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INTRODUCTION |
The insertion of membrane proteins is a complex process in which
two major obstacles must be overcome: the transfer of hydrophobic regions into the bilayer with the correct final topology and the efficient translocation of hydrophilic domains to the trans
side of the bilayer. Many such proteins are believed to insert
post-translationally into bacterial, mitochondrial, and chloroplast
membranes, and a variety of in vivo and in vitro
approaches have been used to study the underlying mechanisms. In
several cases it has been shown that the proteins follow an
"assisted" pathway that involves protein transport machinery in
both the soluble phase and the target membrane. Escherichia
coli has been a popular model system, and a range of membrane
proteins has been shown to rely on the cytosolic signal recognition
particle (SRP),1 which is a
complex of 4.5 S RNA molecule and Ffh protein, a homolog of the 54-kDa
component of eukaryotic SRPs that target proteins to the endoplasmic
reticulum (1-5). It is generally believed that this factor docks with
the SecYEG translocon in the inner membrane, and this has been
confirmed recently for at least one SRP substrate (6-8). A further
component, FtsY, has been suggested to serve as a soluble SRP receptor
that transfers SRP substrates to the SecYEG complex (6, 9).
Other membrane proteins appear to use simpler insertion mechanisms that
require none of the known protein transport machinery characterized to
date for the targeting of either hydrophobic or soluble proteins. In
E. coli, two single-span proteins, the coat proteins of the
M13 and pf3 phages, have been shown to insert into the inner membrane
without the aid of SRP, SecA, or the membrane-bound Sec apparatus (10,
11). These proteins may thus insert spontaneously into the bilayer. A
similar mechanism may apply to other bacterial inner membrane proteins,
but none has been characterized in vitro, and the in
vivo analyses have tended to offer a less direct mode of analysis.
The chloroplast thylakoid membrane has also been employed as a model
system for the study of membrane protein biogenesis, in part because
the component photosynthetic proteins have been characterized
intensively and partly because the membrane itself has been shown to
insert or import a wide range of proteins in vitro (for
review see Ref. 12). This may reflect the relative ease with which
these membranes can be isolated in a purified form. The chloroplast is
prokaryotic-like in many respects, having probably evolved from an
endosymbiotic cyanobacterium, and it contains a Sec system for the
transport of proteins into the lumen (13-15) as well as a stromal SRP
molecule containing a homolog of the 54-kDa protein (cpSRP54 (16)). As
with bacteria, two distinct pathways have been identified for the
insertion of membrane proteins, one of which is assisted, and the other
of which is SRP/Sec/
pH-independent. One imported multispanning
membrane protein, the major light-harvesting chlorophyll-binding
protein of photosystem II (Lhcb1, but usually termed LHCII or LHCP) has
been shown to require SRP together with GTP for insertion into the
membrane (17), and proteolysis of thylakoids blocks this insertion
process (18, 19) indicating the involvement of protein-targeting
apparatus (probably the Sec apparatus, but this remains to be
confirmed). In broad terms this targeting pathway therefore resembles
the bacterial SRP-dependent pathway for membrane proteins,
but significant mechanistic differences may exist because no RNA
molecule has been identified in stromal SRP, and the SRP54 subunit
instead forms a complex with a novel 43-kDa subunit (20).
A very different SRP/Sec-independent pathway has been identified for a
subset of thylakoid membrane proteins: CF0II and the X and
W subunits of photosystem II (PsbX, PsbW). Each of the mature proteins
contains a single transmembrane span, and each is synthesized in the
cytosol with a bipartite presequence in which a typical "envelope
transit" signal is followed by a cleavable signal peptide. These
proteins insert into thylakoids in the absence of SRP, SecA, nucleoside
triphosphates (NTPs) or a
pH (21-23), and there is strong evidence
that the Sec apparatus is likewise not involved: proteolysis of
thylakoids abolishes Sec-dependent transport of lumenal
proteins yet has no effect on the insertion of these membrane proteins
(19). It has been suggested that these proteins may insert
spontaneously into the membrane, and the signal peptides probably serve
an unusual function by simply providing an additional hydrophobic
region that assists insertion through the formation of a loop
intermediate (24). In this respect the insertion of these proteins may
resemble that of M13 coat protein, which is the only other membrane
protein known to be synthesized with a signal peptide but inserted by a
Sec-independent process (10).
To date, Sec/SRP-independent insertion processes have only been
characterized for simple single-span membrane proteins, and the
SRP-dependent process has been characterized only for LHCP. In this study we have analyzed the insertion of four additional members
of the extended light-harvesting chlorophyll
a/b-binding protein (CAB) family. We show that
two members resemble LHCP in that stromal factors, NTPs, and protein
transport machinery are all absolutely essential for integration. In
contrast, two other members, early light-inducible protein 2 (Elip2)
and photosystem II subunit S (PsbS), can insert efficiently in the
complete absence of these factors, suggesting a possible spontaneous
insertion mechanism. A portion of the population of Elip2 and PsbS
molecules does, however, appear to utilize the assisted pathway
described above, indicating that some thylakoid proteins are capable of following parallel insertion pathways.
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MATERIALS AND METHODS |
Synthesis of Precursor Proteins--
EST cDNA clones
encoding Arabidopsis CAB proteins were obtained from the
Arabidopsis stock center at Ohio State University. Full-length cDNAs were characterized, and both strands of each cDNA were fully sequenced; the full sequence details of these clones together with those encoding other CAB proteins will appear elsewhere.2 The stock numbers
for the clones are 93I7T7, 37A1T7, VCVCD09, and 137M5T7 for cDNAs
encoding Lhca1, Lhcb5, Elip2, and PsbS, respectively. The precursor
proteins were prepared in vitro by transcription of the
cDNAs using T7 or T3 RNA polymerase followed by translation in a
wheat germ lysate in the presence of [35S]methionine.
Other precursors were synthesized as detailed in Ref. 19, and a
cDNA encoding petunia pLHCP was kindly provided by Dr. Paul Viitanen.
Biochemicals and Suppliers--
Apyrase (grade VI, potato),
nigericin, proteinase K, thermolysin, trypsin (type XIII, bovine
pancreas), and trypsin inhibitor (type I-S, soybean) were all obtained
from Sigma. Pea seeds (Pisum sativum var. Kelvedon Wonder)
were obtained from Nickerson-Zwaan.
Integration Assays--
Isolated thylakoid membranes were
prepared from intact pea chloroplasts, as described in Ref. 25.
Thylakoid membranes were washed twice with ice-cold 10 mM
Hepes-KOH, pH 8.0, and 5 mM MgCl2 (HM) and were
then resuspended in HM or stromal extract to a concentration of 0.5 mg
ml
1 chlorophyll. Stromal extract was prepared by lysing
intact chloroplasts in HM at 1.0 mg ml
1 chlorophyll.
Integration assays (50 µl) contained 20 µg of chlorophyll and 5 µl of in vitro-translated precursor protein mixture
(treated with 0.1 mg ml
1 final concentration of
puromycin). Where appropriate, the thylakoids and translation mixture
were preincubated on ice in the presence of inhibitors. Apyrase was
used to deplete the assay of NTPs as detailed in Hulford et
al. (26). Assays were performed in an illuminated water bath
(intensity 150 µmol photons m
2 s
1,
26 °C) for 20 min and were terminated by the addition of 1 ml of
ice-cold HM followed by reisolation of the thylakoid membranes for
direct analysis, treatment by protease, or urea extraction. All samples
were analyzed by SDS-polyacrylamide gel electrophoresis and
fluorography. Insertion efficiencies were quantitated by measurement of
the protease-resistant degradation product using a PhosphorImager and
are shown quantitated as the percentage of available precursor in the
insertion reaction.
Urea Extraction--
The technique was adapted from Breyton
et al. (27). Thylakoid membranes (equivalent to 10 or 20 µg of chlorophyll) were washed in 1 ml of 20 mM
Tricine-NaOH, pH 8.0, centrifuged at 17,000 × g for 5 min at 4 °C and the supernatant carefully removed. The pellet was
resuspended in 100 µl of freshly made 6.8 M urea and 20 mM Tricine-NaOH, pH 8.0, and incubated for 10 min on the
bench (22-24 °C). The sample was then subjected to two freeze-thaw
cycles (solid CO2/room temperature) before being
centrifuged at 50,000 rpm (135,000 × g) for 15 min at
4 °C in a Beckman TL100 benchtop ultracentrifuge, using the TLA100.3
rotor. The first 80-µl supernatant was withdrawn carefully to avoid
contamination with the pellet, and the remaining supernatant was
discarded (this inevitably resulted in the loss of a very small
quantity of the pellet). The pellet was resuspended once more in 100 µl of 6.8 M urea and 20 mM Tricine-NaOH, pH
8.0, and the extraction process was repeated. Equivalent amounts of the
first supernatant and the second pellet were analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. The second round of extraction rarely resulted in the removal of any extra material from the thylakoid membranes.
Trypsin Treatment of Thylakoid Membranes Before Integration
Assays--
The protocol was as described in Ref. 19, with the
following modifications. After activation of the chloroplast ATPase by illuminating intact chloroplasts in the presence of dithiothreitol and
Ca2+, isolated thylakoids in HM were incubated with or
without 60 µg ml
1 trypsin for 10 min on ice. Both
samples were washed with HM buffer containing 120 µg
ml
1 trypsin inhibitor, and the membranes were reisolated
by centrifugation at 50,000 rpm (135,000 × g) for 6 min at 4 °C in a Beckman TL100 ultracentrifuge, using the TLA100.3
rotor. The thylakoids were washed twice further with HM and 60 µg
ml
1 trypsin inhibitor and similarly reisolated before
being resuspended in stromal extract (from chloroplasts lysed at 1.0 mg
ml
1 chlorophyll) or HM buffer. Each assay contained (in
50 µl) thylakoids equivalent to 20 µg of chlorophyll in stromal
extract or HM, 60 µg ml
1 trypsin inhibitor, 0.5 mM MgATP, and 10 µl of puromycin-treated in
vitro translation mixture (which also contains ATP at 1.2 mM). The assay was set up in a darkroom under a green
safelight, and the incubation was carried out for 30 min at
26-27 °C. Incubations were terminated by the addition of 1 ml of
ice-cold HM followed by centrifugation at 17,000 × g
for 15 min at 4 °C. There were no discernible differences in the
recovery of membranes between trypsin-treated and control samples. One
half of each sample was analyzed directly, the other half by
thermolysin treatment (0.2 mg ml
1 thermolysin, 2.5 mM CaCl2, 40 min on ice). Samples were analyzed by SDS-polyacrylamide gel electrophoresis and fluorography.
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RESULTS |
Structures of CAB Family Proteins--
CAB proteins form a diverse
family of pigment-binding proteins that apparently originated through
internal gene duplication from an ancestral single-span protein (for
review, see Ref. 28). Most plant CAB proteins have three transmembrane
spans, and the structure of the most abundant member (LHCP) has been
solved by electron crystallography (29). In this report we have studied the insertion of four Arabidopsis thaliana CAB proteins;
these are the Lhca1 protein (Lhca corresponds to the light-harvesting complex of photosystem I), Lhcb5 (Lhcb = light-harvesting complex of photosystem II), PsbS, and Elip2. Elips are early light-inducible proteins that appear very rapidly after the onset of illumination and
then decline rapidly in abundance. Their function is unclear, but roles
in transient binding of chlorophyll molecules or in energy dissipation
have both been suggested (28). All of the proteins exhibit diagnostic
conserved transmembrane regions (the first and third regions in the
Lhca1, Lhcb5, and Elip2 sequences (28)). PsbS is an unusual component
of the photosystem II core complex which is believed to contain four
transmembrane spans (30, 31). The full sequence details will be
presented elsewhere.2
Assisted Insertion Mechanisms for Lhca1 and Lhcb5--
In this
study we used assays for the insertion of proteins into isolated
thylakoids, and as criteria for correct insertion we used protease
protection assays and urea washing of the membranes. To characterize
the protease resistance of the authentic mature proteins we used
thylakoids containing radiolabeled mature protein that had been
imported into intact chloroplasts. Once inside the organelles, many
membrane proteins have been found to insert efficiently and correctly
into the thylakoid membrane, and incorrect insertion has not been
documented to date. Fig. 1 shows
chloroplast import assays using the precursors of Lhca1 and Lhcb5. The
precursors are imported and sorted efficiently to the thylakoids
(lanes T); incubation of these membranes with thermolysin
(lanes T+) reveals that Lhca1 is wholly resistant to
digestion, whereas Lhcb5 is clipped to a slightly smaller size. The
translation products are quantitatively digested to low molecular mass
peptides by the same concentrations of protease (lanes Tr+),
confirming that the observed protease resistance of the mature proteins
is caused by their transmembrane configurations.

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Fig. 1.
Import of Lhcb5 and Lhca1 into intact
chloroplasts. Precursors of Lhcb5 and Lhca1 (pLhcb5, pLhca1) were
synthesized by transcription-translation (lanes Tr) and
incubated with intact pea chloroplasts. After incubation, samples of
the chloroplasts were analyzed immediately (lane C) and
after thermolysin treatment (C+); other samples of
thermolysin-treated chloroplasts were lysed and centrifuged to generate
stromal (S) and thylakoid (T) fractions.
Lanes T+, the thylakoid fraction was treated with 0.2 mg/ml
thermolysin for 40 min on ice; lanes Tr+, samples of
translation product were incubated with thermolysin under the same
conditions.
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Fig. 2 shows assays for the insertion for
these two proteins into thylakoids, in which we tested the requirement
for added stromal extract or the effects of pretreating the incubations with apyrase. Apyrase hydrolyzes all available NTPs and totally blocks
targeting by the SRP- and SecA-dependent pathways, both of
which depend entirely on NTPs (26, 32, 33). Incubation of both
pre-Lhca1 and pre-Lhcb5 with thylakoids and stromal extract leads to
the generation of mature protein because of the action of
stromal-processing peptidase, which removes the envelope transit domain. Under these conditions, a significant portion of the population of mature protein molecules is resistant to further digestion. The
appearance of the protease-resistant Lhca1 and Lhcb5 depends almost
entirely on the presence of stromal extract and is totally abolished by
preincubation with apyrase. These data indicate strongly that the
protease-resistant protein represents inserted protein and that
insertion requires stromal factors and NTPs. Only one point remains
unclear; mature Lhcb5 generated in chloroplast import assays is
digested slightly by thermolysin, whereas the same concentration of
protease does not digest the inserted protein in thylakoid insertion
assays. One possibility is that this difference reflects differing
states of assembly of the protein in the two types of assay, but
further work will be required to test this. Other tests have confirmed
that the protease-resistant Lhca1 and Lhcb5 are indeed inserted into
the membrane; the proteins are recovered only in the pellet fraction
after extensive urea washes as used below for PsbS and Elip2 (not
shown). In general, therefore, Lhca1 and Lhcb5 appear to resemble LHCP
in that both stromal factors and NTPs are required for insertion into
thylakoids. Future studies should reveal whether the same stromal
factors are involved in each case.

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Fig. 2.
Insertion of Lhcb5 and Lhca1 into thylakoids
requires stromal extract and NTPs. pLhca1 and pLhcb5 were
incubated with isolated, washed pea thylakoids (as detailed under
"Materials and Methods") in the presence or absence of added stroma
as indicated. Other samples were preincubated with apyrase. After
incubation, samples were washed once and analyzed immediately or after
treatment with thermolysin (therm) under the conditions used
in Fig. 1. Lanes Tr+, thermolysin-treated translation
mixture as in Fig. 1.
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PsbS and Elip2 Are Able to Insert by an SRP/Sec-independent
Mechanism--
Our primary aim in this study was to determine whether
all of the CAB family members use similar insertion pathways, and PsbS and Elip2 were found to be good test subjects because of their ability
to insert efficiently into isolated thylakoids. Again, acquisition of
protease resistance was used as a criterion for membrane insertion.
Fig. 3A shows chloroplast
imports of pre-Elip2 in the absence or presence of nigericin, a proton
ionophore that dissipates the thylakoidal
pH. This compound markedly
inhibits the insertion of LHCP leading to its appearance in the stroma (32). However, no effect is observed on the import characteristics of
Elip2, and the protein is found almost entirely in the thylakoid fraction in both panels. Incubation of the thylakoids with 50 µg/ml
proteinase K (lanes T+) leads to the appearance of two
degradation products (DP1 and DP2). Assays for the import of Elip2 into
thylakoids (Fig. 3B) show that the thylakoid-associated
protein is converted to the same two products by proteinase K, and
comparison with authentic mature Elip2 from a chloroplast import shows
that the DP1:DP2 ratio is identical when generated by even 100 or 150 µg/ml proteinase K. Both concentrations of proteinase K digest the
translation product essentially to completion. These data indicate that
DP1 and DP2 are diagnostic of correct insertion and that Elip2 has inserted correctly into the isolated thylakoids. Insertion is more
efficient in the presence of stromal extract but nevertheless occurs
efficiently in the complete absence of stroma.

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Fig. 3.
Import of Elip2 into intact chloroplasts and
insertion into isolated thylakoids. Panel A, the
precursor of Elip2 (pre-Elip2) was incubated with intact
chloroplasts and samples subsequently fractionated and analyzed as
detailed for Lhcb5 and Lhca1 in Fig. 1. Lanes T+, thylakoid
membranes were incubated with 50 µg/ml proteinase K for 30 min on
ice. Import incubations were carried out under control conditions or in
the presence of 2 µM nigericin. Panel B,
pre-Elip2 translation product (Tr) was incubated with
isolated pea thylakoids in the absence or presence of stroma as
indicated ( stroma, +stroma). After incubation, samples of washed
thylakoids (lanes T) were treated with 100 or 150 µg/ml
proteinase K as indicated above the lanes. The same proteinase K
treatments were carried out on thylakoids (T) from a
chloroplast import reaction (chloro.) carried out as in
panel A and on translation mixtures (transl.)
incubated with stroma.
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The effects of apyrase and the presence/absence of stromal extract are
shown in Fig. 4, and the efficiency of
insertion was quantitated by measurement of the
[35S]methionine in the degradation products. Insertion
efficiencies are calculated on the assumption that the degradation
products contain the same number of labeled residues as mature size
protein (because the sites of cleavage are not known). The figures
given are therefore certain to be underestimates because analysis of the primary sequence shows that methionine residues must be lost when
Elip2 is converted to this size of degradation product by cleavage at
either the NH2 or COOH terminus (not shown). In the absence
of stroma, insertion of Elip2 is again observed as demonstrated by the
appearance of DP1 and DP2. The presence of stroma again enhances the
insertion efficiency approximately 2-fold, and the presence of apyrase
reduces import efficiency to about the same level as observed in the
absence of stroma. These data strongly suggest that a proportion of the
molecules use an assisted pathway that depends on stromal factors and
NTPs, whereas just over half of the inserting molecules do so in the
complete absence of stroma/NTPs. The lower panel of Fig. 4
is a control to verify the effectiveness of apyrase in this particular
experiment. i33K is an intermediate size construct that is imported
into the thylakoid lumen by the ATP-dependent Sec system
(26), and the data show that the appearance of protease-protected,
mature 33-kDa protein is stimulated by the presence of stromal extract
but completely blocked by apyrase treatment.

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Fig. 4.
Elip2 can insert into thylakoids in the
absence of stromal extract or NTPs. Pre-Elip2 and i33K
(lanes Tr) were incubated with thylakoids as in Fig. 3 in
the absence or presence of stromal extract (SE) as
indicated. Incubations were also carried out in the presence of stromal
extract after pretreatment with boiled apyrase (BAp) or
active apyrase (Ap) as indicated above the lanes. After
incubation, samples of washed thylakoids were analyzed directly or
after incubation ( or +, respectively) with either 50 µg/ml
proteinase K for 30 min on ice (for Elip2) or 200 µg/ml thermolysin
under the same conditions (for i33K imports). Lane Tr+,
Elip2 translation product incubated with the proteinase K under the
same conditions. The symbols are as in Fig. 3. Insertion
efficiencies are shown quantitated as detailed under "Materials and
Methods."
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Similar tests were carried out on PsbS. Fig.
5 shows a chloroplast import assay in
which the precursor protein (pPsbS) is efficiently imported, processed
to the mature size, and localized in the thylakoid membrane (lane
T). Incubation of the thylakoids with 50 µg/ml proteinase K
converts the 24-kDa mature protein to a series of degradation products
with masses of between 12 and 15 kDa. The larger product (DP1) becomes
less prominent at higher concentrations of proteinase K (100 and 150 µg/ml), but the remaining products (DP2-4) are relatively resistant
even to these very high concentrations of protease. The translation
product is completely digested by all three concentrations of
proteinase K, and labeled digestion products are only observed
comigrating with the dye front (df). Incubation of pPsbS
with isolated thylakoids in either the presence or absence of stromal
extract leads to the appearance of the same four DPs (Fig. 5,
lower panel), and the relative intensities of the four bands
are essentially identical when thylakoids are digested from chloroplast
or thylakoid import assays. This finding represents strong evidence
that PsbS is able to insert into thylakoids. Calculation of the
insertion efficiency is made more difficult by the presence of only a
single methionine in the mature protein (the presequence contains two),
hence some degradation products may not be apparent in this type of
assay. However, even taking this into account, our experience is that PsbS inserts into thylakoids with relatively high efficiency (e.g. slightly more efficiently than Elip2 or pLHCP).

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Fig. 5.
Import of PsbS into chloroplasts and
insertion into thylakoids. Top panel, pPsbS (lane
Tr) was incubated with pea chloroplasts, and samples were analyzed
and fractionated as detailed in Figs. 1 and 3. Samples of either the
translation product or thylakoids from an import reaction (as indicated
above lanes) were incubated with proteinase K for 30 min on ice at
concentrations indicated above the lanes in µg/ml. df, dye
front. Lower panel, pPsbS was incubated with isolated
thylakoids in the presence or absence of stroma, and samples of the
washed thylakoids (T) were incubated with proteinase K at 50 or 100 µg/ml. Samples of thylakoids from a chloroplast import
reaction (chloro.) were treated in the same manner.
Symbols are as in the top panel.
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The effects of apyrase are shown in Fig.
6. As with Elip2, insertion is stimulated
by the presence of stroma, and apyrase reduces this enhanced level of
insertion to the figure observed in the absence of stroma. The presence
of stromal extract leads to the processing of the precursor protein to
the mature size but insertion proceeds in the absence of any apparent
processing in the "
stromal extract" incubation, indicating that
the full precursor is competent for insertion. The data indicate that, in this experiment, just over half of the PsbS inserts by an assisted mechanism with the remainder able to insert in the absence of NTPs or
stromal factors.

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Fig. 6.
Effects of apyrase on the insertion of pPsbS
into thylakoids. pPsbS (lane Tr) was incubated with
thylakoids in the absence or presence of stromal extract
(SE). Further incubations carried out in the presence of
stromal extract were preincubated with boiled apyrase (BAp)
or active apyrase (Ap) as indicated. Samples of thylakoids
were washed once and analyzed immediately ( ) or after incubation with
100 µg/ml proteinase K (+). Symbols are as in Fig.
5.
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Although protease resistance is used widely as an indication that
membrane insertion has taken place, we sought an alternative criterion,
and urea washing has proven to be very useful. Breyton et
al. (27) have shown that this form of washing is highly effective at removing extrinsic, non-inserted proteins from thylakoid membranes, and we have found the same to be true in tests on newly inserted thylakoid membrane proteins (34). Fig.
7A shows a Coomassie-stained gel that illustrates the effects of urea washing on some of the major
thylakoid proteins. Known extrinsic proteins such as the 33-kDa and
23-kDa oxygen-evolving complex proteins are quantitatively removed and
recovered in the supernatant, whereas LHCP, the major visible staining
band, is resistant and hence found in the pellet fraction. As a control
for thylakoid insertion assays we used LHCP as shown in Fig.
7B. Incubation of the petunia precursor protein (pLHCP) with
thylakoids and stromal extract leads to the appearance of mature LHCP
(lane T), and digestion of the membranes with thermolysin
(T+) yields resistant mature-size protein that is diagnostic
of correct insertion (19, 35). Urea washing of undigested membranes
generates pellet and supernatant fractions, each of which contains both
pLHCP and mature LHCP. When the import reaction is preincubated with
apyrase, insertion is completely blocked, and no protease-resistant
LHCP is apparent in the T+ lane, as found
previously (19). Notably, the thylakoid-associated LHCP is almost
entirely urea-extractable, and virtually no protein is found in the
pellet fraction. This result demonstrates that urea washing is highly
effective at discriminating between inserted and non-inserted LHCP.

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Fig. 7.
Use of urea washing to identify inserted CAB
proteins. Panel A, pea thylakoids (T) were
washed with urea, and samples of the pellet (P) and
supernatant (S) fractions were analyzed. Indicated on the
Coomassie-stained gel are the and subunits of the
CF1CF0-ATPase, the lumenal 33-kDa and 23-kDa
proteins of the oxygen-evolving complex and LHCP. Panel B,
petunia pLHCP was incubated with pea thylakoids under control
conditions or after pretreatment with apyrase. After incubation,
samples of washed thylakoids (T), thylakoids treated with
200 µg/ml thermolysin (T+), and samples of the pellet
(Pel) and supernatant (Sn) fractions were
analyzed after washing of non-protease-treated thylakoids with urea.
Panel C: left, experiment identical to that in
panel B except that pElip2 was used as precursor;
right, samples of thylakoids after the insertion reaction
(T), the same thylakoids after incubation with 100 µg/ml
proteinase K (T+), and the pellet/supernatant fractions
after urea washing of protease-treated thylakoids. Panel D,
as in panel C, left, except using pPsbS as
substrate. Lanes Tr+ show protease-treated translation
products in all cases.
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Similar tests on Elip2 and PsbS are shown in panels C and
D. In both cases, a significant proportion of the population
of thylakoid-associated protein is found to be resistant to urea extraction and hence inserted. In the Elip2 experiment we also treated
an aliquot of the membranes with proteinase K after the import
incubation (as described in Fig. 4), and the right panel shows that the two DPs are completely resistant to urea extraction, confirming that they are fully integrated into the membrane. Import incubations carried out after apyrase treatment show a slightly greater
proportion of protein in the supernatant fraction, consistent with the
lowering of insertion efficiency observed above.
A Functional Sec System Is Not Required for the Insertion of
Elip2--
Elip2 and PsbS can clearly insert into thylakoids in the
absence of SRP, SecA or NTPs, but as a final test we sought to
determine whether the membrane-bound transport machinery (probably
SecYEG) is required for their insertion. This type of analysis is
complicated by the finding that insertion of LHCP is highly dependent
on the thylakoidal proton motive force (32), and in previous studies on
petunia LHCP we found that almost no insertion took place in the
presence of nigericin (19). Even mild proteolysis of thylakoids blocks
their ability to generate any
pH by photosynthetic
electron transport (19), and tests for the involvement of surface-bound transport apparatus therefore require that a
pH is generated by
other means. We have shown that this can be achieved by driving the
CF1CF0-ATP synthase in reverse in the dark
through the addition of ATP because this protein is highly resistant to
trypsin digestion (19). This procedure was used in the experiment shown
in Fig. 8. In the control experiments, a
pH of 2.1 was generated, and this is sufficient to support the
insertion of pLHCP into the membrane. In other tests (not shown) it was
found that these thylakoids were also able to import p23K with high
efficiency; because this protein is targeted by the
pH-dependent pathway this finding provides further
evidence that a high
pH was generated. The Sec substrate, i33K, is
also imported and processed to the mature size under these conditions
(Fig. 8). A
pH of equal magnitude was generated by the
trypsin-treated thylakoids, but the import of i33K is blocked
indicating that the membrane-bound Sec machinery has been destroyed.
Identical results were found in earlier tests on i33K (19). Similar
results are obtained using pLHCP as substrate; no insertion is apparent
using the trypsin-treated thylakoids, underlining the importance of
translocation machinery as found previously (18, 19). Although neither
Elip2 nor PsbS requires a
pH for insertion (Fig. 3 and data not
shown) they were imported under identical conditions, and the
lower panel shows that Elip2 inserts in both the control and
trypsin-treated thylakoids. Insertion efficiency is lowered by the
trypsin treatment, consistent with the data shown above indicating that
a proportion of the population of molecules (roughly half) is targeted
by an assisted mechanism. Similar tests have been carried out on PsbS,
but these tests have been complicated by the fact that pPsbS is
exquisitely sensitive to proteolysis. Although the thylakoids are
washed extensively after the trypsin treatment, the minute quantities
that remain with the thylakoids are able to digest the pPsbS molecules
to a significant extent. Insertion of pPsbS is nevertheless observed (not shown), but an alternative proteolysis regime will be required before a more quantitative assessment of insertion efficiency is
possible.

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Fig. 8.
Proteolysis of thylakoids blocks the Sec- and
SRP-dependent pathways but does not block insertion of PsbS
or Elip2. Pea chloroplasts were incubated in the light together
with dithiothreitol and CaCl2 to activate the
CF1CF0-ATP synthase, after which thylakoids
were prepared as detailed under "Materials and Methods." Samples
were incubated on ice for 15 min ± 60 µg/ml trypsin after which
the thylakoids were washed three times and used for import reactions in
the dark in the presence of 0.5 mM ATP and stromal extract.
Import incubations were carried out using wheat i33K, petunia pLHCP,
and Elip2 as substrates. After incubation, samples were analyzed
immediately or after protease treatment of thylakoids as detailed in
previous figures.
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DISCUSSION |
Previous work on the insertion of thylakoid membrane proteins has
focused primarily on a very small number of proteins: one single
multispanning protein (LHCP) and a series of single-span proteins that
are synthesized with signal peptides. The data from these studies
showed that the requirements of these proteins differ in significant
respects, in that the insertion of LHCP is totally dependent on stromal
factors (including cpSRP54 but also probably others), NTPs, and protein
transport machinery in the thylakoid membrane (17, 19, 26, 32), whereas
CF0II, PsbX, and PsbW require none of these factors for
efficient insertion (19, 21-23). It has remained unclear whether these
requirements simply reflect either the structural differences between
multi- and single-spanning proteins or that these particular
single-span proteins are synthesized with signal peptides. In this
study we have addressed this question through the analysis of a number
of multispanning proteins, and the data indicate that such proteins can
insert by both types of mechanism. Two of the proteins, Lhca1 and
Lhcb5, appear to resemble LHCP in that they likewise depend absolutely
on stroma, NTPs, and protein transport apparatus. We have yet to
determine whether SRP is involved in the insertion of these proteins,
but this appears highly likely on the basis of these data.
Elip2 and PsbS have very different characteristics. These proteins can
clearly insert in the absence of the above factors, and we therefore
propose that these proteins insert spontaneously into the thylakoid
membrane, as has been suggested for CF0II, PsbX, and PsbW.
We would, however, stress that this notion is based on the lack of
requirement for any of the known targeting factors, and further tests
are certainly required to determine whether any unidentified factors
are involved, whether soluble or membrane-bound. For example, the wheat
germ translation system could conceivably contain soluble
chaperone-type molecules, and proteolysis of thylakoids does not digest
every thylakoid membrane protein.
This is the first direct demonstration that such complex multispanning
proteins can insert in the absence of SecA, SRP, and NTPs, and,
although the sample number is still small, these data raise the
possibility that this may be a mainstream pathway for multispanning
proteins as well as for single-spanning proteins of the
CF0II type. At the same time, a proportion of the
population of Elip2 and PsbS molecules can be targeted by an
alternative pathway when these factors are available. These molecules
may of course be targeted by the SRP pathway, and this possibility will
be addressed in future studies. The precise conditions of our in
vitro assays are different from those found in vivo,
and it is difficult to predict from these data alone the extent to which Elip2 and PsbS insert via the "spontaneous" pathway in intact chloroplasts. However, A. thaliana knockout strains have
recently been generated in which cpSRP54 is totally undetectable (36), and the insertion of LHCP and other CAB family members is seriously affected in these seedlings during the early stages of growth. Notably,
the insertion of PsbS is completely unaffected, providing firm evidence
that cpSRP is not required for the efficient insertion of this protein
in vivo as well as in vitro and supporting the possibility that PsbS is spontaneously inserted in vivo.
These data have interesting implications for the role and mechanism of
cpSRP. Cross-linking studies (5, 37) have suggested that SRPs in
general bind preferentially to regions of particularly high
hydrophobicity, and stromal SRP was found to bind tightly to the third,
most hydrophobic region within pea LHCP but not to the other two spans
(37). Accordingly, this was proposed to be the SRP binding signal, and
the data suggest that the SRP pathway might be particularly important
for highly hydrophobic proteins. At the very least, our data show that
hydrophobicity does not correlate with an absolute
requirement for SRP. Hydropathy analysis of the proteins
analyzed in this study (not shown) indicates that transmembrane segment
III of pea LHCP is indeed more hydrophobic than segments I or II in
this protein, and this span reaches a figure of greater than 2.0 on the
GES scale (38). However, the corresponding regions within Elip2 and
PsbS (spans III and IV, respectively) are only slightly less
hydrophobic, and it is notable that other regions within these proteins
are very hydrophobic indeed, particularly the span II regions in both
proteins (this region in PsbS is by far the most hydrophobic among any
in the five proteins in this sample, according to this type of
prediction). If, as seems likely, these regions can bind to cpSRP
in vitro, our data argue that SRP cross-linking does not
necessarily reflect an essential role for this targeting
factor. At the same time, these may be the regions that provoke binding
by SRP and which thereby initiate the targeting of some Elip2 and PsbS
molecules by the assisted pathway.
In this study we have shown that two further members of the CAB family,
Lhca1 and Lhcb5, are able to use a pathway that requires stroma, NTPs,
and protein translocation machinery. If this involves SRP (as seems
likely, but which remains to be addressed) the data for Lhca1 may have
other implications for the SRP pathway. Lhca1 was chosen for analysis
because all three transmembrane spans are of relatively low
hydrophobicity, yet this protein is totally dependent on stroma and
NTPs for integration. If cpSRP is indeed required for the targeting of
this protein, this would indicate that the cpSRP binding signal is
probably more complex than a simple region of high hydrophobicity.
Irrespective of the mechanism by which SRP selects its substrates, the
striking observation is that Elip2 and PsbS can insert in the absence
of any known targeting machinery, whereas three other members of the
same family are completely reliant on each of these factors. In
particular, they are totally unable to insert in the absence of stroma.
A possible explanation for these data might be that three of the
proteins (LHCP, Lhcb5, and Lhca1) are simply import-incompetent in the
absence of cpSRP (perhaps being prone to aggregation), whereas Elip2
and PsbS are more stable in solution. However, this explanation may be
far too simplistic, and an important future aim should be to determine
whether the SRP requirement correlates with other structural features
in this family of membrane proteins.
The spontaneous insertion pathway is of significant interest, and,
given the operation of broadly similar SRP- and
Sec-dependent pathway in bacteria, we predict that
bacterial membrane proteins will emerge with similar insertion
mechanisms. Further work on this topic is certainly merited to unravel
the crucial early events in this particular insertion process, and it
will be especially important to determine whether soluble factors other
than SRP are involved in the soluble phase of the pathway. The lack of requirement for ATP or GTP precludes several obvious candidate "chaperone" proteins such as FtsY or members of the Hsp60 or Hsp70 family, and it is therefore possible that this type of protein can
maintain insertion-competence without the aid of soluble factors. Very
little is known about this element of the insertion pathway, or indeed
about the actual insertion event, but proteins such as Elip2 and PsbS
may be good subjects for this form of analysis.