Báo cáo Y học: Unique structural determinants in the signal peptides of ‘spontaneously’ inserting thylakoid membrane proteins pptx

Unique structural determinants in the signal peptides
of ‘spontaneously’ inserting thylakoid membrane proteins
Christophe Tissier, Cheryl A. Woolhead and Colin Robinson
Department of Biological Sciences, University of Warwick, Coventry, UK
A series of thylakoid membrane proteins, including PsbX,
PsbY and PsbW, are synthesized with cleavable signal pep-
tides yet inserted using none of the known Sec/SRP/Tat/
Oxa1-type insertion machineries. Here, we show that,
although superficially similar to Sec-type signal peptides,
these thylakoidal signal peptides contain very different
determinants. First, we show that basic residues in the
N-terminal domain are not important, ruling out electro-
static interactions as an essential element of the insertion
mechanism, and implying a fundamentally different target-
ing mechanism when compared with the structurally similar
M13 procoat. Second, we show that acidic residues in the
C-domain are essential for the efficient maturation of the
PsbX and PsbY-A1 peptides, and that even a single substi-
tution of the )5 Glu by Val in the PsbX signal peptide
abolishes maturation in the thylakoid. Processing efficiency
is restored to an extent, but not completely, by the highly
hydrophilic Asn, implying that this domain is required to be
hydrophilic, but preferably negatively charged, in order to
present the cleavage site in an optimal manner. We show that
substitution of the PsbX C-domain Glu residues by Val leads
to a burial of the cleavage site within the bilayer although
insertion is unaffected. Finally, we show that substitution of
the Glu residues in the lumenal A2 loop of the PsbY poly-
protein leads to a block in cleavage on the stromal side of the
membrane, and present evidence that the PsbY-A2 signal
peptide is required to be relatively hydrophilic and unable to

adopt a transmembrane conformation on its own. These
data indicate that, rather than being merely additional
hydrophobic regions to promote insertion, the signal pep-
tides of these thylakoid proteins are complex domains with
uniquely stringent requirements in the C-domain and/or
translocated loop regions.
Keywords: chloroplast biogenesis; membrane protein inser-
tion; signal peptides; thylakoid.
Most chloroplast thylakoid proteins are nuclear-encoded in
plants and are therefore inserted into the membrane post-
translationally after import from the cytosol (reviewed in
[1,2]). Many of these proteins are synthesized with an
N-terminal ÔtransitÕ peptide that mediates interaction with
the envelope-localized import machinery and transport into
the stroma, after which this presequence is removed by the
stromal processing peptidase. In these cases, insertion into
the thylakoid membrane involves targeting determinants
located in the mature protein, and this has been experi-
mentally confirmed for one thylakoid protein, the major
light-harvesting chlorophyll-binding protein, Lhcb1 [3,4].
Further studies on this protein have shown it to integrate
using a complex pathway involving stromal signal recogni-
tion particle (SRP), FtsY, GTP and membrane-bound
translocation machinery that includes the protein Albino3
[5–9]. The Alb3 protein is related to the Oxa1p and YidC
proteins that play important roles in the insertion of
proteins into the bacterial plasma membrane and mitoch-
ondrial inner membrane, respectively [10,11]. In general, this
insertion pathway resembles that used by at least some
plasma membrane proteins in bacteria, which also involves

SRP and FtsY (reviewed in [12]). This is perhaps unsur-
prising as chloroplasts are widely accepted to have evolved
from endosymbiotic cyanobacteria.
Other thylakoid membrane proteins are inserted by a
different pathway that contrasts markedly with the highly
complex SRP-dependent pathway. The proteins PsbX,
PsbW, CFoII and PsbY are synthesized with N-terminal
bipartite presequences in which the first domain specifies
import from the cytoplasm across the chloroplast envelope,
after which it is removed by a stromal processing peptidase.
The second domain resembles typical ÔsignalÕ peptides,
containing three distinct domains: an N-terminal charged
region (N-domain), hydrophobic core region (H-domain)
and more polar carboxyterminal region (C-domain) ending
with an Ala-Xaa-Ala consensus region. Signal peptides
usually specify translocation by Sec-type translocation
systems in the endoplasmic reticulum, bacterial plasma
membrane or thylakoid membrane, and have been studied
in detail in these systems (reviewed in [13,14]). However,
these thylakoid proteins have been shown to insert into the
thylakoid membrane in the absence of SRP, SecA, nucleo-
side triphosphates or (dpH, thereby excluding all the ÔassistedÕ
modes of insertion into thylakoids reported to date [15–17].
Furthermore, proteolysis of thylakoids destroys the mem-
brane-bound Sec and twin-arginine translocase machineries
but has no effect on the insertion of these proteins [18]. In
the absence of identifiable translocation factors it has been
suggested that these proteins insert spontaneously into the
thylakoid membrane. A similar mechanism was originally
proposed for M13 procoat, which is also synthesized with a

Correspondence to C. Robinson, Department of Biological Sciences,
University of Warwick, Coventry CV4 7AL, UK,
Fax: + 44 2476523701, Tel.: + 44 2476523557,
E-mail:
Abbreviations: SRP, signal recognition particle, SPP, stromal
processing peptidase, TPP, thylakoidal processing peptidase.
(Received 10 January 2002, revised 9 April 2002,
accepted 19 April 2002)
Eur. J. Biochem. 269, 3131–3141 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02943.x
cleavable signal peptide and which inserts into the Escheri-
chia coli plasma membrane by an SRP/Sec-independent
pathway. However, this protein is now known to be highly
dependent on YidC for insertion [11], whereas recent studies
have shown that PsbX, PsbW and PsbY do not rely at all on
the thylakoidal YidC homolog, Alb3 [19]. Because none of
the known thylakoidal protein transport machinery is
required for the insertion of these proteins, it has been
suggested that they may insert spontaneously into the
thylakoid membrane.
The initial stages of this spontaneous insertion mechan-
ism involve binding of the intermediate-size protein to the
membrane, after which both hydrophobic domains (one in
the signal peptide, the other in the mature protein) insert
into the membrane and form a transmembrane loop
intermediate [20]. As a result, the hydrophilic, negatively
charged domain is translocated across the membrane to the
lumen where further processing by a thylakoid processing
peptidase (TPP) removes the remaining presequence leaving
the mature protein inserted in the membrane with a lumenal
N-terminus and a stromal C-terminus. The presence of the

hydrophobic signal peptide has been shown to be essential
inthecaseofCF
o
II [21] but the important features within
this class of signal peptide have not been studied in any
systematic manner and, because Sec- and Tat-type signal
peptides interact with proteinaceous binding sites, it is
possible that these thylakoid signal peptides may possess
unique characteristics that are essential for their correct
functioning. In this study we have analyzed the importance
of charged residues in the insertion and proteolytic
processing of PsbX, PsbW and PsbY. We show that basic
residues in the N-domain are not important for either
process whereas acidic residues in the C-domains of several
of the signal peptides play important roles in the processing
of precursor forms to the mature size. These requirements
are completely unlike those of M13 procoat, which also
bears a signal peptide, or the Sec-type signal peptides of
translocated proteins.
MATERIALS AND METHODS
Construction and expression of truncated pre-PsbW
proteins
A cDNA clone encoding the precursor form of Arabidopsis
PsbW, pPsbW [16] was amplified using inverse PCR to
generate intermediate-size and ÔshortÕ (see Results section)
versions truncated at the N-terminus (iPsbW and sPsbW).
For iPsbW, the forward and reverse primers were ATGGG
TAAGAAGAAGGGAGGA and TCTCTTTGCTCGGA
CGCG, respectively. For sPsbW, the forward and reverse
primers were ATGGAGACAAAGCAAGGAAAC and

TCTCTATTTGCTCGGACGCG. All constructs were
synthesized in vitro by transcription of cDNAs followed
by translation in a wheat germ lysate (Promega Biotech) in
the presence of [
35
S]methionine.
Mutagenesis of pPsbX and pPsbY
cDNA clones encoding Arabidopsis pPsbX [22] and pPsbY
[23] were subjected to site-specific mutagenesis using the
Stratagene Quikchange
TM
kit according to the manufac-
turer’s instructions. All mutants were fully sequenced to
verify the mutagenesis results, and the precursor proteins
were synthesized as described above, except that the PsbX
data were obtained using [
3
H]leucine, as were some of the
pPsbY data (see text).
Import reactions
Chloroplast import reactions were carried out using intact
pea chloroplasts from 8- to 9-day-old-seedlings as described
previously [22,23]. Urea washes were carried out as accord-
ing to [19] using a method modified from that detailed in
[24]. For time course analysis, precursor proteins were
imported into chloroplasts for 10 min, after which the
organelles were centrifuged (1 min in a microcentrifuge),
and the pellet resuspended in 1 mL of import buffer (50 m
M
Hepes/KOH, 330 m

M
sorbitol) and further incubated.
Sonication studies were carried out using a Branson 1210
water bath sonicator at 0 °C. Thylakoid import reactions
were carried out as in [16], after which samples were
analyzed immediately or after washing twice with 1 mL
20 m
M
Hepes/KOH, 5 m
M
MgCl
2
.
RESULTS
Electrostatic interactions are not essential in the early
stages of the PsbW insertion process
Studies on M13 procoat (reviewed in [12]) have demonstra-
ted the importance of electrostatic interactions between
basic residues in the protein and the negatively charged
membrane surface. These data indicated that basic residues
were essential in both the extreme N-terminal region of the
signal peptide and the C-terminal region of the mature
protein. Removal of either set of charges led to a block in
insertion, strongly indicating that the electrostatic interac-
tions were required during the early stages of the insertion
process, probably to bind the protein stably to the
membrane surface. pPsbW resembles procoat in several
respects, as detailed above, and similarly forms a loop
intermediate during insertion [20] but the early stages of the
insertion process are poorly understood and it is unclear

how this protein binds to the thylakoid membrane prior to
insertion. The thylakoid membrane is also negatively
charged due to the presence of sulfolipids and an electro-
static interaction seemed possible. However, although the
N-terminal region of the pPsbW signal peptide is positively
charged, the C-terminal region of mature PsbW is devoid of
basic residues, ruling out an electrostatic interaction with the
membrane surface. This region is in fact highly negatively
charged due to the presence of a series of five acidic residues
(see Fig. 1A). This protein is thus an attractive model
system in which to address this issue because we needed only
to test the importance of the basic residues in the N-terminal
region. This was achieved by simply truncating the protein
to remove increasing numbers of basic residues.
The overall structure of pPsbW is shown in Fig. 1. The
first Ôenvelope transitÕ domain has been omitted from the
pPsbW sequence which starts at residue 31. The precise site
of cleavage by the stromal processing peptidase has yet to be
identified but the likelihood is that it lies just before or after
(or within) the KKK sequence in the N-terminal region of
the signal peptide. Irrespective of the precise site of cleavage,
the N-terminal domain is positively charged and we reduced
3132 C. Tissier et al. (Eur. J. Biochem. 269) Ó FEBS 2002
the overall charge in this region by synthesizing an
intermediate-size protein (iPsbW) that lacks the envelope
transit domain and a smaller protein (sPsbW) that lacks any
basic residues in the signal peptide.
The effects of the truncations were tested by assaying for
the insertion of in vitro synthesized proteins into isolated
thylakoids. Because the TPP active site is on the lumenal

face [25], maturation is clear evidence of insertion and
Fig. 1B shows that all of the proteins insert into pea
thylakoids and become processed to the mature size. The
truncated proteins insert with slightly lower efficiencies
(sPsbW insertion efficiency is down to 45% of that of wild-
type protein) but the truncations, clearly, by no means block
insertion. It should be noted that even the sPsbW form still
carries a single positive charge at its N-terminus, due to the
protonated amino group. Nevertheless, we conclude that
electrostatic interactions are not as important for PsbW
insertion as for procoat insertion.
The translocated loop regions of spontaneously-inserting
proteins contain negative charges in either the mature
protein or the signal peptide
Four thylakoid membrane proteins have been shown to be
synthesized with cleavable signal peptides but inserted by
spontaneous mechanisms, and comparison of the translo-
cated loop regions shows that they are all negatively charged
(Fig. 2A). In the cases of PsbW and CF
o
II, the charges lie in
the N-terminal region of the mature protein, but PsbX
differs in that two Glu residues are located in the C-terminal
region of the signal peptide. The polyprotein, PsbY, also
contains acidic residues in this region of each signal peptide.
Other types of signal peptide (e.g. those specifying Sec-
or Tat-dependent translocation) rarely contain negative
charges in the C-terminal region and we considered it
possible that this feature may have evolved in the PsbX/
PsbY signal peptides in order to enhance the overall

insertion process. Constraints on the functions of the
mature proteins may have precluded the presence of acidic
residues in the N-terminal regions of the mature proteins.
Accordingly, we sought to test whether this characteristic is
important in the spontaneous insertion process by making
site-specific mutations in the signal peptides, focusing
primarily on PsbX as a simple model system but then
extending the studies to encompass PsbY. The mutations
are shown in Fig. 2B. In brief, the Glu residues were
substituted by hydrophobic residues such as Val, or by
highly hydrophilic but neutral residues such as Asn
(attempts to replace one of the Glu residues by Gln were
unsuccessful, for unknown reasons). The importance of net
charge in the loop region was also tested.
Acidic residues in the signal peptide are important
for efficient maturation of pPsbX.
As an initial test for the importance of the two Glu residues
(positions )5and)2, relative to the TPP cleavage site) we
Fig. 2. Primary sequences of signal peptides and translocated regions
within spontaneously-inserting proteins, and structures of PsbX mutants.
(A) The figure shows the sequences of the signal peptides and
N-terminal regions of the mature proteins of Arabidopsis PsbX,
Arabidopsis PsbW and spinach CF
o
II. Also shown are the two signal
peptides within the Arabidopsis PsbY polyprotein and the N-terminal
regions of the two single-span mature proteins generated after insertion
and processing (PsbYA1 and PsbYA2). TPP cleavage sites are denoted
by asterisks and hydrophobic regions are shown underlined. (B)
Sequences of PsbX mutants generated in this study. The hydrophobic

regions in the signal peptides and mature protein are shown under-
lined. The sequence of the wild-type protein (WT) is given at the top;
the )5and)2 (relative to TPP cleavage site) Glu residues targeted for
mutagenesis and the nomenclatures of the mutants reflect the residues
present at these positions. The efficiency of cleavage by TTP is given in
the right hand column, calculated according to the ratio of interme-
diate: mature protein in the total chloroplast samples (lanes C in Figs 3
and 4).
Fig. 1. N-terminally truncated pPsbW constructs insert into isolated
thylakoids. (A) The primary sequence of Arabidopsis thaliana pPsbW is
shown, starting from Leu31. The TPP cleavage site is denoted by an
asterisk and the hydrophobic regions in the signal peptide and mature
protein are underlined. Note that the site of cleavage by stromal pro-
cessing peptidase is not known. Basic residues in the N-terminal region
of the signal peptide are shown in bold, as are a series of five acidic
residues in the extreme C-terminal region of the mature protein. Shown
underneath the pPsbW sequence are the truncated presequences of an
intermediate-size PsbW construct (iPsbW) and a shortened construct
lacking all basic residues in the signal peptide (sPsbW). (B) pPsbW,
iPsbW and sPsbW were synthesized in vitro by transcription–transla-
tion (lanes Tr) and incubated with isolated pea thylakoids. After
incubation, samples were analyzed of the thylakoids either directly
(lanes T) or after washing as detailed in Materials and methods (lanes
W). The mobility of mature-size PsbW is indicated by open arrowhead,
precursor forms by closed arrows.
Ó FEBS 2002 Signal peptides of thylakoid membrane proteins (Eur. J. Biochem. 269) 3133
made a mutant in which both were substituted by Val. The
import and sorting characteristics of this mutant, PsbX/VV,
and wild-type PsbX were analyzed by incubating the
precursor proteins (PsbX/VV and pPsbX, respectively) with

intact chloroplasts and subsequently determining the intra-
organellar locations and cleavage products (shown in
Fig. 3). Wild-type pPsbX is efficiently imported, targeted
to the thylakoids (lane T) and processed primarily to the
mature size, as found previously [22]. PsbX/VV is imported
with similar efficiency and the protein is likewise targeted to
the thylakoids, but only the intermediate form (iPsbX/VV)
is found within the organelles. This intermediate is of
precisely the same size as a mutant analyzed previously [20]
in which the terminal processing site was altered to prevent
cleavage by TPP. Clearly, the thylakoid-associated PsbX/
VV corresponds to the product generated by the stromal
processing peptidase. These data demonstrate that the
intermediate form is either unable to insert into the
membrane, or that it does insert but can not be processed
by TPP.
We then carried out single-residue substitutions to
determine whether either of the two Glu residues is more
important in this context. Accordingly, we analyzed
mutants in which only one of the Glu residues was
substituted by Val. The results (Fig. 3, lower panels) show
that substitution of the )2 Glu by Val (PsbX/EV) again
affects maturation but to a lower extent. In the total
chloroplast fraction (lane C) the intermediate- and mature-
size bands are of approximately equal intensity, whereas the
mature-size PsbX protein predominates in the thylakoid
fraction (lane T). These findings suggest that the protein is
gradually converted to the mature size during the import/
fractionation procedure (the chloroplast fraction is removed
and processed for electrophoresis well before the other

samples, which require protease treatment and, in the case
of the stroma/thylakoid samples, fractionation after lysis).
This was confirmed by time-course analyses, which show
gradual conversion to the mature size (data not shown;
similar examples are shown below). The presence of Val at
the )2 position thus slows down maturation to a consid-
erable extent, but does not block it. In contrast, the final
panel in Fig. 3 shows that substitution of the )5GlubyVal
(PsbX/VE) completely blocks maturation as found with the
double Val mutant.
The imported PsbX mutants described in Fig. 3 are
found exclusively in the thylakoid fraction which suggests
that insertion has taken place. However, to confirm this
point we carried out urea washes of the thylakoids because
this procedure is highly effective at removing extrinsic
membrane-associated proteins [19,24]. Figure 4 shows that
this procedure is sufficiently harsh to remove even some of
the fully inserted mature size wild-type PsbX, because some
is found in the supernatant fraction (lane Sn) after the
extraction procedure. This is apparently because single-span
proteins are more easily removed from the thylakoids by
urea [19]. However, most of the mature-size PsbX is found
in the membrane pellet fraction (lane pel) and the same
applies to the intermediate size iPsbX/VV, which is equally
resistant to extraction. As with the double Val mutant, urea
washes confirmed that the imported mature-size single-Val
mutants are fully integrated into the thylakoid membrane
(data not shown). Accordingly, we propose that the protein
cannot be cleaved by TPP, and this could be due to one of
two reasons: first, the processing site may have been altered

such that TPP can no longer recognize the cleavage site, or
secondly, the processing site may be intact but TPP may be
unable to reach it.
Hydrophilic Asn residues can partially compensate
for loss of negative charge in the C-domain of PsbX
The above data indicate that substitution of the )5Gluhas
far more dramatic consequences than alteration of the )2
residue, which suggests that the )5 Glu is significant either
because a negative charge is important in this region, and/or
because the presence of a very hydrophilic residue is
important for maturation. The )5 Glu effectively caps the
H-domain and the VE mutant thus contains a longer
hydrophobic region that now extends to the )2Glu(see
Fig. 2). We investigated these possibilities by substituting
the )5and)2 glutamates with asparagine, which is highly
hydrophilic but uncharged. According to the Kyte–Doolit-
tle and GES hydrophobicity scales, Asn is almost as
hydrophilic as Glu [26,27]. The )5and)2 substitutions
(see Fig. 2) are termed PsbX/NE and PsbX/EN, respect-
Fig. 3. Substitution of the )5 Glu by Val blocks maturation of imported
PsbX. pPsbX, PsbX/VV, PsbX/VE and PsbX/EV were incubated with
intact pea chloroplasts, after which samples were analyzed of the
chloroplasts (lanes C), chloroplasts after thermolysin treatment (C+)
and the stromal (S) and thylakoid (lanes T) fractions after lysis of
thermolysin-treated chloroplasts. Lanes TR: translation products. int
denotes intermediate form generated by stromal processing peptidase.
Fig. 4. Appearance of iPsbX/VV results from a block in processing and
not insertion. Wild-type pPsbX and the PsbX/VV mutant were
imported into chloroplasts as in Fig. 4 and the thylakoid fraction
isolated (lane T). Samples were subjected to two washes with 4

M
urea
and samples analyzed of the supernatant (Sn) and pellet (Pel) fractions.
Other symbols as in Fig. 3.
3134 C. Tissier et al. (Eur. J. Biochem. 269) Ó FEBS 2002
ively, according to whether the first or second Glu is
substituted by Asn, and the double mutant is PsbX/NN.
Import assays using the EN and NE single mutants are
shown in the upper panel of Fig. 5. As with the other
mutants analyzed in this study, these proteins are efficiently
imported and targeted to the thylakoid membrane. No
stromal intermediates are present and the thylakoid-associ-
ated proteins are as resistant to urea-extraction as authentic
PsbX (not shown). These data indicate that the mutations
have no detectable effect on insertion efficiency. Both
mutants are also processed to the mature size but it is
notable that maturation is not as efficient as for the wild-
type protein. Whereas PsbX is invariably found almost
exclusively as the mature form after import into chloro-
plasts, the intermediate-size forms of both single-Asn
mutants are apparent in the thylakoid fractions indicating
an inhibitory effect on maturation by TPP.
This effect is exacerbated in the PsbX/NN mutant that
contains Asn at both the )5and)2 positions. In this case,
a much greater proportion of the imported protein is
present as the intermediate form (iPsbX/NN) at the end of
the import/fractionation procedure. In this experiment we
also carried out a time-course analysis in which the PsbX/
NN mutant and wild-type pPsbX were imported for
10 min, after which the chloroplasts were washed to

remove nonimported protein and samples were analyzed at
various times thereafter to follow the maturation of the
imported protein (lower panels of Fig. 5). Repeat tests
using wild-type pPsbX showed that protein is found only
as the mature protein at even early time-points (see bottom
panel). In contrast, PsbX/NN is found primarily as the
intermediate form at early time points and this form is only
gradually converted to the mature size during the subse-
quent 60 min All of the imported protein was found to be
inserted in the thylakoid fraction at each time-point (not
shown), demonstrating that the double Asn mutations slow
down processing by TPP but do not block this process. We
conclude from these experiments that hydrophilic Asn
residues at the )5and)2 positions enable processing by
TPP to occur, but with less efficiency than when Glu
residues are present.
Valine substitutions at the )5 and )2 positions may lead
to burial of the cleavage site within the membrane
Several of the PsbX mutants shown above exhibit slow
maturation kinetics within the chloroplast, despite being
inserted into the thylakoid membrane. We propose that this
stems from an inability of TPP to actually access the
cleavage site, rather than an alteration of the site such that
TPP can reach the site but not carry out cleavage. Two
points should be emphasized. Firstly, the identity of the )5
and )2 residues varies enormously among thylakoidal
signal peptides, and many different residues are found at
these two positions. Asparagine, in particular, is common in
the C-domain and is often found at the )5and)2 positions.
Almost any residue appears to be tolerated at the )2

position and it is unlikely in the extreme that valine should
pose a problem. In general, the important determinants for
TPP cleavage appear to be short-chain residues at the )3
and )1 residues [28], and a helix-breaking residue is also
commonly found in the region of )4to)6. Other signal
peptidases exhibit broadly similar preferences [29].
In a second line of investigation, we analyzed the
positioning of the translocated loop regions of several PsbX
derivatives, by comparing their sensitivities to digestion by
elastase (Fig. 6). The experiment involved importing wild-
type PsbX (which is cleaved exclusively to the mature size)
and three mutant forms. The first mutant (PsbX/A74T)
contains threonine at the )1 position in place of alanine and
previous studies on this mutant [20] showed that this
mutation has no effect on insertion efficiency but cleavage
by TPP is blocked, leading to the formation of a loop
intermediate with the TPP site exposed on the lumenal side
of the membrane. The other two mutants analyzed were
PsbX/NN, which has a reduced rate of maturation and
PsbX/VV, which is completely blocked in maturation. The
aim here was to determine whether this block was due to
alteration of the TPP site, such that the peptidase can no
longer cleave, or inaccessibility of the site. Control tests
(Fig. 6A) confirmed that all of these proteins are sensitive to
elastase when not inserted into membranes; elastase cleaves
pPsbX to yield a primary degradation product (denoted by
Fig. 5. Asn can partially compensate for the
absence of Glu at the )5 and )2 positions. (A)
PsbX/NE, PsbX/EN and PsbX/NN were
imported into pea chloroplasts and the

organelles analyzed and fractionated as des-
cribed in Fig. 3 for other mutants. (B) PsbX/
NN and pPsbX were imported into chloro-
plasts for 10 min, after which the organelles
were washed to remove nonimported protein.
Theorganelleswerethenfurtherincubated
and samples analyzed directly at times (in min)
indicated above the lanes.
Ó FEBS 2002 Signal peptides of thylakoid membrane proteins (Eur. J. Biochem. 269) 3135
asterisk) that is in fact slightly larger than mature PsbX.
Importantly, all of the PsbX forms are cleaved to the same
products and the mutants are not cleaved with lower
efficiency. The PsbX/VV mutant, which is of particular
interest in this experiment, is indeed cleaved with higher
efficiency than wild-type pPsbX.
After import of the proteins into chloroplasts, samples of
the thylakoids were analyzed without further treatment
(lanes T), after incubation with elastase (lanes T-el) or after
incubation with elastase and concommitant sonication in a
water bath, such that the protease can enter the lumenal
space (lanes T-son). The data using wild-type PsbX show
that the mature-size protein is not cleaved in any of the
samples, as expected because this small protein is essentially
buried within the bilayer with only short domains exposed
on either face. With PsbX/A74T, the thylakoid sample
contains primarily intermediate-size protein, and elastase
treatment without sonication has little effect. Again, this is
unsurprising because the regions exposed to the stromal face
(C-terminus of the mature protein and N-terminus of the
signal peptide) are short. However, when sonicated the

protease efficiently cleaves the intermediate to a product of
similar mobility to the mature protein, indicating that the
loop region is exposed to the lumen. The PsbX/NN mutant
behaves similarly; most of the protein is of mature-size by
the end of the experiment but the intermediate is again
resistant to proteolysis in the absence of sonication but
sensitive when sonicated. In contrast, the PsbX/VV mutant
is almost totally resistant to digestion under all conditions
and only a very minor proportion is cleaved when the
sample is sonicated. The loop region is thus inaccessible to
elastase on either side of the membrane and must therefore
be buried in the bilayer to a much greater extent than is the
case with the wild-type protein.
Acidic residues are important for efficient processing
of the PsbY polyprotein
PsbY is an unusual protein that is synthesized with two
cleavable signal peptides [23]. After insertion into the
thylakoid membrane, TPP cleaves twice on the lumenal
face to release the two signal peptides and an unidentified
protease cleaves on the stromal face of the membrane to
complete the process and generate the two single-span
mature proteins, denoted A1 and A2 [30]. Mutagenesis
studies [31] have clearly demonstrated that the cleavage on
the stromal face occurs at a late stage in the overall insertion
process. As shown in Fig. 7, both of the signal peptides (SP1
and SP2) contain acidic residues in the C-domain, and the
A2 loop also contains Glu at the +3 residue, relative to the
cleavage site. We tested the importance of these residues by
substituting the A1 Glu with Val (PsbY-A1/V) and both A2
Glu residues with Val (PsbY-A2/VV); the precise structures

of these mutants are shown in Fig. 7B.
Fig. 7. Structure of pPsbY and mutant derivatives. (A) The full
sequence of Arabidopsis thaliana pPsbY is shown [23]. The N-terminal
envelope transit domain specifies import into the chloroplast, after
which it is removed by a stromal processing peptidase (SPP). This
domain is indicated but note that the precise site of cleavage by SPP is
not known; we assume that this occurs before the hydrophobic domain
of the first signal peptide. Also shown (in bold) are the two signal
peptides (denoted SP1 and SP2) preceding A1 and A2. The TPP
cleavage sites are denoted by asterisks. The approximate cleavage site
between the C-terminus of A1 and the signal peptide of A2 is denoted
by +? (the identity of the responsible protease is not known). The Glu
residues mutated in this study are shown italicized and in larger font.
Also shown in this manner are the Met residues at the C-terminal ends
of the A1 and A2 proteins, which were also mutated (see text). (B)
Structure of PsbY mutants in which Glu residues in the vicinity of
either the A1 or A2 TPP cleavage sites were mutated.
Fig. 6. The loop region of PsbX/VV is inaccessible to digestion by
elastase after insertion into the thylakoid membrane. (A) pPsbX, PsbX/
A74T, PsbX/NN and PsbX/VV (lanes Tr) were incubated with
0.2 mgÆmL
)1
elastase for 45 min on ice (lanes El). (B) The same
mutants were imported into chloroplasts and the thylakoid fraction
isolated and analyzed as in previous figures (lanes T). Other samples of
the thylakoids were incubated with 0.2 mgÆmL
)1
elastase for 45 min
on ice (T-el) or were treated in the same manner except that the
samples were incubated in a sonicating water-bath for 15 min at 0 °C

andthenfurtherincubatedfor30minonice(lanesT-son).ÔIntÕ
denotes intermediate form of protein, asterisk denotes elastase degra-
dation product. Lanes Tr: translation products.
3136 C. Tissier et al. (Eur. J. Biochem. 269) Ó FEBS 2002
The import data using the PsbY-A1/V mutant are shown
in Fig. 8A. In the control import using wild-type pPsbY
(left hand panel), the precursor protein is imported and
converted to a close doublet of mature A1 and A2 proteins,
as found previously [23,31]. The PsbY-A1/V mutant is also
targeted to the thylakoid membrane and the appearance of
the A2 protein is unaffected. However, the A1 protein is
apparent in the thylakoid sample analyzed at the end of the
import/fractionation procedure (lane T) but is present in
low amounts in the chloroplast samples (lanes C and C+).
Instead, two larger intermediates are present (denoted ÔintsÕ).
These polypeptides were found to accumulate when the
processing of A1 was blocked in an earlier study [30] in
which alteration of the )1 residue was shown to block
cleavage by TPP. This suggests that the presence of the
valine in PsbY-A1/V has likewise slowed down processing
by TPP. We suspected that the near-absence of the
intermediate bands in the thylakoid sample (lane T) may
result from slow but continuing cleavage by TPP during the
course of the experiment (as described earlier). This is
confirmed in Fig. 8B, which shows time-course analyses
similar to that described above for the PsbX/NN mutant.
After a 15-min import incubation the chloroplasts were
washed to remove nonimported protein and the organelles
were further incubated for the times (in min) shown above
the lanes. The import reaction using wild-type pPsbY shows

that essentially all of the imported protein is present as
mature A1 and A2 at the earliest time-points. However,
after import of pPsbY-A1/V the A1 protein is virtually
absent at the initial time-point and the two intermediate
bands are instead present. These decline over the subsequent
10–20 min and the A1 protein appears. These data show
that the presence of the valine at the )5 position, relative to
the TPP cleavage site, leads to a substantial inhibition of
processing at the A1 site.
The data obtained using the double Val substitution in
the A2 cleavage region are shown in Fig. 9. Here, the
substitutions lead to very different effects. The import of
PsbY-A2/VV is shown in Fig. 9A, together with a thylakoid
sample from a control import (lane Con) using wild-type
pPsbY. This mutant is imported and targeted to the
thylakoid membrane, but surprisingly the appearance of
the A2 protein is unaffected and it is the A1 protein which is
absent. A larger intermediate is instead apparent, which
was assumed to contain the A1 protein (labeled A1int).
Fig. 8. The presence of Val in place of Glu inhibits processing of the A1
signal peptide. (A) pPsbY and PsbY-A1/V were imported into
chloroplasts and samples were analyzed of the chloroplasts, protease-
treated chloroplasts, stroma and thylakoids as described in Fig. 3 for
PsbX proteins. The full precursor form of PsbY is denoted ÔPreÕ,theA1
and A2 mature proteins are indicated and intermediate-size forms of
the PsbY-A1/V mutant are denoted ÔintsÕ. (B) pPsbY and the PsbY-
A1/V mutant were imported into chloroplasts for 10 min, after which
the chloroplasts were washed once in import buffer (see Materials and
methods) and further incubated for times (in min) indicated above the
lanes.

Fig. 9. Glu fi Val substitutions near the A2 processing site blocks
cleavage of the A1 signal peptide. (A) PsbY-A2/VV was imported into
chloroplasts and the organelles were fractionated and analyzed as
detailed in Fig. 8A. Lane ÔConÕ shows the chloroplast fraction from a
control import carried out with wild-type pPsbY. The mobilities of the
A1 and A2 proteins are indicated, together with an intermediate form
of the A1 protein (A1-int). (B) The left hand panel shows the import
characteristics of a pPsbY mutant (A2-met) in which the A2 methi-
onine is replaced by leucine. The panel shows translation products
(lanes Tr) carried out using [
3
H]leucine (leu) or [
35
S]methionine (met)
and the chloroplast samples after import of each of these labeled
polypeptides (lanes ÔimpÕ, with the identity of the radiolabel indicated
below). The right hand panel shows identical analyses of the PsbY-A2/
VV mutant carried out with these radiolabeled amino acids.
Ó FEBS 2002 Signal peptides of thylakoid membrane proteins (Eur. J. Biochem. 269) 3137
However, it was deemed important to verify this point,
firstly because this result was completely unexpected, but
also because we considered it possible that both the A1 and
A2 bands may have shifted in the gel due to complex effects
arising from the mutations. We therefore used alternative
means to identify the A1- and A2-containing proteins
unambiguously. Analysis of the protein sequence (see
Fig. 7) reveals that the A1 and A2 mature proteins each
contain a single methionine towards the C-terminus of the
peptide (shown underlined and italicized). The methionine
at the end of A2 was substituted with leucine in both wild-

type pPsbY (ÔPsbY-A2-metÕ) and the PsbY-A2/VV mutant
(mutant PsbY-A2/VV-met). The proteins were synthesized
in the presence of either [
3
H]leucine or [
35
S]methionine and
the import of these proteins is shown in Fig. 9B. Translation
products (Tr) and the thylakoid samples from the import
reactions (lanes imp) are shown above the lanes, together
with the radiolabeled amino acid used in the translation (leu
or met). In the control import with PsbY-A2-met (panel A2-
met) the A1 and A2 proteins are both apparent as expected
when the protein is synthesized in the presence of [
3
H]leu-
cine, but the A2 protein is now absent when a [
35
S]methi-
onine-labeled translation product is used (lane imp met).
This confirms that the A2 protein does indeed contain a
single methionine residue as predicted from the sequence. In
the case of the PsbY-A2/VV-met mutant, the two polypep-
tides observed in Fig. 9 A are again observed in the
[
3
H]leucine-labeled sample and the identity of the lower
band as A2 protein is again confirmed by the finding that
thebandisabsentinthe[
35

S]methionine-labeled sample,
while the A1int band is still present. This result confirms
that the double valine substitution in the A2 cleavage site
region does not actually affect cleavage of A2, but instead
leads to a complete block in the processing of A1 to the
mature size. The A1int polypeptide is too small to contain
three transmembrane spans (the three-span intermediates
are characterized in [28]) and, because cleavage on the
stromal surface is known to occur last and the A1 TPP
cleavage site is completely unaffected, this polypeptide
almost certainly comprises A1 plus the A2 signal peptide.
DISCUSSION
Previous studies have shown that a series of thylakoid
membrane proteins are synthesized with cleavable signal
peptides, yet are inserted by mechanisms that do not rely
on any of the known translocation machinery, either in the
soluble phase or at the membrane surface. It has been
suggested that these signal peptides provide an additional
hydrophobic region that helps to drive the insertion
process, perhaps through the formation of a Ôhelical
hairpinÕ that might provide the required driving force to
flip the N-terminus of the mature protein across the
thylakoid membrane. Intriguingly, these signal peptides
resemble those of Sec-dependent lumenal proteins to a
marked degree, and one of this class of signals can even
function as a Sec-type signal for a lumenal passenger
protein in chloroplasts [21]. However, the data shown here
point to defining features in some of these peptides that are
essential for their correct functioning and which are not
apparent in other forms of signal peptide. Our data also

lead to new ideas on the biogenesis of the unique PsbY
polyprotein.
The studies on the PsbW truncations focused on the role
of basic residues in the N-domain, because previous work
on M13 procoat and Sec-type signal peptides has shown
that basic residues in the N-domain play essential roles in
insertion/translocation [12,13,32]. In fact, our data indicate
quite clearly that these play no important function in the
insertion of PsbW, because their removal inhibits insertion
to only a minor extent. When considered in conjunction
with other data on this group of thylakoid proteins, it is now
very interesting to compare and contrast their insertion
mechanism with that of procoat. Initial models for the
insertion of these thylakoid proteins were based heavily on
that of procoat insertion. M13 procoat and pPsbW are very
similar indeed in structural terms, in that they possess a
single transmembrane span in the mature protein, are
synthesized with rather similar cleavable signal peptides and
the intervening loop regions (which are flipped across the
membrane) are of similar lengths and overall charge. Both
proteins form loop intermediates prior to cleavage by signal
peptidase but it is now clear that their insertion require-
ments are completely different in almost every sense.
Previous work has shown procoat to rely heavily on the
protonmotive force (reviewed in [12]) whereas pPsbW is
DlH
+
- independent, as are the other thylakoid proteins in
this group [15,16]. Procoat is also totally dependent on YidC
for efficient insertion [11] whereas the thylakoid proteins do

not require the homologous Alb3 protein [19]. We have now
shown that these proteins differ in the means by which they
initiate insertion; electrostatic forces play a central role in
the early stages of the procoat insertion mechanism [12]
whereas pPsbW contains no basic residues in the C-terminal
region and our data show that basic residues in the
N-terminal region are not important for insertion into
thylakoids. pPsbW must therefore interact with the thylak-
oid membrane by other means. Basic residues in the
N-domain are also highly important for the functioning of
Sec-type signal peptides, possibly to promote interaction
with anionic phospholipids or SecA [13,32,33], and it
therefore appears that the signal peptides of these Sec-
independent thylakoid proteins function in fundamentally
different ways, despite the superficial similarities.
The other studies on PsbX and PsbY focused on the
C-domain, prompted by the presence of acidic residues in
this region. Acidic residues are not important in any of the
domains within Sec-type signal peptides and are generally
uncommon, especially in the C-domain which is generally
five or six residues in length and polar but uncharged [13]. In
contrast, our results point to an important function for
acidic residues in the translocated regions of these Sec/SRP/
Alb3-independent thylakoid membrane proteins. In some
cases (e.g. CF
o
II) the extreme N-terminus of the mature
protein is highly negatively charged, and we believe that
additional acidic residues in the signal peptide are probably
unnecessary. In other cases (for example PsbX and PsbY-

A1), acidic residues are not present in the N-terminus of the
mature protein and in these cases the signal peptides contain
conserved acidic residues in the C-domain. Our data
indicate that these residues are very important for the
correct maturation of the inserted protein. Substitution of
the )5and)2 Glu residues by Val leads to a complete block
in the maturation of PsbX, although insertion appears not
to be affected. The )5 Glu, in particular, appears to be
important because the presence of Val at this position alone
3138 C. Tissier et al. (Eur. J. Biochem. 269) Ó FEBS 2002
is equally detrimental. Processing efficiency is restored to
some extent by Asn at the )5 position. For reasons that are
presently unclear, the translocated loop region is cleaved
most efficiently when carrying negative charges although
other hydrophilic residues can substitute to some extent.
On the basis of these data we propose that the translo-
cated region needs to bear a negative charge in order for the
TPP cleavage site to be presented in an optimal manner. A
model for the effects of these mutations on the PsbX
insertion mechanism is as follows. Insertion of the wild-type
protein leads to the formation of a loop intermediate [20]
and the hydrophilic nature of the loop region is essential for
correct presentation to TPP. We believe that the presence of
negative charges close to the TPP site serves to distance the
site from the membrane interior and enable processing to
occur. The presence of Val at the )5siteleadstoa
lengthening of the hydrophobic region which then becomes
buried in the membrane interior. This premise is supported
by studies on the PsbY-A1V mutant, which contains no
negative charges in the TPP cleavage site region. Processing

of this mutant is again significantly impaired although not
tothesameextentasinsomeofthePsbXmutants.
These studies are reminiscent of some observations made
with Sec-type signal peptides [34–36], where alteration of the
C-domain or H/C boundary can also affect processing by
signal peptidase. However, in the vast majority of these
cases, processing was not blocked but rather occurred
elsewhere, or the mutations made were far more drastic than
those generated in PsbX. It should be emphasized that a
near-complete block in processing occurred after only a
single substitution ()5 Glu to Val) and processing is
drastically affected in the PsbX/NN mutant despite the
presence of a highly polar C-domain of the correct length.
Overall, these mutations have far more drastic consequences
than similar mutations made in Sec-type signal peptides,
and we conclude that this may be due to one or both of the
following reasons: (a) our studies are on membrane proteins
rather than hydrophilic translocated proteins, and the
cleavagesiteregionmaythereforebemorehighlycon-
strained in the membrane because the mature protein is not
pulled across the bilayer; and/or (b) the unusual lipid
composition of the thylakoid membrane (primarily galacto-
lipid rather than phospholipid [37]) may require that the
translocated loop is more effectively presented to the signal
peptidase when acidic residues are present, for unknown
reasons.
The third aspect of this study concerned the PsbY-A2
signal peptide, but very different results were obtained in
this case. Here, the substitution of two Glu residues in the
translocated loop by Val does not block cleavage by TPP,

indicating that the Glu in the C-domain of this signal
peptide is not as important. Possibly, the presence of three
helix-breaking proline residues upstream (see Fig. 7) is
sufficient to maintain the TPP site away from the mem-
brane, or other effects may operate in this case. However,
these mutations do have dramatic effects and in this case it is
cleavage at the A1-SP2 site on the stromal surface that is
completely inhibited. In fact, the studies on this mutant
fortuitously provide important information on the biogen-
esis of the PsbY polyprotein. In previous work on PsbY
[31], we noted that blockage of the TPP cleavage reaction at
either the A1 or A2 sites led to the accumulation of a three-
membrane-span intermediate, indicating that cleavage on
the stromal side of the membrane had failed to occur in each
case. Inhibition of TPP cleavage at both sites led to the
stable accumulation of a four-span intermediate. Clearly,
cleavage at the stromal site occurs at a late stage prompting
the question: why is this protease unable to recognize this
site until both cleavages by TPP have occurred? The present
study provides further information on this issue; lengthen-
ing the H-domain of the A2 signal peptide leads to the stable
accumulation of a two-span intermediate containing A1 and
the A2 signal peptide (SP2). Our interpretation is that the
unidentified protease on the stromal surface can only cleave
when SP2 is released from a transmembrane state to adopt a
flexible orientation in the membrane. Our model for the
overall process is as follows (see Fig. 10).
Stage 1. The PsbY polyprotein inserts into the membrane
in the double loop formation shown in Fig. 10, and TPP
cleaves at one of the two sites. Most probably, TPP can

cleave at either site first but for simplicity it is shown as
cleaving at the SP1-A1 site. This releases SP1 which is
rapidly degraded.
Stage 2. TPP cleaves at the SP2-A2 site, releasing A2 as a
single-span mature protein and generating the A1-SP2
intermediate (Stage 2).
Stage 3. SP2 is now far more flexible, either because it is no
longer tethered at the lumenal face by charged residues or
because it is not bound to its partner polypeptide region,
A2. The stromal loop region is more accessible and cleavage
in this loop can now occur.
One possibility is that this final cleavage can only
occur when the A1 and SP2 regions are unconstrained
by cognate partner polypeptide regions (A1-SP1,
A2-SP2). First, the PsbY-A2V mutant can be cleaved
at both positions by TPP but the A1-SP2 intermediate
accumulates as a stable species (see lower panel of
Fig. 10). In our view, this is most likely because the SP2
Fig. 10. Model for the maturation of PsbY. 1. After insertion, PsbY
forms a double loop intermediate with two signal peptides (SP1, SP2)
and two regions (A1 and A2) destined to become single-span mature
proteins. 2. TPP cleaves SP2 which is rapidly degraded; SP2 continues
to be held in a transmembrane form due to interactions with A2. TPP
then cleaves after SP2 yielding the mature A2 protein. 3. SP2 is now
more flexible and the A1–SP2 junction on the stromal surface can be
accessed by an unknown protease (hence the question mark) com-
pleting the maturation process. In the case of the PsbY-A2/VV mutant,
SP2 is now more hydrophobic and able to maintain a transmembrane
conformation, preventing cleavage on the stromal side.
Ó FEBS 2002 Signal peptides of thylakoid membrane proteins (Eur. J. Biochem. 269) 3139

H-domain is now significantly longer and is effectively a
true membrane-spanning region. The H-domains of the
signal peptides of these thylakoid membrane proteins are
much shorter and are generally less hydrophobic than
true membrane-spanning regions and, we believe, can
only adopt transmembrane conformations when tethered
to genuine transmembrane spans.
Further evidence for this proposed model comes from
considerations of the stabilities of SP1 and SP2. It is notable
that the A1-SP2 intermediate is highly stable, as are the
PsbX and PsbW loop intermediates generated in a previous
study [20]. Clearly, the signal peptides are completely
resistant to proteolysis when bound to genuine transmem-
brane spans. In complete contrast, the signal peptides
cannot be detected in even low amounts when released
during normal insertion reactions, despite being as large as
some of the mature proteins (e.g. PsbX and PsbW are only 4
and 6 kDa, respectively). Tricine gels readily resolve these
small mature proteins but the complete absence of the
cleaved signal peptides, even immediately after insertion [16]
means that they are degraded very rapidly indeed. We
propose that this is due solely to their low hydrophobicity,
which precludes the maintenance of transmembrane con-
figurations and instead leads to other positions in the
membrane, or even release from the membrane [14], upon
which they are degraded by proteases that perhaps specif-
ically target peptides that are unable to adopt transmem-
brane conformations.
In summary, we have shown that the signal peptides of
these spontaneously-inserting proteins have evolved with

specific and unusual properties that are especially important
for correct proteolytic cleavage following insertion. In the
cases of PsbX and PsbY-A1, the hydrophobicity of the
C-domain is critical for correct maturation and negative
charges in particular appear to be favored. In the case of
PsbY-A2, the negative charge in the translocated loop plays
a key role in defining the hydrophobicity of the A2 signal
peptide, which is of necessity low in order to facilitate the
movements that allow the final cleavage on the stromal
surface. In general, these signal peptides are not merely
additional hydrophobic regions but are rather exquisitely
structured extensions whose properties complement those of
the N-terminal regions of the mature proteins.
ACKNOWLEDGEMENTS
This work was supported by Biotechnology and Biological Sciences
Research Council grant C09633 to C. R.
REFERENCES
1. Dalbey, R.E. & Robinson, C. (1999) Protein translocation into
and across the bacterial plasma membrane and the plant thylakoid
membrane. Trends Biochem. Sci. 24, 17–22.
2. Robinson, C., Thompson, S.J. & Woolhead, C. (2001) Multiple
pathways used for the targeting of thylakoid proteins in chlor-
oplasts. Traffic 2, 245–251.
3. Lamppa, G.K. (1988) The chlorophyll a/b-binding protein inserts
into the thylakoids independent of its cognate transit peptide.
J. Biol. Chem. 263, 14996–14999.
4. Viitanen, P.V., Doran, E.R. & Dunsmuir, P. (1988) What is the
role of the transit peptide in thylakoid integration of the light-
harvesting chlorophyll a/b protein? J. Biol. Chem. 263, 15000–
15007.

5. Li,X.,Henry,R.,Yuan,J.,Cline,K.&Hoffman,N.E.(1995)A
chloroplast homologue of the signal recognition particle subunit
SRP54 is involved in the post-translational integration of a protein
into thylakoid membranes. Proc. Natl Acad. Sci. USA 92, 3789–
3793.
6. Kogata, N., Nishio, K., Hirohashi, T., Kikuchi, S. & Nakai, M.
(1999) Involvement of a chloroplast homologue of the signal
recognition particle receptor protein, FtsY, in protein targeting to
thylakoids. FEBS Lett. 329, 329–333.
7. Tu, C.J., Schuenemann, D. & Hoffman, N.E. (1999) Chloroplast
FtsY, chloroplast signal recognition particle, and GTP are
required to reconstitute the soluble phase of light-harvesting
chlorophyll protein transport into thylakoid membranes. J. Biol.
Chem. 274, 27219–27224.
8. Mori,H.,Summer,E.J.,Ma,X.&Cline,K.(1999)Component
specificity for the thylakoidal Sec and delta pH-dependent protein
transport pathways. J. Cell Biol. 146, 45–55.
9. Moore, M., Harrison, M.S., Peterson, E.C. & Henry, R. (2000)
Chloroplast Oxa1p homolog albino3 is required for post-transla-
tional integration of the light harvesting chlorophyll-binding
protein into thylakoid membranes. J. Biol. Chem. 275, 1529–1532.
10. Hell, K., Neupert, W. & Stuart, R.A. (2001) Oxa1p acts as a
general membrane insertion machinery for proteins encoded by
mitochondrial DNA. EMBO J. 20, 1281–1288.
11. Samuelson, J.C., Chen, M., Jiang, F., Moeller, I., Wiedmann, M.,
Kuhn, A., Phillips, G.J. & Dalbey, R.E. (2000) YidC mediates
membrane protein insertion in bacteria. Nature 406, 637–641.
12. Dalbey, R.E. & Kuhn, A. (2000) Evolutionarily related insertion
pathways of bacterial, mitochondrial, and thylakoid membrane
proteins. Annu. Rev. Cell Dev. Biol. 16, 51–87.

13. Izard, J.W. & Kendall, D.A. (1994). Signal peptides: exquisitely
designed transport promoters. Mol. Microbiol. 13, 765–773.
14. Martoglio, B. & Dobberstein, B. (1998) Signal sequences: more
than just greasy peptides. Trends Cell Biol. 10, 410–415.
15. Michl, D., Robinson, C., Shackleton, J.B., Herrmann, R.G. &
Klo
¨
sgen, R.B. (1994) Targeting of proteins to the thylakoids by
bipartite presequences: CF
o
II is imported by a novel, third path-
way. EMBO J. 13, 1310–1317.
16. Kim, S.J., Robinson, C. & Mant, A. (1998) Sec/SRP-independent
insertion of two thylakoid membrane proteins bearing cleavable
signal peptides. FEBS Letts. 424, 105–108.
17. Lorkovic, Z.J., Schro
¨
der, W.P., Pakrasi, H.B., Irrgang, K D.,
Herrmann, R.G. & Oelmu
¨
ller, R. (1995) Molecular characterisa-
tion of PSII-W, the only nuclear-encoded component of the
photosystem II reaction centre. Proc. Natl Acad. Sci. USA 92,
8930–8934.
18. Robinson, D., Karnauchov, I., Herrmann, R.G., Klo
¨
sgen,R.B.&
Robinson, C. (1996) Protease-sensitive thylakoidal import
machinery for the Sec-, DpH- and signal recognition particle-
dependent protein targeting pathways, but not for CF

o
II
integration. Plant J. 10, 149–155.
19. Woolhead, C.A., Thompson, S., Moore, M., Tissier, C., Rodger,
A.,Henry,R.&Robinson,C.(2001)DistinctAlb3-dependentand
-independent pathways for thylakoid membrane protein insertion.
J. Biol. Chem. 276, 40841–40846.
20. Thompson, S.J., Kim, S.J. & Robinson, C. (1998) Sec-independent
insertion of thylakoid membrane proteins: analysis of insertion
forces and identification of a loop intermediate. J. Biol. Chem. 273,
18979–18983.
21. Michl, D., Karnauchov, I., Bergho
¨
fer, J., Herrmann, R.G. &
Klo
¨
sgen, R.B. (1999) Phylogenetic transfer of organelle genes to
the nucleus can lead to new mechanisms of proteinintegration into
membranes. Plant J. 17, 31–40.
22. Kim, S.J., Robinson, D. & Robinson, C. (1996) An Arabidopsis
thaliana cDNA encoding PSII-X, a 4.1 kDa component of pho-
tosystem II: a bipartite presequence mediates SecA/DpH-inde-
pendent targeting into thylakoids. FEBS Letts. 390, 175–178.
3140 C. Tissier et al. (Eur. J. Biochem. 269) Ó FEBS 2002
23. Mant, A. & Robinson, C. (1998) An Arabidopsis cDNA encodes
an apparent polyprotein of two non-identical thylakoid membrane
proteins that are associated with photosystem II and homologous
to algal ycf32 open reading frames. FEBS Letts. 423, 183–188.
24. Breyton,C.,deVitry,C.&Popot,J L.(1994)Membraneasso-
ciation of cytochrome b

6
f subunits. J. Biol. Chem. 269, 7597–7602.
25. Kirwin, P.M., Elderfield, P.D., Williams, R.S. & Robinson, C.
(1988) Transport of proteins into chloroplasts. Organisation,
orientation and lateral distribution of the plastocyanin processing
peptidase in the thylakoid network. J. Biol. Chem. 263, 18128–
18132.
26. Kyte, J. & Doolittle, R.F. (1982) A simple method for displaying
the hydropathic character of a protein. J. Mol. Biol. 157, 105–132.
27. Engelman, D.M., Steitz, T.A. & Goldman, A. (1986) Identifying
nonpolar transbilayer helices in amino acid sequences of
membrane proteins. Annu. Rev. Biophys. Biophys. Chem. 15,
321–353.
28. Shackleton, J.B. & Robinson, C. (1991) Transport of proteins into
chloroplasts. The thylakoidal processing peptidase is a signal-type
peptidase with stringent substrate requirements at the -3 and -1
positions. J. Biol. Chem. 266, 12152–12156.
29. Dalbey, R.E. & von Heijne, G. (1992) Signal peptidases in pro-
karyotes and eukaryotes – a new protease family. Trends Biochem.
Sci. 17, 474–478.
30.Gau,A.E.,Thole,H.H.,Sokolenko,A.,Altschmied,L.,
Hermann, R.G. & Pistorius, E.K. (1998) PsbY, a novel manga-
nese-binding, low-molecular-mass protein associated with photo-
system II. Mol. Gen. Genet. 260, 56–68.
31. Thompson, S.J., Robinson, C. & Mant, A. (1999) Dual signal
peptides mediate the Sec/SRP-independent insertion of a thyla-
koid membrane polyprotein, PsbY. J. Biol. Chem. 274, 4059–4066.
32. Sasaki, K., Matsuyama, S. & Mizushima, S. (1990) In vitro kinetic
analysis of the role of the positive charge at the amino-terminal
region of signal peptides in translocation of secretory protein

across the cytoplasmic membrane in Escherichia coli. J. Biol.
Chem. 265, 4358–4363.
33. Demel, R.A., Goormaghtigh, E. & de Kruijff, B. (1990) Lipid and
peptide specificities in signal peptide–lipid interactions in model
membranes. Biochim. Biophys. Acta 1027, 155–162.
34. Nothwehr, S.F., Hoeltzli, S.D., Allen, K.L., Lively, M.O. &
Gordon, J.I. (1990) Residues flanking the COOH-terminal
C-region of a model eukaryotic signal peptide influence the site of
its cleavage by signal peptidase and the extent of coupling of its
co-translational translocation and proteolytic processing in vitro.
J. Biol. Chem. 265, 21797–21803.
35. Nothwehr, S.F. & Gordon, J.I. (1990) Structural features in the
NH2-terminal region of a model eukaryotic signal peptide influ-
ence the site of its cleavage by signal peptidase. J. Biol. Chem. 265,
17202–17208.
36. Carlos, J.L., Paetzel, M., Brubaker, G., Karla, A., Ashwell, C.M.,
Lively, M.O., Cao, G., Bullinger, P. & Dalbey, R.E. (2000) The
role of the membrane-spanning domain of type I signal peptidases
in substrate cleavage site selection. J. Biol. Chem. 275, 38813–
38822.
37. Bruce, B.D. (1998) The role of lipids in plastid protein transport.
Plant Mol. Biol. 38, 223–246.
Ó FEBS 2002 Signal peptides of thylakoid membrane proteins (Eur. J. Biochem. 269) 3141