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INTRODUCTION |
Early light-induced proteins
(Elips)1 are among the first
light-induced proteins that accumulate transiently in developing plastid membranes (1, 2). In mature green plants, Elips and Elip-like
proteins are induced in response to various stress conditions (reviewed
in Ref. 3).
The Elips in higher plants are nucleus-encoded proteins
synthesized on cytoplasmic ribosomes in their precursor forms (pElips) and post-translationally imported into chloroplasts (1, 4). Prior to
insertion into the thylakoid membranes pElips are processed to their
mature forms by a stromal processing peptidase (5), which cleaves the
N-terminal leader sequence (6). A spontaneous insertion mechanism into
thylakoid membranes has been reported for Elips in barley (6) and
Arabidopsis thaliana (7). The insertion of these proteins
into membranes occurred in the complete absence of signal recognition
particle, SecA activity, nucleoside triphosphates, or a functional Sec
system (7).
The Elips are polytopic thylakoid membrane proteins with three
predicted transmembrane
-helices, where the helices I and III show
very high homology to the corresponding regions of the light-harvesting
chlorophyll a/b-binding proteins (Lhcps) of photosystem I (PSI) and II (PSII) (4). Recently, Elip-related proteins with two (8)
or one (9) predicted transmembrane helix have been described from
Arabidopsis. In barley, two multigene families of Elips were
reported (2, 4): the high molecular mass Elips (pElips between 24 and
27 kDa) and the low molecular mass Elips (pElips between 16 and 18 kDa). While the high molecular mass pElips are processed to mature
products of different sizes between 18.5 and 18.0 kDa, the low
molecular mass pElips give end products of the same size of 13.5 kDa.
Recent purification of Elips from light-stressed pea leaves has shown
that these proteins bind chlorophyll (Chl) a and lutein (10). However, very unusual pigment-binding characteristics were
reported for isolated Elips, such as a weak excitonic coupling between
Chl a molecules and an extremely high lutein content as compared with other Chl-binding proteins (10). A similar weak excitonic
coupling between Chls was previously reported for the Chl
a/b-binding 22-kDa protein (PSII-S) from PSII (11).
Interestingly, this protein is the only known Cab family member present
in etiolated seedlings and stable in the absence of Chls (12).
No definite function has been yet described for Elips in higher plants.
It was proposed that these proteins could act as ligand chaperones
required for transient binding of pigments during biogenesis or
turnover of Chl-binding proteins (13-15). Such a function would be
essential for the coordination between pigment biosynthesis and their
ligation as well as for reducing toxic effects of nonbound Chl molecules.
During greening of etiolated plants, accumulation of Elips in plastid
membranes occurs only at the time when the abundance of their
transcripts has already considerably declined (1, 2). This suggests the
existence of a post-transcriptional control in Elip expression.
In this work, the post-translational regulation of the expression of
low molecular mass Elips from barley was investigated using an in
vitro insertion system into plastid membranes. We showed that the
efficiency of the pElip processing depended on the developmental status
of etioplasts and increased with the etioplast age. Furthermore, the
processing of pElips was not influenced during early stages of
chloroplast differentiation in light. The stable insertion of Elips
occurred within greening plastid membranes but not in Chl-free
etioplast membranes. Since stromal or membrane factors are not required
for Elip insertion (6, 7), we investigated whether the quantity and/or
quality of pigments play a role during this process. We demonstrated
that the stable insertion of Elips into etioplast membranes was
promoted by the addition of Chl a but not Chl b
or xanthophyll zeaxanthin and that this process did not depend on light
intensity. However, when pElips were posttranslationally imported into
plastids isolated from greening leaves of barley, amounts of membrane
inserted Elips increased in a light intensity-dependent manner. This indicates that in addition to Chl a other
factors that are induced and/or regulated by a high intensity light
also control the accumulation of Elips in plastid membranes.
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EXPERIMENTAL PROCEDURES |
Plant Material--
For isolation of intact etioplasts, barley
seedlings (Hordeum vulgare cv. Apex) were grown on
vermiculite in complete darkness for 8 days at 25 °C. The apical
segments of primary leaves (4-5 cm) were detached and kept floating on
water at 4 °C prior to the isolation of etioplasts. All
manipulations on dark-grown plants were performed in complete darkness
without safety lights.
For isolation of pigments, barley plants were grown for 8 days on
vermiculite at 25 °C at a light intensity of 100 µmol·m
2·s
1
provided by white fluorescent lamps under a light regime of 12 h
of dark/12 h of light. Primary leaves were carefully removed from the
coleoptiles and were either directly frozen in liquid nitrogen and
stored at
70 °C or exposed to a high light intensity (2000 µmol·m
2·s
1)
for 6 h prior to storage.
Preparation of Radioactively Labeled pElip--
The low
molecular mass Elip clone HV60 (4, 6) was used for in vitro
transcription and translation as described (6). Translation mixtures
were diluted 3-5-fold with plastid suspension buffer (50 mM Hepes/KOH, pH 8.0, 330 mM sorbitol, and 8 mM methionine) and centrifuged for 30 min at 200,000 × g at 4 °C. The supernatant was used for in
vitro integration assays.
Extraction of Pigments--
Total pigments (Chls and
carotenoids) were extracted with 80% acetone as described (14). The
pigment mixture was separated by thin layer chromatography (TLC Silica
Gel 60, Merck), and the identities of pigments were proven by a
comparison of their spectral characteristics. Individual pigments were
re-extracted from the TLC plates with 80% acetone (Chls a
and b), hexane (
-carotene), or ethanol (lutein,
violaxanthin, zeaxanthin, antheraxanthin, and neoxanthin) as described
(12), dried under vacuum, and stored at
70 °C in aliquots.
Pigments were resuspended in ethanol/ether (1:1, v/v) to the
appropriate concentration prior to addition to the integration assays.
Isolation of Etioplasts and Integration Assay--
Intact
etioplasts were isolated according to Ref. 16. Integration assays were
performed as described (6) with some modifications. Isolated intact
plastids were osmotically disrupted in lysis buffer (10 mM
Hepes/KOH, pH 8.0, 10 mM methionine, and 5 mM
MgCl2) at a concentration of 100 mg of protein/ml. The
plastid lysate (100 µl) was combined with 15 µl of 0.1 M Mg-ATP (pH 8.0), 35 µl of Elip translation products,
and 2 µl of ethanol/ether solution with or without dissolved
pigments. The assays were incubated at 25 °C at a light intensity of
10 µmol·m
2·s
1
for 1 h and the integration reaction was stopped by the addition of 350 µl of ice-cold lysis buffer. Plastid membranes were pelleted by centrifugation at 8,000 × g for 10 min at 4 °C,
and corresponding supernatants were subjected to centrifugation at
40,000 × g for 20 min to remove residual membranes
prior to the precipitation of proteins with 5% trichloroacetic acid.
Integration of Elips into plastid membranes was verified by protease
protection assays and washes of membranes with a chaotropic salt or
alkali treatment.
Trypsin treatment of plastid membranes was performed at a protein
concentration of 100 mg of protein/ml and 80 µg/ml trypsin for 30 min
on ice as described (6). As a control, an aliquot of the membrane
suspension was incubated under the same experimental conditions in the
absence of trypsin.
Washes with 0.1 M Na2CO3 or 0.1 M NaOH were performed at a protein concentration of 0.5 mg
of protein/ml for 20 min at room temperature in the presence of trypsin
inhibitor (Sigma). Plastid membranes and extracted peripheral membrane
proteins were separated by centrifugation at 40,000 × g for 20 min at 4 °C, and the supernatant was used for
precipitation with 5% trichloroacetic acid (end concentration). Plastid membranes and precipitated extracted proteins were analyzed by
SDS-polyacrylamide gel electrophoresis and/or immunoblotting as
described below.
In Vitro Import--
In vitro import of low molecular
mass pElip was performed as described (4) using plastids isolated from
etiolated barley leaves exposed to light for 6 h. After import,
plastids were separated into stroma and membrane fractions (4), and the
protein composition of each fraction was analyzed by SDS-polyacrylamide
gel electrophoresis as described below.
Assay of Proteins--
Proteins were separated by
SDS-polyacrylamide gel electrophoresis according to Ref. 17. Equal
amounts of protein (50 µg) were loaded on the gels. The gels treated
for fluorography (18) were dried and exposed to x-ray film at
70 °C. For quantification, signals linear in intensity with
exposure time (A600 < 0.8) were scanned at 600 nm (Personal Densitometer, Molecular Dynamics) using the ImageQuant 3.3 program.
Immunoblotting was carried out using polyvinylidene difluoride
membranes with 45-µm pores (Millipore Corp.) according to Ref. 19. Blots were incubated with polyclonal antibodies raised against LhcpII or subunit
of the CF1-ATPase complex, and the signal was
detected by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech).
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RESULTS |
Stable Insertion of Elips into Plastid Membranes Depends on the
Stage of the Thylakoid Greening and Not on the Developmental Stage of
Etioplasts--
Insertion of low molecular mass Elips into membranes
was investigated using plastids isolated from etiolated barley leaves or leaves exposed to light for 3 or 6 h. After exposure to light, the pigment content in plastids increased considerably with the time of
illumination. While etioplasts contained no detectable amounts of Chls
and 0.007 mg of total carotenoids (calculated per 100 mg of membrane
proteins), these amounts increased to 0.117 mg of chlorophylls and
0.021 mg of total carotenoids or to 1.65 mg of chlorophylls and 0.052 mg of total carotenoids after 3 or 6 h of illumination, respectively.
The results (Fig. 1) demonstrated that
the greening stage of plastids did not significantly change the
processing efficiency of pElips. Comparable amounts of mature Elips
were found under all conditions tested (Fig. 1A,
lanes S). Furthermore, independently of the
greening stage of plastids, both pElips and Elips were almost equally
distributed between soluble and membrane fractions prior to the trypsin
treatment (Fig. 1A, compare lanes S
and M
). When a membrane insertion of Elips was verified by
a protease protection assay, the 10-kDa trypsin-resistant Elip fragment
was detected in the membranes isolated from plastids exposed to light. The amounts of these fragments were higher in the membranes isolated from 6-h than from 3-h illuminated barley seedlings (Fig.
1A, compare lanes M+ in the
middle and right panels). As reported before (6) the trypsin-protected Elip fragment resulted from the
cleavage of the 2-4-kDa peptide from the stroma-exposed N terminus of
this protein. Such Elip fragments were not generated when trypsin
treatment was performed in the absence of added membranes (not shown).
A relatively weak appearance of the mature Elips and their tryptic
fragments in fluorograms as compared with pElips resulted from the loss
of 4 out of 10 labeled [35S]methionine residues after
processing of pElips.

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Fig. 1.
Insertion of Elips into plastid membranes
isolated from barley seedlings at different stages of greening.
A, The low molecular mass Elip precursors (pElips, clone
HV60) were translated in vitro in the presence of
[35S]methionine, and translation products
(lane Tls) were incubated with lysed plastids
(equivalent to 10 mg of proteins) isolated from etiolated barley leaves
exposed to light (100 µmol·m 2·s 1)
for 0, 3, or 6 h. Integration assays were then fractionated into
soluble (lanes S) and membrane (lanes
M) fractions, and the membrane fraction was subjected to
trypsin treatment (+), and as a control a mock treatment ( ) was
performed. Equal amounts of proteins (50 µg) of each fraction were
separated on 17% SDS-polyacrylamide gels, and radioactively labeled
proteins were visualized by fluorography. Bars indicate
positions of pElips, mature forms of Elips, and the 10-kDa
trypsin-resistant Elip fragment (Tryptic fragment).
B, the trypsin-treated thylakoid membranes (lanes
1) were subjected to washes with 0.1 M NaOH or
0.1 M Na2CO3 as described under
"Experimental Procedures" and separated into soluble
(lanes 2) and membrane (lanes
3) fractions, containing either peripheral or integral
membrane proteins, respectively. As references, the distribution of the
subunit of the CF1 ATPase complex (CF1-a) and the major
Chl a/b-binding protein of PSII (Lhcb2) was
assayed by immunoblotting.
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In contrast to greening plastid membranes, protease-protected Elip
fragments were not detected in membranes isolated from dark-grown
barley leaves (Fig. 1, lane M+ in left
panel). The complete degradation of Elips by trypsin
suggested that these proteins were not protected by a lipid bilayer but
only associated with the membrane surface and thus accessible to the
trypsin digestion. Interestingly, small amounts of pElips and Elips
were found to be resistant to the proteolytic treatment, and these
bands are visible, to various extents, in most of the experiments. We
cannot explain at this point whether this effect resulted from the
shielding of potential protease cleavage sites by certain protein
conformations. This effect was also present when higher trypsin
concentrations or longer incubation times were applied (not shown). The
same phenomenon was reported for LhcpII (20).
As an additional proof for an integral membrane location of Elips and
their trypsin-resistant fragments, washes of membranes with a
chaotropic salt or alkali were performed. These treatments are known to
remove proteins that are only loosely associated with membranes as well
as removing peripheral membrane proteins (21). Upon the treatment of
membranes with 0.1 M NaOH or 0.1 M
Na2CO3, both Elips and their tryptic fragments
remained in the membrane fraction, confirming their integral location
(Fig. 1B, upper panel). In contrast,
pElips were extracted from the membranes, both with NaOH and
Na2CO3, which proved that the presence of
pElips in the membrane was a result of unspecific association of these proteins with the membrane surface rather than a specific binding. A
similar nonspecific association of pElips with membranes was reported
to occur during the insertion of Elips into isolated thylakoid
membranes (6).
The distribution of Lhcb2, an integral antenna protein of PSII, and a
peripheral located subunit
of the CF1-ATPase complex was assayed
under the same treatment conditions and is shown as a reference (Fig.
1B, lower panels).
The failure of etioplast membranes to accumulate stably inserted Elips
might be related to the developmental state of the plastid. To prove
whether the developmental stage of etioplasts can influence the
insertion of Elips into membranes, three sets of etioplasts, isolated
from basal, middle, or apical segments of barley leaves, were used for
our studies. The basal segments contained meristematic tissue with
proplastids, whereas cells at the leaf tip were fully differentiated
and contained mature etioplasts (22). The results revealed (Fig.
2) that only the efficiency of pElip
processing and not the insertion of Elips was influenced by the
differentiation state of plastids. While in plastids isolated from
basal segments of leaves only 12% of pElips were found to be processed
to their mature forms, in plastids isolated from middle or apical
segments, ~25 or 40% of pElips were processed, respectively. This
calculation was based on the quantification of the radioactive label
incorporated into pElips and Elips. The numbers were corrected for the
loss of label that occurred during pElip processing (4). Independent of
the developmental stage of etioplasts, no stable insertion of Elips
into membranes was detected (Fig. 2).

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Fig. 2.
Insertion of Elips into etioplast membranes
isolated from developmentally different segments of a barley leaf.
Insertion assays were performed as described in Fig. 1 using lysed
plastids (equivalent to 10 mg of protein) isolated from basal, middle,
or apical segments of 8-day-old etiolated barley leaves.
Bars indicate positions of pElips and mature forms of
Elips.
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Stable Insertion of Elips into Etioplast Membranes Is Stimulated by
the Addition of Pigments in a Concentration-dependent
Manner--
The possibility that the insertion of Elips into plastid
membranes might depend on pigment content and/or composition was tested
using etioplast lysates complemented with Chls and carotenoids extracted from green barley leaves. Since it was reported that in
mature green plants Elips are stable only under light stress conditions
and degraded rapidly after lowering of light intensity (13-15), two
sets of pigments, isolated from low light (LL)-treated or from high
light (HL)-treated plants, were used for our studies. In addition to
quantitative differences in amounts of particular carotenoids, the HL
mixture of pigments contained zeaxanthin instead of violaxanthin, which
was present in the LL pigment mixture. Zeaxanthin is a constituent part
of the protective xanthophyll cycle and is formed in thylakoid
membranes from violaxanthin under conditions of light stress (23). The
insertion assays of Elips in etioplast membranes complemented with LL
or HL pigment mixtures are shown in Fig.
3. In the absence of added pigments
(control assays), no protease-resistant Elip fragments were detected
(Fig. 3, left panel). Comparable amounts of
trypsin-protected Elip fragments were obtained in etioplast membranes
regardless of which pigment mixture had been added to insertion assays
(Fig. 3, middle and right panels).
Since the LL pigment mixture did not contain zeaxanthin, it can be
concluded that the presence or the absence of zeaxanthin did not
influence the insertion of Elips into membranes under conditions
tested.

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Fig. 3.
Insertion of Elips into etioplast membranes
complemented with pigments isolated from control or light
stress-treated green barley leaves. The low molecular mass pElips
translated in vitro (lane Tls) were
incubated with lysed plastids (equivalent to 10 mg of protein) isolated
from 8-day-old etiolated barley leaves in the presence or absence of 65 µg of total pigments extracted from green barley leaves grown at low
light intensity (+LL, 100 µmol·m 2·s 1)
or exposed to light stress (+HL, 2,000 µmol·m 2·s 1)
for 6 h. Both pigment mixtures contained 37 µg of Chl
a, 13 µg of Chl b, and 15 µg of total
carotenoids. The carotenoid composition in the HL pigment mixture was
3.3 µg of -carotene, 4 µg of lutein, 3 µg of zeaxanthin, 1 µg of antheraxanthin, 1.6 µg of neoxanthin, and 2.1 µg of
unidentified carotenoids. The LL pigment mixture contained 3.1 µg of
-carotene, 2.4 µg of lutein, 4.5 µg of violaxanthin, 1 µg of
antheraxanthin, 1.5 µg of neoxanthin, and 2.5 µg of unidentified
carotenoids. After incubation, samples were treated as described in
Fig. 1.
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It was reported for the recombinant LhcpII expressed in
Escherichia coli that this protein, when reconstituted
in vitro with pigments, was partially resistant against
proteolytic attack by trypsin, and this protease resistance could be
achieved by a ligation to pigments and not by a membrane insertion
(24). Washes of etioplast membranes with Na2CO3
or NaOH confirmed that Elips and trypsin-resistant Elip fragments were
intrinsically located in the membrane (not shown).
To test whether the stable insertion of Elips into etioplast membranes
is regulated by the quantity of pigments, insertion assays were
complemented with pigment mixtures added at increasing concentrations.
The results revealed (Fig. 4) that in the
absence of pigments a stable insertion of Elip into etioplast membranes did not occur (Fig. 4, left panel). Traces of
trypsin-resistant Elip fragments were detected when 6.5 µg of total
pigments were added to the integration assay. The amount of
trypsin-resistant Elip fragments increased almost linearly with
increasing pigment concentrations (Fig. 4, middle and
right panels).

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Fig. 4.
Insertion of Elips into etioplast membranes
complemented with increasing concentrations of pigments. The low
molecular mass pElips translated in vitro (lane
Tls) were incubated with lysed plastids (equivalent to 10 mg
of protein) isolated from 8-day-old etiolated barley seedlings and
complemented with increasing concentrations of pigments extracted from
green barley leaves exposed to light stress for 6 h. The 130 µg
of total pigments contained: 75 µg of Chl a, 25 µg of
Chl b, and 30 µg of total carotenoids (composed of 6.5 µg of -carotene, 8 µg of lutein, 6 µg of zeaxanthin, 2 µg of
antheraxanthin, 3.2 µg of neoxanthin, and 4.3 µg of unidentified
carotenoids). Aliquots of the pigment mixture of identical composition
but in lower concentrations were added to parallel assays. After
incubation, samples were treated as described in Fig. 1.
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In Contrast to in Vitro Import, Stable Insertion of Elips into
Etioplast Membranes Complemented with Pigments Did Not Depend on Light
Intensity--
In vitro import of pElips into isolated
greening plastids performed under various light regimes demonstrated
that the amount of membrane-integrated Elips increased with an
increment in the light intensity (Fig.
5A). 5-6-Fold higher amounts
of Elips were detected in membranes when the import assays were
performed at 1,000 µmol·m
2·s
1
than at 1 µmol·m
2·s
1.

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Fig. 5.
In vitro import and insertion of
Elips into etioplast membranes complemented with pigments and exposed
to increasing light intensities. A, the low molecular
mass pElips translated in vitro (lane
Tls) were incubated at increasing light intensities with
plastids isolated from etiolated barley leaves exposed to light for
6 h. Stroma and membrane fractions were isolated and analyzed by
SDS-polyacrylamide gel electrophoresis followed by fluorography.
B, the low molecular mass pElips translated in
vitro (lane Tls) were incubated with lysed
plastids (equivalent to 10 mg of protein) isolated from 8-day-old
etiolated barley seedlings and complemented with 65 µg of pigments
containing 37 µg of Chl a, 13 µg of Chl b,
and 15 µg of total carotenoids (composed of 3.3 µg of -carotene,
4 µg of lutein, 3 µg of zeaxanthin, 1 µg of antheraxanthin, 1.6 µg of neoxanthin, and 2.1 µg of unidentified carotenoids).
Insertion assays were incubated in rotating tubes at various light
intensities for 1 h. After incubation, samples were treated as
described in Fig. 1
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To test whether the insertion of Elips into etioplast membranes is a
light intensity-dependent process, assays complemented with
pigments were incubated at various light intensities in rotating tubes
to minimize the shading effect of membranes. The results showed (Fig.
5B) that the stable insertion of Elips into membranes was
light intensity-independent between 1 and 500 µmol·m
2·s
1.
Illumination of assays with light intensities between 500 and 1000 µmol·m
2·s
1
resulted in slightly reduced amounts of trypsin-protected Elip fragments. This effect might result from an increased generation of
free radicals and a photooxidative damage of membrane components during
illumination of the etioplast lysate in the presence of free Chls.
Chl a Alone Is Sufficient for the Stable Insertion of Elips into
Etioplast Membranes--
To test which of both Chls is crucial for the
stable insertion of Elips into etioplast membranes, integration assays
were complemented with increasing concentrations of isolated Chl
a (Fig. 6A), Chl
b (Fig. 6B), or a combination of both (not
shown). The results revealed that the stable insertion of Elips into
etioplast membranes was strictly dependent on the presence of Chl
a (Fig. 6A). Furthermore, the amounts of
trypsin-resistant Elip fragments increased with increasing
concentrations of this pigment (Fig. 6A). The addition of
Chl b, either alone (Fig. 6B) or in combination with Chl a (not shown) did not promote accumulation of Elips
within plastid membranes (Fig. 6B). The results revealed
that the stable integration of Elips was not influenced by the presence
of Chl b.

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Fig. 6.
Insertion of Elips into etioplast membranes
complemented with purified Chl a or b
added at increasing concentrations. The low molecular mass
pElips translated in vitro (lane Tls)
were incubated with lysed plastids (equivalent to 10 mg of protein)
isolated from 8-day-old etiolated barley seedlings and complemented
with increasing concentrations of Chls. A, insertion assays
were performed in the presence of Chl a. B, insertion assays
were performed in the presence of Chl b. After incubation,
samples were treated as described in the legend to Fig. 1.
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DISCUSSION |
We have shown that a stable insertion of Elips into etioplast
membranes did not occur in the absence of Chls. External addition of
Chl a promoted this process, thus indicating that etioplasts contain the whole machinery, which is required for the insertion of
Elips into membranes and that the only missing component in this system
is Chl a. This observation supports the concept that the
stabilization of Elips by pigments in thylakoid membranes is a part of
a post-translational regulation of expression of these proteins. At
which particular step this control occurs is not yet known. One
possibility is that Elips are inserted into etioplast membranes but due
to the absence of Chls they are degraded by "cleaning proteases."
It was reported that the LhcpII is rapidly degraded by protease(s) in
the absence of Chls in the etiolated mutant of Chlamydomonas
reinhardtii y-1 (25) and in etiolated higher plants (26, 27).
Another possibility is that Chls may play an active role during the
insertion process itself. Earlier studies showed that the recombinant
LhcpII expressed in E. coli was targeted to the bacterial
inner membrane by the addition of a bacterial signal peptide (28).
Therefore, it was concluded that Chls are not essential for LhcpII to
become embedded in a lipid bilayer, but the process of insertion was
observed to be inefficient in the absence of photosynthesic pigments.
This function of pigments in the refolding of LhcpII was suggested as
the driving force for translocating parts of this protein across the
membranes (20, 29).
Our data presented in Fig. 6, A and B,
demonstrated that the stable insertion of Elips into etioplast
membranes could be obtained by the addition of Chl a while
Chl b had no effect on Elip insertion. In this respect,
Elips differed from the LhcpII, which could be inserted into etioplast
membranes reconstituted with Chl b as the only Chl component
(20). Furthermore, the replacement of Chl b by Chl
a in reconstitution assays led to the absence of stably
inserted forms of LhcpII (20). The absence of Chl b in the
chlorina-f2 mutant of barley also led to the
depletion of the major Lhcb1 and one of the minor Chl
a/b-binding proteins, the Lhcb6 (called also CP24), but not
that of the Lhcb4 (called also CP29) (30). It was suggested that the
stability of these proteins in the absence of Chl b may
depend on the Chl a/b ratio. This ratio was relatively low
(between 0.9 and 1.6) for Lhcb1 and Lhcb6 and very high (around 3.0)
for Lhcb4 (29, 31). More recent studies showed that the six major
Lhcb1-6 proteins did not accumulate in the null chlorina
ch1-3 allele in Arabidopsis that completely
lacked Chl b (32). This mutation has been shown to be
stronger than chlorina-f2 in barley and influenced
the gene encoding Chl a oxygenase, an enzyme converting Chl
a into Chl b, leading to the ch1
mutant phenotype. Based on these data, it can be expected that if Elips
bind any Chl b molecules their content should be very low,
and this would explain why integration of Elips did not depend on
the presence of this pigment.
The recent purification of Elips from light stress-treated pea leaves
confirmed experimentally that these proteins bind pigments. Chromatographic and spectroscopic analysis of pigments bound to purified Elips revealed the presence of Chl a and lutein,
while Chl b and other carotenoids were not detected (10).
However, it was suggested that some pigments could easily dissociate
from Elips and be lost during protein purification. Analysis of the deduced amino acid sequence of Elips (4) and its comparison with the
electron crystallographic structure reported for the LhcpII (33, 34)
demonstrated that low and high molecular mass Elips from barley possess
four conserved Chl-binding residues located in helices I and III. Based
on the data presented for Lhcb4, these Chl-binding sites are selective
for Chl a, whereas the peripheral sites located in helix II
and in a short amphipathic C-terminal
-helix have mixed Chl
a/Chl b specificity (35). Since peripheral
Chl-binding sites are missing in Elips, these proteins might truly bind
only Chl a. Another possibility is that Chl b
might not be bound by specific amino acid residues but rather held in
place by pigment-pigment interactions as was reported for Lhcb4 (36)
and thus be less crucial for the stable Elip insertion into the membrane.
It was shown in Figs. 4 and 6, A and B, that the
stable insertion of Elips into etioplast membranes was more efficient
with increasing concentrations of Chl a added to assays. It
is unlikely that the availability of Chls represented a limiting step
for stable insertion of Elips into etioplast membranes reconstituted with lower pigment concentrations. Rather, it can be assumed that some
of Chl molecules were damaged due to photooxidation or aggregated during incubation of the integration assays and that this pigment fraction was not available for ligation with Elips. This explanation is
supported by the observation that the direct addition of pigments to
the membrane is ~100 times less efficient in promoting insertion of
LhcpII than the in situ synthesis of pigments from their
precursors (20).
It is known that not only Chls but also carotenoids play an important
role in the stabilization and folding of Chl-binding proteins (37-40).
It was proposed that Elips (41) and the Cbr (carotene
biosynthesis-related) protein, an algal homolog of higher plant Elips
(42), may represent zeaxanthin-binding proteins and that the stability
of the Cbr might be regulated by the binding of this pigment (42). Our
data demonstrated that comparable amounts of Elips were stably
integrated into the membrane in the presence or in the absence of
externally added zeaxanthin. Based on the structural model of LhcpII
(33), two xanthophyll-binding sites, the L1 and L2, have been located
in the center of the complex, forming an internal cross-brace
interacting with helices I and III. It was shown that the L1 and L2
sites in LhcpII have the highest affinity for lutein but can also bind
violaxanthin or zeaxanthin with lower affinity (40). In
vitro reconstitution of Lhcb1 protein overexpressed in bacteria
demonstrated that zeaxanthin and
-carotene were bound to L1 and L2
sites only when violaxanthin and lutein were either absent or present
in limiting amounts (40). Since etioplast membranes of barley contained
significant amounts of lutein and violaxanthin, this could explain why
the absence of zeaxanthin did not limit the stable insertion of Elips
into the membrane.
The spectroscopic analysis performed on purified native Elips from pea
suggested that Chls bound to these proteins did not interact with each
other (10). The weak association of pigments with the protein and their
low excitonic coupling supported the idea that Elips may represent a
group of Chl-binding proteins with function(s) different from light
harvesting (reviewed in Refs. 3 and 41). Recently, it was shown (44)
that the PSII-S protein contributes to photoprotective energy
dissipation rather than photosynthetic light harvesting. It was
proposed that Elips in higher plants (41) and the Cbr in algae (45) may
have a similar function and/or act as transient pigment-binding
proteins (13-15). The Elips could act as ligand chaperones required
for transient binding of pigments during biogenesis or turnover of "typical" Chl-binding proteins. Such a function would be supported by our results demonstrating that amounts of Elips accumulated in
membranes after in vitro import into greening plastids were enhanced at high light intensities, which are known to promote protein
turnover in thylakoid membranes (44-48).