) and, since this treatment does not affect PsaK
insertion at all, we conclude that Alb3 is not required for PsaK biogenesis. This finding is
significant because the homologous YidC protein plays a central role in the insertion of several
SRP/Sec-independent membrane proteins in E. coli [19]. This factor has come to be regarded as a
novel form of translocase in its own right, since the related Oxa1 protein also plays an important
role in membrane protein biogenesis in yeast [17,18] and there is no evidence as yet for additional
subunits in the mirochondrial Oxa1p complex. Our data thus indicate that PsaK is unique (to date)
because it is the only Alb3/YidC-independent membrane protein among those analyzed in bacteria
and plant thylakoids
In general, the topology adopted by PsaK is consistent with the ‘positive-inside’ rule [30] which
states that positively-charged residues are found more frequently on the cis side of the membrane.
The stroma-exposed loop region contains 4 basic residues whereas the C-terminal lumenal tail
contains only one and the N-terminal region does not contain any [24]. However, it is presently
unclear why insertion is stimulated to some extent by the thylakoidal ∆pH. In bacteria and
mitochondria, the insertion of many membrane proteins is stimulated by the proton motive force
[1,17,18] and the same applies to the Alb3-dependent insertion of Lhcb1 in thylakoids [9,11,12].
However, these effects probably reflect the harnessing of the ∆µH+ by the translocation machinery
(the Sec and/or Oxa1-type apparatus) and these factors are not required for PsaK insertion. Possibly,
there is a mildly stimulatory electrophoretic effect on the translocation of the C-terminal region of
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PsaK, which contains a single acidic residue. However, this point remains to be investigated in
detail.
The Sec/Alb3-independent nature of the insertion mechanism raises the strong possibility that
insertion of PsaK occurs spontaneously upon reaching the thylakoid membrane, and such
mechanisms have been postulated before on theoretical grounds and following studies on
membrane-interactive peptides and toxins (reviewed in [32]). However, further work is required to
address this possibility, and one argument against this possibility is that PsaK may then be able to
interact with other membranes (eg the envelope) in the absence of a specific and dedicated targeting
system. Possibly, PsaK is predisposed to insert only into thylakoid-type lipids, and the thylakoid
membrane is indeed very unusual in terms of lipid composition, being composed primarily of
galactolipids which are chemically very different to phospholipids (reviewed in [33]). Further work
is certainly required to determine whether such a lipid-based sorting process operates for PsaK
insertion, or whether novel forms of translocation apparatus are involved.
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Acknowledgements
This work was supported by an Engineering and Physical Sciences Research Council studentship to
C.W. and EPSRC Biosciences Interface Network grant GR/M91105 to A.R. and C.R., and by
Biotechnology and Biological Sciences Research Council grant C07900 to C.R. and by National
Science Foundation grant MCB-9807826 to R. H.
Abbreviations
AMP-PNP (5’adenylylimidodiphosphate); GMP-PNP (5’guanylylimidodiphosphate); LHC protein
(light-harvesting chlorophyll a/b-binding protein); SDS-PAGE (SDS polyacrylamide gel
electrophoresis); Tricine (N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine).
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[33]. Douce, R. and Joyard, J. (1996). In D.R. Ort, C.F Yocum, eds. Advances in Photosynthesis,
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Figure Legends
Figure 1 Schematic diagram of the ‘horse-shoe’ topology adopted by PsaK.
The protein forms two transmembrane alpha helices, with both N- and C-termini in the thylakoid
lumen. The positively-charged loop region on the stromal side of the membrane is accessible to the
protease trypsin, yielding two degradation products corresponding to the bulk of the two
transmembrane helices and their small lumenal tails. Recent crystallographic data indicate that one
of the transmembrane spans of cyanobacterial PsaK crosses the membrane at a tilted angle (N.
Krauss, personal communication), and therefore may form a more open ‘horse-shoe’ than
represented here. The Figure also shows the complete amino acid sequence of pPsaK with the
transit peptide (T. peptide) given on the upper line and the transmembrane domains underlined and
methionine residues shown in bold. Potential trypsin-sensitive basic residues in the stromal loop
region are shown in bold italics. The sequence of the mature PsaK construct is also indicated (with
the introduced initiation methionine shown in parentheses).
Figure 2. Barley pPsaK is efficiently imported by intact pea chloroplasts.
12.5 µl in vitro-translated pPsaK (lane Tr) was incubated with intact pea chloroplasts equivalent to
50 µg chlorophyll for 20 min in the light. After the incubation, chloroplasts were washed, reisolated
and fractionated, then analyzed by SDS-PAGE and fluorography. Lane C, total washed
chloroplasts; lane C+, thermolysin-treated chloroplasts; lane S, stromal extract; lane T, thylakoid
membranes; lane Turea, thylakoid membranes subjected to severe washing with 6.8 M urea/20 mM
Tricine-NaOH, pH 8; lane 1, trypsin-digestion of the thylakoid membranes from lane T; lane 2, the
proteolysed membranes from lane 1 subjected to urea-extraction; lane 3, the membranes from lane
Turea digested with trypsin; pPsaK, the precursor of PsaK, PsaK, the mature protein; TM1 and
TM2, the degradation products corresponding to transmembrane spans 1 and 2 of the mature protein
respectively.
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Figure 3 Insertion of pPsaK and PsaK into isolated pea thylakoids.
A: 5 µl in vitro-translated pPsaK (lane Tr) was incubated with isolated pea thylakoids equivalent to
20 µg chlorophyll for 20 min in the light. After the incubation, the thylakoids were washed with
HM and TB (see Experimental Procedures) and then urea-extracted, before digestion with trypsin
(lane T+). Samples were analysed by SDS-PAGE and fluorography.
B: 5 µl in vitro-translated PsaK (lane Tr) was incubated with isolated pea thylakoids (20 µg
chlorophyll) in the presence of stromal extract (SE), in the absence of stromal extract (HM), in the
absence (-) or presence (+) of 2 U apyrase, and in the presence of 0.5 mM AMP-PNP or 0.5 mM
GMP-PNP (as indicated above the lanes). After a 20 minute incubation in the light, the membranes
were washed with HM and TB, before being subjected to extraction with urea (lower panel, +Urea),
the urea-resistant protein being denoted as PsaK. Next the urea-washed membranes were digested
with trypsin (upper panel, + Trypsin), where the characteristic degradation products are denoted by
TM1 and TM2. An aliquot of the translation mixture alone was also digested with trypsin (lane
Tr+). The samples were analysed by SDS-PAGE and fluorography. Insertion efficiencies (beneath
the lower panel, quoted as %), relative to the HM buffer control (100%) were calculated by using a
phosphorimager to measure the densities of the urea-resistant mature PsaK bands.
C: As a control, pea pLhcb1 (lane Tr) was translated in vitro and incubated with isolated pea
thylakoids, in the absence (HM) or presence (SE) of stromal extract, and in the absence (-) or
presence (+) of 2 U apyrase, for 20 min in the light. After the incubation, the thylakoids were
washed with HM and TB, before being subjected to both urea-extraction and digestion by trypsin,
which yielded the normal degradation product (marked DP) when correct insertion occurred. An
aliquot of the translation mixture alone was also digested with trypsin (lane Tr+).
Figure 4. Insertion of PsaK is stimulated by the thylakoidal ∆µH+.
4A. Intact pea chloroplasts equivalent to 50 µg chlorophyll were incubated with 12.5 µl in vitro-
translated pPsaK (lane Tr) in the absence (Control) and presence (+ Nigericin) of the proton
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ionophore nigericin (at 2 µM final concentration), for 20 min in the light. After the import
incubation, the chloroplasts were washed, fractionated and analysed by SDS-PAGE and
fluorography. Lanes C, total washed chloroplasts; lanes C+, thermolysin-treated chloroplasts; lanes
S, stromal extract (prepared, as always, in the presence of 10 mM EDTA, to prevent residual
thermolysin activity from degrading any stromal intermediates); lanes T, thylakoid membranes;
lanes T+, trypsin-treated thylakoid membranes; pPsaK, precursor of PsaK; iPsaK, an intermediate
form of PsaK; PsaK, mature protein; TM1 and TM2, degradation products corresponding to
transmembrane spans 1 and 2 respectively.
4B.Isolated pea thylakoids equivalent to 20 µg chlorophyll were incubated with 5 µl in vitro-
translated PsaK (lane Tr), in the presence (SE) and absence (HM) of stromal extract, and in the
presence (N) and absence (C) of 2 µM proton ionophore nigericin, for 20 min in the light. After the
insertion incubation, the membranes were washed with HM and TB, before being extracted with
urea (upper panel, +Urea). After urea-extraction, the membranes were digested with trypsin (lower
panel, + Trypsin). Urea-resistant mature protein is marked PsaK, while the protease degradation
products corresponding to transmembrane spans 1 and 2 are marked TM1 and TM2 respectively.
After the experiment the samples were analysed by SDS-PAGE, and insertion efficiencies (shown
below the lower panel, in %) measured by a phosphorimager to calculate the amounts of urea-
resistant mature PsaK relative to the control sample (HM, C).
Figure 5. Pre-digestion of the thylakoid membranes with trypsin does not prevent insertion of PsaK.
Thylakoid membranes were digested with 60 µg/ml trypsin (Trypsin) or buffer (Control) and the
chloroplast ATPase was subsequently activated to generate a ∆pH in the dark, as described in detail
in reference 31. The activated membranes (20 µg chlorophyll) were incubated with 5 µl in vitro-
translated PsaK, pLhcb1 or p23K (lanes Tr) in the dark for 30 min, with all manipulations being
carried out under a dim, green, safe light. After the incubation, the membranes were washed with
HM and reisolated. PsaK and Lhcb1 samples were washed further with TB, before being subjected
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to urea-extraction (lanes T) and then trypsin-digestion (lanes T+). 23K samples were analysed
directly after washing with HM (lanes T) or after digestion with 0.2 mg/ml thermolysin for 40 min
on ice (lanes T+). All samples were analysed by SDS-PAGE and fluorography. The insertion
efficiencies of PsaK (values in %, relative to the control sample) were measured using a
phosphorimager. PsaK, mature protein; TM1 and TM2, trypsin degradation fragments
corresponding to transmembrane helices 1 and 2 of PsaK; pLhcb1, precursor of Lhcb1; DP, trypsin
degradation fragment of inserted Lhcb1; p23K, precursor of 23K; 23K, mature protein.
Figure 6. Anti-Alb3 antibodies inhibit the insertion of Lhcb1 but not PsaK. Isolated pea thylakoids
were pre-incubated with anti-Alb3 antibodies (Alb3), pre-immune serum (PI) or import buffer
(HM) for 2h on ice, as detailed in reference 15. After this period the thylakoids were incubated with
pLhcb1 or PsaK as indicated and analyzed by protease treatment as described in Fig. 3.
Figure 7. Independent insertion of the C-terminal transmembrane span of pPsaK. The full precursor
of PsaK (pPsaK) or the mature-size construct (PsaK) were incubated either with thylakoids that had
been washed 3 times in HM buffer to remove stromal extract (denoted as 'Thyl') or with thylakoids
that had been treated with 0.15 mg/ml proteinase K for 30 min on ice and then washed 3 times with
HM buffer (denoted as 'PK-Thyl'). After incubation, samples of the thylakoids were analysed
directly (-) or after trypsin treatment as in previous Figures (+). Lanes Tr: translation products.
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Figure 1.
Stroma
Thylakoid membrane
Thylakoidlumen
+ Trypsin 0.2 mg/ml
N C NC
TM1 TM1TM2 TM2
Radiolabeled methionine residue
TM Transmembrane span
++
+ -
-
-
+
T. peptide: MASQLSAMTSVPQFHGLRTYSSPRSMATLPSLRRRRSQGIRCPsaK: (M)DYIGSSTNLIMVTTTTLMLFAGRFGLAPSANRKATAGLKLEA RESGLQTGDPAGFTLADTLACGAVGHIMGVGIVLGLKNTGVLDQIIG
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Figure 2
- 6.2
- 8.2
- 16.9- 14.4
- 10.8
Tr C C+ S T Turea 1 2 3
pPsaK -
PsaK -
TM2TM1
kDa
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Figure 3
Tr T T+
Tr Tr+ - + - +
SE HM GMP-PNP AMP-PNP------------ ------------ ------------- ------------
SE HM SE HM
- TM2- TM1
pPsaK -
PsaK -
PsaK -
+ Trypsin
+ Urea
A B
Efficiency: 83 41 100 71 117 109 93 85
pLhcb1 -
- DP
- + - +Tr Tr+-------- --------- HM SEC
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Figure 4A
Tr C C+ S T T+ C C+ S T T+------------------------------- -----------------------------
Control + Nigericin
- pPsaK
- PsaK
- TM2- TM1
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Figure 4B
Tr N C N C------------- -------------
SE HM
- PsaK
- TM2- TM1
+ Urea
+ Trypsin
Efficiency: 42 55 75 100
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Figure 5
pLhcb1 -
p23K -
- 23K
- DP
PsaK -- TM2- TM1
Tr T T+ T T+ --------- -------- Control Trypsin
Efficiency: 100 70
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Figure 6
Tr. HM PI Alb3 HM PI Alb3------------------ ---------------------Washed Prot. Treated
100% 89% 11%
- DP
Tr. HM PI Alb3 HM PI Alb3------------------ ---------------------Washed Prot. Treated
100% 93% 109%
- TM2- TM1
pLhcb1 -
PsaK -
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Figure 7
- PsaK
- TM2- TM1
pPsaK -
Tr - + - + Tr - + - +
--------------------- ----------------------pPsaK PsaK
Thyl: + + + +PK-thyl: + + + +
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Alexandra Mant, Cheryl A. Woolhead, Misty Moore, Ralph Henry and Colin Robinsonfunctional Albino3
occurs in the absence of signal recognition particle, nucleoside triphosphates or Insertion of PsaK into the thylakoid membrane in a 'horse-shoe' conformation
published online July 12, 2001J. Biol. Chem.
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