From the Department of Biological Sciences, University of Warwick,
Coventry CV4 7AL, United Kingdom
A group of membrane proteins are synthesized with
cleavable signal sequences but inserted into the thylakoid membrane by
an unusual Sec/SRP-independent mechanism. In this report we describe a
key intermediate in the insertion of one such protein, photosystem II
subunit W (PSII-W). A single mutation in the terminal cleavage site
partially blocks processing and leads to the formation of an
intermediate-size protein in the thylakoid membrane during chloroplast
import assays. This protein is in the form of a loop structure: the N
and C termini are exposed on the stromal face, whereas the cleavage
site has been translocated into the lumen. In this respect the
insertion of this protein resembles that of M13 procoat, which also
adopts a loop structure during insertion, and we present preliminary
evidence that a similar mechanism is used by another thylakoid protein,
PSII-X. However, whereas the negatively charged region of procoat is
translocated by an apparently electrophoretic mechanism using the
µH+, the corresponding region of PSII-W is
equally acidic but insertion is
µH+
independent. We furthermore show that neutralization of this region has
no apparent effect on the insertion process. We propose that a central
element in this insertion mechanism is a loop structure whose formation
is driven by hydrophobic interactions.
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INTRODUCTION |
The biogenesis of biological membranes requires the synthesis of
numerous hydrophobic proteins and their insertion into membrane bilayers, and intensive efforts have been made to understand the mechanisms involved. The majority of bacterial, chloroplast, and mitochondrial membrane proteins are inserted post-translationally, and
interest has centered on the mechanisms by which these proteins are
initially maintained in a soluble form, and how the hydrophobic regions
are then transferred from an aqueous milieu into the apolar regions of
the membrane bilayer. Some elements of the insertion events have been
characterized in detail, one example being the influence of positive
charges on overall topology, since these residues have a strong
tendency to remain on the cis side of the membrane (1). It
has also proved possible to recognize two broad categories of insertion
mechanism, which can be regarded as "assisted" and "unassisted"
according to whether protein translocation machinery is relied upon. In
bacteria, for example, the secretion (Sec) apparatus used for the
translocation of periplasmic proteins is also required for the
insertion of some membrane proteins (2-5). In addition, another
element of the export machinery, signal recognition particle
(SRP),1 is
involved in the insertion of a range of membrane proteins (6-8). Other
proteins, however, are integrated by Sec-independent mechanisms and
these may well insert spontaneously into the plasma membrane (9-12).
However, whereas the overall requirements have been detailed for
several membrane proteins, the actual insertion mechanisms are poorly
understood in most cases. In vitro reconstitution assays
favored for the study of protein translocation have been of limited use
because most hydrophobic proteins are inherently unstable in aqueous
solution. Few proteins have been examined in genuine mechanistic
detail, although the coat proteins of the M13 and Pf3 phages are
notable exceptions that have been shown to integrate into the
Escherichia coli membrane by Sec-independent, possibly
spontaneous mechanisms (reviewed in Ref. 9).
Our knowledge of membrane protein insertion is particularly vague in
the case of thylakoid proteins. Most thylakoid proteins are imported
after synthesis in the cytosol, and at least four distinct pathways
have been identified for their subsequent targeting into and across the
thylakoid membrane (reviewed in Refs. 13 and 14). Two of the pathways
apply to thylakoid lumen proteins, which are imported by means of
bipartite presequences containing envelope transit and thylakoid
transfer signals in tandem. After import into the stroma, the transfer
signal directs transport across the thylakoid membrane by either an
ATP-dependent, Sec-related mechanism, or a very different
mechanism that relies on the thylakoidal
pH. After translocation,
the stromal intermediates are processed to the mature forms by a
lumen-facing thylakoidal processing peptidase, TPP (15). The thylakoid
transfer signals of these proteins are all similar in overall structure
to bacterial signal peptides, but specify translocation by only one of
these mechanisms (16-18).
Two further pathways have been identified for integral thylakoid
membrane proteins. The multispanning membrane protein, LHCP, is
synthesized with an envelope transit signal only, and hence integrates
into the thylakoid membrane by means of information contained in the
mature protein (19, 20). The integration process requires GTP and a
stromal homologue of the 54-kDa protein of signal recognition particles
(21), and is thus likely to be similar in certain respects to the
SRP-dependent pathway identified in bacteria. However, a
very different mechanism has been demonstrated for three other
thylakoid proteins: subunit II of the integral CFo
component of the thylakoidal ATP synthase (CFoII) and
subunits X and W of photosystem II (PSII-X and PSII-W). These proteins are synthesized with bipartite presequences that strongly resemble typical Sec-type signal peptides (22-24), yet they integrate into thylakoid membranes in the absence of stromal factors, nucleoside triphosphates, or a
pH (25, 26). Furthermore, mild proteolysis of
thylakoids blocks the Sec-, SRP-, and
pH-dependent
mechanisms but has no effect on the insertion of CFoII,
PSII-W, and PSII-X (26, 27). It has therefore been proposed that they
insert spontaneously into the thylakoid membrane. This particular
insertion mechanism is highly unusual in that no other mainstream
insertion process studied to date involves the use of cleavable signal
peptides for "unassisted" membrane insertion, and M13 procoat is in
fact the only other protein known to use this type of mechanism (see below). In this report we have analyzed this integration pathway in
greater detail in order to elucidate mechanistic details of the
membrane-insertion events. We describe a loop intermediate that is
central to the insertion process and we present evidence that
hydrophobic interactions are the driving force for insertion.
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EXPERIMENTAL PROCEDURES |
Synthesis of Precursor Proteins--
Precursors of
Arabidopsis thaliana PSII-W and PSII-X were synthesized by
transcription in vitro of cDNA clones followed by translation of capped transcripts in the presence of
[3H]leucine as described (24, 26). Mutated forms were
prepared by oligonucleotide-directed site-specific mutagenesis of
cDNAs using an Amersham International kit according to the
manufacturer's instructions.
Import Assays and Topological Analysis--
Intact chloroplasts
were prepared from leaves of 8-9-day-old pea seedlings (Pisum
sativum, var. Feltham First) by Percoll pad centrifugation and
used in import assays as described (24-26). Proteolysis of thylakoid
membranes was carried out in 10 mM Hepes, pH 8.0, 5 mM MgCl2 (HM buffer) unless otherwise specified
in the figure legends, using thylakoid suspensions at 1 mg/ml
chlorophyll. Sonication involved the use of a sonicating water bath at
0 °C. Carbonate washing of thylakoids was carried out as detailed by Michl et al. (25).
Sequence Analysis--
The sequences of PSII-X proteins were
extracted from data base entries for the plastid genomes of
Odontella sinensis, Cyanophora paradoxa, and
Porphyra purpurea (accession numbers Z67753, U30821, and
U38804, respectively).
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RESULTS |
Cleavage by TPP Is Not Required for the Insertion of
PSII-W--
Pre-PSII-W and pre-PSII-X share overall structural
similarities with M13 procoat and appear likewise to insert into their target membrane without the aid of translocation machinery. However, there are significant differences in terms of insertion requirements (see below) and we have set out to investigate the thylakoid proteins in greater detail. Our first aim was to determine whether the cleavable
signal-type peptides of the thylakoid proteins are used for a similar
purpose: the formation of a loop intermediate in which the hydrophobic
region (H-domain) spans the membrane. This is probably the most
important role of the M13 signal peptide and our priority was to
determine whether such an intermediate is a core feature in the
biogenesis of PSII-W and/or PSII-X. We addressed this question by
inhibiting the cleavage step catalyzed by TPP (whose active site is in
the thylakoid lumen; Ref. 15), in order to probe the location and
topology of this protein immediately prior to the final maturation
step. Fig. 1 shows the structures of the
relevant sections of both precursors, including the signal peptide and
the mature region of the mature protein. TPP is known to depend on the
presence of short-chain residues at the
3 to
1 positions in the
substrate, relative to the processing site, and Ala at the
1 position
is essential with even Ser and Gly unable to support efficient cleavage
(28). We therefore introduced Thr at this position in both pre-PSII-W
and pre-PSII-X, in the expectation that processing would be drastically
affected without substantially altering the characteristics of the
translocated region.

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Fig. 1.
Structural features of CFoII
(C),PSII-W (W), PSII-X (X), and M13
procoat (Pc). Top panel, the figure illustrates
the full sequence of M13 procoat, spinach CFoII, and PSII-W
and PSII-X from A. thaliana; the thylakoid
proteins start with the signal-type peptides (the N-terminal envelope
transit peptides are omitted). The extreme C-terminal region of
CFoII has also been omitted. Hydrophobic domains are
underlined, acidic residues are shown italicized and the TPP
cleavage site is denoted by an asterisk. The figure also
shows (in bold) the changes introduced into two pre-PSII-W
mutants, W/E88Q and W/D82N,E83Q, and the locations of the individual
sections within iPSII-W (membrane, lumen, or stroma exposed) deduced in
this study. Bottom panel, alignment of PSII-X sequences from
Arabidopsis (A. thal), O. sinensis
(O. sinen), C. paradoxa (C. para), and
P. purpurea (P.purp). The Arabidopsis
sequence includes the signal peptide of the presequence region; the
remaining sequences are given in full. Identical residues are denoted
by asterisks; conserved residues by dots.
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The import characteristics of the PSII-W mutant (W/A78T) are shown in
Fig. 2. The wild-type precursor protein
is imported into the chloroplasts, processed to the mature size, and
fractionation tests confirm that this polypeptide is located
exclusively in the membrane fraction (lane T). Protease
treatment of the thylakoid membranes (lane T+) results in
digestion of the exposed C-terminal region (as found in previous
studies on PSII-W (29)), and the production of a slightly smaller
degradation product. The mutant protein, on the other hand, is
converted to a mixture of mature- and intermediate-size proteins, the
latter form presumably resulting from the action of stromal processing
peptidase, which cleaves the envelope transit domains from most
bipartite presequences. Importantly, this iPSII-W form is also found
only in the thylakoid fraction and further tests show that it is
completely resistant to carbonate extraction, because after this
treatment both the intermediate and mature bands are recovered only in
the pellet fraction (lane P). Since this procedure
effectively removes extrinsic proteins from the thylakoid membrane
(25), we can exclude the possibility that the action of TPP is required
for the insertion of this protein. Thermolysin treatment of the iPSII-W
results in a mobility shift that is comparable to that of mature
PSII-W, confirming that at least one section of the intermediate is
exposed on the stromal face of the thylakoid membrane.

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Fig. 2.
Stable insertion of PSII-W occurs in the
absence of cleavage by thylakoidal processing peptidase.
Pre-PSII-W (top panel) or pre-PSII-W containing an
Ala78 Thr mutation (W/A78T) were imported into intact
chloroplasts. After import, samples were analyzed of the total
chloroplast fraction (lane C), protease-treated chloroplasts
(lane C+), and of the stromal (S) and thylakoid
(T) fractions after lysis. Lane T+,
protease-treated thylakoids (200 µg/ml thermolysin for 30 min on
ice). Lanes Tr, translation products. In the case of the
W/A78T import, further samples of the thylakoids were subjected to
carbonate washing (see "Experimental Procedures") and samples were
analyzed of the pellet and supernatant fractions (P, S). Int,
intermediate-size form of PSII-W; DG1 and DG2
denote proteolytic degradation products.
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A Loop Intermediate in the Insertion of PSII-W--
The topology
of the iPSII-W polypeptide was mapped by determining the accessibility
of sites for trypsin and thermolysin cleavage. Fig. 1 shows that the
PSII-W mature protein contains only a single basic residue which is
located in the lumenal N-terminal region (Arg at position 5); the
presequence contains many basic residues but these are all located
prior to the H-domain in the signal peptide. In a loop structure these
sites are predicted to be on the lumenal and stromal sides of the
membrane, respectively. Fig. 3A shows that trypsin cleaves
iPSII-W to a smaller form that remains significantly larger than
mature-size PSII-W; low concentrations of trypsin (e.g. 10 µg/ml) generate a mixture of iPSII-W and a smaller degradation
product, whereas 25 µg/ml is sufficient to fully convert the iPSII-W
to the degradation product. Trypsin therefore cleaves the N terminus of
the iPSII-W signal peptide on the stromal surface of the membrane. A
loop intermediate would also contain the C terminus of the mature
protein on this face of the membrane, and Fig. 3B shows that
this is the case. The C terminus of mature PSII-W is exposed on the
stromal face and the data in Fig. 2 showed that it is cleaved by
thermolysin; Fig. 3B shows that thermolysin also cleaves a
small fragment from iPSII-W, generating a degradation product similar
in mobility to that produced by trypsin (compare lanes Th
and Tp). Significantly, incubation of iPSII-W with both
thermolysin and trypsin generates a much smaller degradation product
(lane Th/Tp) indicating that trypsin and thermolysin cleave
at different ends of the molecule (the N and C termini, respectively).
Lane Tp/son of Fig. 3B provides final
confirmation of a loop structure: thylakoids containing iPSII-W were
incubated with trypsin and the thylakoids were sonicated to allow
access of the protease to the lumenal side of the membrane. Both the
mature PSII-W and the iPSII-W are almost quantitatively converted to a
polypeptide that is smaller than mature PSII-W, indicating that
cleavage has taken place after the Arg at position 5 in the mature
protein, which must therefore be exposed on the lumenal side of the
membrane.

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Fig. 3.
A loop intermediate in the PSII-W insertion
process. The W/A78T mutant described in Fig. 2 was imported into
chloroplasts and the thylakoid fraction isolated. A,
thylakoids were incubated with trypsin (at the concentrations indicated
above the lanes) for 30 min on ice. DG denotes
degradation product generated from the iPSII-W polypeptide.
B, the thylakoids were incubated with 200 µg/ml
thermolysin (Th), 25 µg/ml trypsin (Tp), or 200 µg/ml thermolysin followed by 25 µg/ml trypsin (Th +Tr).
In the latter case, the thylakoids were washed once in 1 ml of HM
buffer between protease treatments. A further sample was incubated with
25 µg/ml trypsin and sonicated for 5 min (Tp + son). All
samples were incubated for 30 min on ice.
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Evidence Favoring a Similar Insertion Process for PSII-X--
A
similar approach was also taken to investigate the insertion of PSII-X
because it is as yet unclear whether the three thylakoid proteins
currently viewed as a group (CFoII, PSII-X, and PSII-W) actually insert by similar mechanisms. The cleavage site of pre-PSII-X was similarly mutated by the introduction of a Thr at the
1 position and the effects examined as for PSII-W. Fig.
4 shows that this mutation likewise
inhibits processing by TPP although the effects are in fact more
drastic; analysis of the thylakoid fraction following an import
reaction shows that hardly any mature PSII-X is detectable, indicating
that the action of TPP is almost totally blocked. Once again, an
intermediate-size form (int) accumulates which is presumed to result
from the action of stromal processing peptidase. This intermediate form
is located exclusively in the thylakoid membrane showing that TPP
action is not required for the insertion of PSII-X.

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Fig. 4.
Cleavage by TPP is not required for the
insertion of PSII-X. Pre-PSII-X containing an Ala78
Thr mutation (X/A74T; lane Tr) was imported into intact
chloroplasts. After import, samples were analyzed of the total
chloroplast fraction (lane C), protease-treated chloroplasts
(lane C+), and the stromal (S) and thylakoid
(T) fractions after lysis. Int, intermediate-size
form of PSII-X. Pre denotes precursor protein.
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Topology studies were carried out on the iPSII-X polypeptide but the
results were not as clear-cut as those obtained with PSII-W. Thylakoids
containing the imported, mature-size wild-type protein were incubated
with thermolysin, Staphylococcus V8 protease (V8), a mixture
of thermolysin/V8, and trypsin, but no cleavage was apparent
(left-hand panel of Fig. 5).
Apparently, only a small region of mature PSII-X protrudes into the
stroma. Unfortunately, the membrane-bound intermediate form is also
resistant to proteolysis as shown in the right-hand panel;
thermolysin and trypsin fail to generate defined cleavage products, and
only V8 of the proteases tested is able to cleave the protein. However,
this takes place only at very high concentrations (200 µg/ml for 60 min) and some penetration into the lumen clearly occurs because the
cleavage product (lane V) is similar in size to the mature
protein. This presumably means that the V8 cleaves at one or both of
the Glu residues in the C-terminal region of the signal peptide, just before the cleavage site (see Fig. 1). However, it is notable that this
cleavage is much more efficient when the thylakoids are sonicated in
the presence of V8 to allow complete access. Under these conditions the
intermediate is quantitatively converted to the degradation product
(lane Vs) providing evidence, albeit circumstantial, that
this region is in the lumen. This would imply the formation of a loop
intermediate as with PSII-W, but further tests will be required to
confirm this point.

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Fig. 5.
Topological analysis of iPSII-X. PSII-X
and the X/A74T mutant were imported into chloroplasts as described in
the legend to Fig. 4 and the thylakoid fractions (denoted ) incubated
with proteases as follows: 200 µg/ml thermolysin (T), 200 µg/ml Staphylococcus V8 (V), thermolysin
followed by V8 (T/V), or 25 µg/ml trypsin (Tp).
Lane Vs, V8 was incubated with thylakoids and the vesicles
were sonicated to allow access of the protease into the lumen. All
protease treatments were for 30 min on ice, except the V8 incubations
that were for 60 min on ice. Int, intermediate form.
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Evidence That Hydrophobic Forces Drive the Insertion of
Pre-PSII-W--
Studies in bacteria have shown that two types of force
can be instrumental in driving protein insertion. Hydrophobic forces appear to be important in all cases, reflecting the loss of free energy
achieved by moving hydrophobic regions into the lipid bilayer, but
electrophoretic forces are also vital in the insertion of both Pf3 coat
protein and M13 procoat. In both cases, insertion depends on the
µH+ (positive outside) which is believed to
drive the translocation of negatively charged residues across the
membrane. The translocated regions of CFoII, pre-PSII-X,
and pre-PSII-W are all similar to that of procoat in terms of charge:
the N-terminal regions of mature coat protein, CFoII, and
PSII-W contain net charges of
3,
3, and
2, respectively, and,
although mature PSII-X contains no negative charges at the N terminus
of the mature protein, the translocated region is nevertheless acidic
due to the two Glu residues in the C-terminal domain of the signal
peptide (see Fig. 1). Although the insertion of the thylakoid proteins
is unaffected by dissipation of the thylakoidal
µH+ in each case (23, 24), we deemed it
important to determine whether insertion might be assisted by a
smaller, standing charge difference across the thylakoid membrane that
might be important in the insertion process even in the absence of a
photosynthetically driven
µH+. This
possibility was tested by mutating acidic residues in the translocated
region of pre-PSII-W as shown in Fig. 1. In one of the mutants (W/E88Q)
the net charge of this region was reduced to
1 and the second mutant,
W/D82N,E83Q contained a neutral translocated region. Fig.
6 shows that both proteins are imported
and efficiently inserted into the thylakoid membrane; the
protease-treated chloroplasts contain only mature-size PSII-W which is
located exclusively in the membrane fraction. The mutations therefore
have no detectable effect on the import characteristics and we conclude
that electrophoretic forces play no role in the insertion of this
protein.

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Fig. 6.
Electrophoretic forces are not required for
the insertion of PSII-W. The pre-PSII-W mutants W/E88Q and
W/D82N,E83Q (see Fig. 1) were imported into intact chloroplasts and the
organelles fractionated as described in the legend to Fig. 2.
Lanes T+, the thylakoid fraction was treated with
thermolysin as detailed in the legend to Fig. 2.
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DISCUSSION |
CFoII, PSII-W, and PSII-X represent an unusual group
of membrane proteins in that no other membrane protein, with the
exception of M13 procoat, has been found to rely on a cleavable signal
peptide for membrane insertion by a Sec- and SRP-independent mechanism. As shown in Fig. 1, the three thylakoid proteins share additional common features with procoat: each contains a single transmembrane span
in the mature protein and the translocated regions are all negatively
charged. It was thus unsurprising that initial models for the
Sec-independent insertion of CFoII (25) were based on the
procoat insertion model, and this study has confirmed that in one
fundamental respect the insertion of PSII-W does indeed resemble that
of procoat. Both proteins adopt a loop intermediate during the
insertion process, strongly suggesting that the function of the PSII-W
signal peptide is to provide a second hydrophobic domain which, in
concert with the transmembrane segment in the mature protein, is then
able to drive the translocation of the hydrophilic intervening region
into the thylakoid lumen. Interestingly, signal-type peptides appear
also to form loop structures when directing soluble proteins across
membranes by means of protein translocation systems (30, 31).
The experimental evidence favoring a Sec/SRP-independent insertion
mechanism (23-27) is consistent with the unusual evolution of the
signal peptides. Genes encoding PSII-X and CFoII have been identified in cyanobacteria and also within the plastid genomes of
several eukaryotic algae/diatoms, such as O. sinensis and
C. paradoxa. None of these cyanobacterial or plastid-encoded
proteins is synthesized with a signal-type peptide, strongly suggesting that the signal peptides were acquired after the transfer of these genes to the nucleus in higher plants. This situation contrasts starkly
with "genuine" Sec-type signal peptides such as those found in the
precursors of lumenal proteins, because the
cyanobacterial/plastid-encoded counterparts of these proteins are
invariably synthesized with classical signal peptides. We therefore
believe that the signal peptides of these membrane proteins differ from
Sec-type signal peptides in terms of both function and origin. However,
we can detect no large-scale structural differences among the
mature proteins that might explain why nuclear-encoded,
imported proteins are now synthesized with signal peptides. Mature
PSII-X proteins, for example, are structurally very similar in higher
plants and cyanobacteria (see Fig. 1). Given these similarities, it may
be simplistic to suggest that the signal peptide functions simply by
providing a critical, additional hydrophobic segment, because the
mature proteins in cyanobacteria and the above mentioned algae can
clearly insert despite possessing only a single hydrophobic region. The
only obvious difference concerns the charge distributions in the
N-terminal regions; these regions are neutral in the cyanobacterial and
plastid-encoded proteins whereas that of Arabidopsis PSII-X is basic due to the Lys at position 10. This could conceivably render
the translocation process more difficult to the extent that the
additional hydrophobic region is required to aid insertion. However, it
is also possible that this unusual insertion mechanism has been forced
on these proteins as a consequence of their more complex insertion
pathway; the proteins may insert co-translationally in cyanobacteria,
in which case the protein may never be free in the aqueous phase and
insertion may be more favorable. It is even possible that the signal
peptide renders the precursor protein more stable in solution, perhaps
by forming a "helical hairpin" in which the signal peptide
partially masks the hydrophobic character of the transmembrane segment.
Structural studies on this type of precursor protein should prove
instrumental in resolving these points.
Although the insertion of these thylakoid proteins is reminiscent of
M13 procoat insertion, there are equally significant differences in the
mechanisms used. The first concerns the initial events in the insertion
process. Gallusser and Kuhn (32) have shown that electrostatic
interactions play a critical role in the insertion of procoat, in which
basic residues in both the extreme N and C termini bind to the
negatively charged membrane surface; removal of either set
of basic residues renders procoat wholly insertion incompetent. Like
procoat, pre-CFoII contains positively charged N and C
termini and a similar mechanism was considered possible for this
protein (25). However, the C-terminal region of pre-PSII-W is extremely
negatively charged (a series of five acidic residues follows the
transmembrane section), precluding any electrostatic binding between
this region and the membrane surface. Further tests are required to
determine whether the basic N-terminal region plays any role through an
electrostatic binding to the thylakoid surface.
A second difference concerns the energetics of insertion. Most
Sec-dependent bacterial proteins are at least partially
dependent on the transmembrane potential for efficient insertion, but
the best characterized Sec-independent proteins (M13 procoat and Pf3 coat) are entirely dependent on the
µH+
(10, 33) In both cases this has been attributed to an essential role of
the membrane potential in driving the translocation of negatively
charged regions through the E. coli plasma membrane by an
electrophoretic mechanism (34). In contrast, dissipation of the
thylakoidal
µH+ has essentially no effect
on the insertion of CFoII, PSII-X, or PSII-W (23, 25, 26).
In the absence of any apparent role for electrophoretic forces, we
propose that hydrophobic interactions between the H-domains in
pre-PSII-W and the membrane interior are the primary driving force for
membrane insertion. It must be emphasized at this point that similar
forces are clearly crucial in the procoat insertion process, because
mutant forms containing positively charged translocated regions can be
inserted even in the absence of a
µH+ (35).
Nevertheless, our data show that the two systems have very different
tolerances and it remains to be determined how such superficially
similar translocated regions can be so potential dependent in E. coli but not in thylakoids. One possibility under consideration is
that these findings reflect differences in the lipid compositions of
the two membranes. The thylakoid membrane is most unusual in that
galactolipids account for over 80% of membrane lipids, whereas
phospholipids predominate in most membranes including those of E. coli. It has been proposed that one of the major thylakoid lipids,
monogalactosyldiacylglycerol, has a pronounced ability to form
non-bilayer structures (36) and we speculate that this property may
facilitate translocation of polar regions across the thylakoid
membrane. In general, the actual insertion events are poorly
characterized for those proteins that insert without the benefit of
proteinaceous translocation apparatus, and structural studies are
clearly required for an understanding of the conformational changes
taking place.