Download Light-dependent Dl Protein Synthesis and Translocation Is

THE JOURNAL OF BIOLOGICAL CHEMISTRY
0 1930 by The American Society for Biochemistry
Vol. 265, No. 21, Issue of July 25, pp. 12563%12568,199O
and Molecular Biology, Inc.
Printed in U.S.A.
Light-dependent
Dl Protein Synthesis and Translocation
by Reaction Center II
REACTION
CENTER
II SERVES
AS AN ACCEPTOR
FOR THE
Dl PRECURSOR*
(Received
Noam
From
Adir,
Susana
the Department
Shochat,
of Biological
and Itzhak
Chemistry,
Is Regulated
for publication,
January
18, 1990)
Ohad
The Hebrew
Light
induces
an irreversible
modification
of the
photosystem
II reaction
center (RCII) affecting
specifically
one of its major
components,
the Dl protein
(Ohad, I., Adir, N., Koike,
H., Kyle, D. J., and Inoue,
Y. I. (1990) J. Biol. Chem. 265, 1972-1979)
which is
degraded
and replaced
continuously
(turnover).
The
turnover
rate of Dl is related
to light intensity.
Evidence is presented
showing
that RCII translocates
from
the site of damage
in the grana (appressed)
domain
of
the chloroplast
membranes
to unappressed
membrane
domains
where
the Dl precursor
protein
(pD1)
is
translated
and becomes
integrated
into RCII. Several
forms of RCII (a, a*, and b) were identified
on the basis
of their electrophoretic
mobility.
pD1 was found only
in the a and b forms in the unappressed
membranes.
Processing
of pD1 occurs
after
its integration
into
RCII. Mature
Dl appeared
mostly in the a form of RCII
and following
its translocation
to the appressed
membrane
domains
also in the Q* form.
Thus the light
intensity-dependent
synthesis
of Dl protein
is related
to the availability
of modified
RCII which serves as an
acceptor
for pD1. The shuttling
of RCII between
the
two membrane
domains
may represent
a control
mechanism of thylakoid
membrane
protein
synthesis.
The chloroplast-encoded
Dl protein of the photosystem
II
reaction center (RCII)’ (1, 2) turns over in the light, but not
in the dark, in higher plants (3) and in green algae (4).
Turnover of Dl is essential for the maintenance
of functional
RCII (5, 6). Dl is synthesized as a precursor protein, pD1, by
polyribosomes
attached to the stroma (exposed face) of the
chloroplast
photosynthetic
membranes
(thylakoids)
(7). In
light-grown
plants the mRNA that encodes pD1 is abundant,
irrespective
of transient
changes in the light regime (8).
Synthesis of pD1, however, occurs only in the light, and thus
it has been proposed that Dl synthesis is controlled
at the
level of translation
(S-10).
The light-dependent
degradation
of Dl does not require
simultaneous
synthesis (4, ll), and thus the two processes
*This
work
was supported
by a grant
awarded
by the Israeli
Academy
of Sciences
(to I. O.), a grant
in cooperation
with K.
Kloppstech,
University
of Hannover,
Federal
Republic
of Germany,
awarded
by the National
Council
for Research
and Development
joint
German-Israel
Program
in Biotechnology,
and Grant
184, B-9 in
cooperation
with R. Herrmann,
University
of Miinchen,
Federal
Republic
of Germany,
awarded
by Sonderforschungs
bereiche.
The
costs of publication
of this article
were defrayed
in part by the
payment
of page charges.
This
article
must
therefore
be hereby
marked
“advertisement”
in accordance
with 18 U.S.C. Section
1734
solely to indicate
this fact.
’ The abbreviations
used are: RCII, reaction
center of photosystem
II; pD1, precursor
of the Dl protein;
SDS, sodium
dodecyl
sulfate.
University
of Jerusalem,
Jerusalem
91904,
Israel
can be separated in time. Recently we have shown (6,12) that
in the green unicellular
alga Chlamydomonas
reinhardtii
y-l,
degradation
of the Dl protein is related to a light-induced
modification
of RCII. This process can be resolved into (i) a
reversible conformational
change affecting the binding site of
the secondary acceptor quinone (Qa) followed by (ii) an irreversible modification
of the Dl protein. The rate of the lightinduced reversible modification
depends on the total amount
of light absorbed (12)) and a link could be established between
this phenomenon
and Dl degradation
and synthesis. The
nature of the mechanism connecting the two phenomena thus
far remains unknown.
RCII is located in the appressed membranes
of the grana
region and is thus segregated from the site of synthesis and
insertion
of pD1 in the unappressed
stroma membrane
domains (7,13). In the present work we considered the possibility that light-modified
RCII might migrate to the unappressed
membranes
where it could serve as an acceptor for pD1
replacing the nonfunctional
Dl. RCII could then translocate
back to the grana as shown so far only for the Dl protein
(13). The results presented here support this hypothesis.
MATERIALS
AND
METHODS
C. reinhardtii
y-l cells were grown
in the light as previously
described
(14). Thylakoids
were isolated
(11) and further
fractionated
into the appressed
grana domain
(10 K fraction),
the intermediate
fraction
enriched
in unappressed
membranes
(40 K fraction),
and the
stromal
unappressed
membranes
(144 K fraction)
using the procedure
of Kyle et al. (15).
The experimental
approach
for the identification
and localization
of protein
precursors
is to use short
radioactive
labeling
(pulse)
followed
by chase of the label or inhibition
of further
protein
synthesis. Based on results of preliminary
experiments
with C. reinhardtii
cells (data not shown),
the only effective
procedure
to block further
chloroplast
protein
labeling and permit
detection
of the Dl precursor
is to use chloroplast
protein
synthesis
inhibitors.
In this procedure
the labeling
of the Dl protein
is effectively
blocked
(see Figs. 2-4).
However,
labeling of cytosolic
synthesized
proteins
continues
during
the chase as shown
in Figs. 2-4. Addition
of cycloheximide
which
could have prevented
this further
labeling was not used so as to avoid
further
disruption
of the normal
cell metabolism.
The precursor
of the Dl protein
(pD1)
was identified
by the
following
procedure.
Cells were incubated
in the light (400 PE mm2
s-l, measured
with a Li-Cor
quantum
radiometer
in the incubation
vessel) for 30-60 min to increase the level of Dl turnover rate (12),
and then labeling was carried
out for 2 min by the addition
of L-[~~S]
methionine
(Du Pont-New
England
Nuclear,
100 &i/ml,
1100 Ci/
mmol).
Chloramphenicol
(D-three,
Sigma, 200 rg/ml)
was added, and
the labeled
cells were harvested
immediately
(pulse).
Part of the
labeled cells was further
incubated
for 30 min (chase).
Termination
of chloroplast-encoded
protein
synthesis
by the addition
of chloramphenicol
occurred
within
less than 2 min as indicated
by the limited
increase
in the radioactivity
of the Dl protein.
For resolution
of pD1 from Dl in thylakoids
or thyalkoid
subfractions, the proteins
were separated
by denaturing
electrophoresis
on a
lo-20%
(w/v) polyacrylamide
gel using the method
of Laemmli
(16)
12563
12564
Light
Regulation
of pD1
Synthesis
which allowed us to discern between pD1 and Dl differing only by l1.5 kDa (13). Separation of thylakoid proteins by SDS-polyacrylamide
gel electrophoresis in the presence of 8 M urea did not improve the
separation of pD1 from Dl. The gels were dried and autoradiographed.
For estimation of the apparent molecular mass of the resolved polypeptides, molecular weight markers (Bio-Rad, low range) were used.
For detection of pD1 and Dl in RCII complexes, isolated thylakoids
or thylakoid subfractions were solubilized in a detergent mixture (w/
v) containing
0.75:0.2:0.05 octyl fi-D-glucoside:nonyl
B-D-glucoside:SDS at a chlorophyll concentration of 1 mg/ml for 10 min at
4 “C. Chlorophyll-protein
complexes were resolved in the first dimension by nondenaturing polyacrylamide (6% w/v) gel electrophoresis
in the presence of the zwitterionic detergent Deriphat-160 (17, 18).
Gel strips were excised and incubated for 15 min at 70 “C in SDS
sample buffer to denature the complexes. The strips were embedded
in stacking gel (pH = 6.8), and the polypeptide composition of each
complex was resolved by denaturing polyacrylamide electrophoresis
using a 14% (w/v) gel in the second dimension. Free low molecular
weight proteins are less hindered during migration through the first
dimensional low concentration gel, and thus their relative electrophoretie mobility is not strictly a function of the logarithm of their
molecular weight as is the case when later separated on the second
dimensional denaturing gel. This results in the migration of the free
polypeptides along an exponential diagonal during the second dimension run. Polypeptides which remained associated in protein complexes during electrophoresis in the first dimension appear in a
vertical line below the diagonal.
Protein immunoblotting
was performed as previously described
(19). For identification of Dl and pD1 in the autoradiograms of Fig.
2 the stained dried gel was rehydrated, removed from the paper, and
soaked in a 6 M urea solution containing 0.1% (w/v) SDS in the
transfer buffer used for blotting (19). The gel was then electroblotted
for 4 h, and the blot was immunodecorated with anti-D1 antibodies
followed by detection with ‘*“I-protein A. The exposure time required
to detect the iodinated protein A was only 20 h as compared with 14
days required for the detection of the L-[““Slmethionine-labeled
proteins present in the gel. Thus double exposure did not interfere with
the results of this experiment. Antiserum against the D2 protein was
produced as described for that of Dl antiserum (19).
RESULTS
Light Enhances Translocation
of RCII to the Unappressed
Membranes--To
test whether exposure of C. reinhardtii
cells
to high light intensities enhances the appearance of RCII in
the unappressed membrane domains, the content of RCII in
the grana, intermediate, and stromal membrane fractions was
assayed by protein immunoblotting. In cells exposed to growth
light intensity, most of the RCII is localized in the granal
fraction as detected by antibodies against the 47-kDa Dl and
D2 polypeptides (Fig. 1, control lanes). Following exposure of
the cells to light intensity known to induce modification of
RCII (6,12) and shorten the tth of the Dl protein from about
8 h at growth light intensity (4) to about 2 h (ll), an increase
in the amount of RCII polypeptides was observed in the
unappressed intermediate fraction (Fig. 1, high light, intermediate lane, compare with control lane). Similar results were
found to occur in the stromal membrane fraction (data not
shown). As a control, the distribution of the apoproteins of
photosystem I reaction center (Fig. 1, PSI) between the two
membrane domains did not change significantly as a result of
the light treatment (Fig. 1). Comparison of the polypeptide
pattern in the control lanes indicates that the unappressed
intermediate fraction is not contaminated with significant
amounts of appressed membranes as suggested by the low
content of RCII polypeptides in the control lane.
Localization
of pD1 Synthesis
in C. reinhardtii
Thylakoids-
Unlike higher plants, thylakoids of C. reinhardtii
are organized into large appressed domains containing only a few
stacked membrane layers interconnected by relatively short
regions of unappressed membranes (20). The average number
of lamellae in a grana stack is about 4-6, and the average
length of the grana stacks containing more than 6 lamellae is
Is Mediated
by RCII
CBB
a
IB
l-3
C
HL
C
HL
MC-02
G
I
FIG. 1. Light-induced
G
translocation
I
of RCII
to unappressed
Cells were exuosed for 90 min to 1600 or 30 ILE
mi s-’ white light (high light (Hi) or low light intensity (control
C)), respectively. Thylakoids were then isolated and fractionated into
the different membrane types. The polypeptides of the appressed
granal (G) and unappressed intermediate (40,000 x g) (Z) fractions
were resolved by SDS-polyacrylamide
gel electrophoresis loading
equal amounts of membranes (3 fig of chlorophyll). The resolved
polypeptides were electrotransferred to nitrocellulose paper. The individual RCII polypeptides (47 kDa, Dl, and D2) and the apoprotein
of photosystem I (PSI) were identified by immunodecoration
with
specific antibodies and “‘I-labeled protein A (ZB). CBB, stained gel
strips. The difference in the detection intensity of the immunodecorated polypeptides (ZB) is ascribed to differences in antisera binding
characteristics.
thvlakoid
domains.
shorter than that of the stacks containing 2-4 lamellae (20).
Thus exposed membrane surfaces where polyribosomes can
bind are found not only in the pure stromal membrane fraction (144 K) as in higher plants (7, 13) but also in the
intermediate fraction (40 K) and to some extent also in the
grana fraction as isolated by the method (15) used in this
work. Therefore, in view of this difference and considering
the results in Fig. 1, it was necessary to separate the grana,
intermediate, and stroma fractions to locate the site of pD1
integration in the membranes. Following a short radioactive
pulse labeling, pD1 could be found only in membrane fractions
consisting of or being enriched in unappressed domains (Fig.
2a, pulse lanes (P) of autoradiogram (A) of the intermediate
and stromal fractions). A small amount of pD1 was already
processed to the mature Dl form and appeared in the intermediate and grana fraction during the radioactive pulse.
These results differ from those obtained with higher plant
systems in which processed Dl appears already at the end of
the radioactive pulse in the stroma fraction (13). After a chase
period of 30 min (Fig. 2, a and b, chase lanes (c), no pD1 was
apparent, and mature Dl could be seen in the grana and
intermediate fractions.
To ascertain that the radioactively labeled band appearing
at the level of the pD1 is indeed pD1, the gel was transferred
to nitrocellulose paper as described under “Materials and
Methods” and immunodecorated with anti-D1 antibodies
(Fig. 2b). The immunoblot detects the steady-state level of
the precursor and mature Dl protein in the cells exposed to
high light prior to (pulse) and after the addition of chloramphenicol (chase). Fig. 2b shows the presence of pD1 in the
stroma and the total membrane fractions of the cells which
were used for the pulse-labeling experiment. During the chase
the precursor protein amount diminishes substantially in
these fractions. Mature Dl protein is present in the intermediate fraction during the pulse period and diminishes as well
during the chase. The amounts of Dl protein in the grana
and total thylakoids are compatible with the amount of
stained protein in the original gels (compare with Fig. 2a,
CBB). The fast processing of pD1 in the unappressed membrane fractions could indicate that the processing enzyme
Light
Regulation
of pD1
Synthesis
Is Mediated
97.466.242.731.0-
42.7-
31.0-
y
‘-J
_-
-I
2..
12565
by RCII
_
f 43
PDI
J
_
I
OIL
DL
e
i
47DI*
D2”
7
21.1 -
-.
14.4-
dcge*“w-eJP-
21.1-
,.k
.
I-
14.4-
97.4 66.2-
* .
42.7CPC
PC
P
c
P
CPC
P
CBB
(b)
c
P
c
.
P
A
31.0--
,-M--I-G,-IY-SY
l
D,
+-F-
-.
-s.
C
*
21.1l4.4-
CElB
CPCPCPCP
FIG. 2. The
Dl precursor
(PDZ) is found
intermediate
thylakoid
membrane
in unappressed
domains.
a,
cells
were radioactively labeled, and thylakoid membranes were prepared
and fractionated as described under “Materials and Methods.” Proteins of equivalent membrane fractions on a chlorophyll basis (5 rg)
were separated by SDS-polyacrylamide
gel electrophoresis. M, total
thylakoid membranes; G, grana fraction; Z, intermediate unappressed
fraction; S, stroma unappressed fraction; P, pulse; C, chase; CBB,
Coomassie Brilliant Blue-stained gel; A, autoradiogram. b, immunoblot of the stained gel after transfer to nitrocellulose paper. After the
pulse pD1 (open circles) is found only in the unappressed membrane
fractions. Some pD1 has already been processed to mature Dl (closed
circles) and translocated to the intermediate and grana fractions.
During the chase all pD1 is processed and translocated to the intermediate and grana fractions.
stroma
and
for the maturation
of pD1 may be enriched
in the interphase between the unappressed and appressed
membrane domains.The Precursor of the Dl Protein Is Integrated in XII-Having
identified pD1 in the unappressed
domains of C. reinhardtii
thylakoids, we addressed the question of whether pD1 is inserted as a free polypeptide in the
membrane or becomes integrated into a protein complex. To
answer this question total thylakoids from the experiment
shown in Fig. 2 were subjected to nondenaturing gel electrophoresis, which preserves the intactness of chlorophyll-protein complexes including RCII (equivalent to CCII+a in Ref.
17) (Fig. 3). The polypeptide compositions of the protein
complexes at the end of pulse (P) and chase (C) are shown in
Fig. 3, left upper and lower panels, respectively. Polypeptides
dissociating from a specific complex appear in a vertical row
deviating from the exponential diagonal migration of free
polypeptides (left upper panel; also see “Materials and Methods”). In this procedure the following components of RCII are
resolved (upper left panel): the 47-kDa polypeptide, Dl and
D2 identified by blotting with specific antibodies, and cytochrome bssg identified by heme staining (21) in the first
dimension nondenaturing gel (data not shown). The 43-kDa
reaction center II polypeptide, which binds chlorophyll and
forms the RCII core antenna, migrates as a separate entity
(CP43 (17)) (Fig. 3).
Autoradiography of the second dimension denaturing gels
shows that all labeled Dl or pD1 as identified in Fig. 2 are
found only in RCII complexes (Fig. 3, P and C panels, arrowhead and large black arrow, respectively). These labeled polypeptides are found in a position identical to the Coomassie
system responsible
FIG.
3. pD1
and
mature
A
Dl
are
found
only
in reaction
cen-
Equal amounts of total thylakoids from cells labeled as in Fig.
2 (3 rg) were resolved into chlorophyll-protein
complexes, and their
constituent polypeptides were separated by two-dimensional gel electrophoresis (see “Materials and Methods”): P, pulse; C, chase; A,
autoradiogram; CBB, stained gels in which the RCII polypeptides, 47
kDa, 43 kDa, Dl, and D2, are marked by different arrows; proteinchlorophyll complexes resolved in the first dimension nondenaturing
electrophoresis are marked. At the end of the pulse, pD1 (arrowhead)
is seen in a position corresponding to RCII resolved into two forms
(RCII a and RCII b) as detected by the Coomassie Brilliant Blue
stain; at the end of the chase, mature Dl protein (large black arrow)
is seen in RCII a.
ter
II.
Blue-stained RCII (compare with CBB panels, Fig. 3), off
diagonal. When the thylakoids were heat-denatured prior to
the first dimension electrophoretic separation, all proteins
appeared on a diagonal line (data not shown). These results
could be explained only if pD1 is integrated into RCII located
in the unappressed membrane domains and is processed after
its integration into the RCII complex.
It has been previously shown that the D2 protein turns over
in the light at lower rates than Dl (11). Some radioactively
labeled D2 (corresponding to the D2 stained band (Fig. 3,
CBB)) is also present in RCII as expected (Fig. 3, P panel,
open arrow). A third labeled band, positioned 2-3 kDa below
D2, which does not correspond to the visibly stained polypeptide band is identified as Dl. This identification is based on
protein immunoblotting (Fig. 2b; see also Fig. 1, Dl). The
nature of this band is not yet clear. Its reactivity with the Dl
antibodies could indicate that this band, representing only a
small amount of protein, could be a degradation product of
Dl. However, the fact that this band is already labeled during
the pulse does not support this interpretation. Thus this band
could represent a different conformer of Dl occurring in the
presence of SDS.
Different Forms of RCII Shuttle between the Appressed and
Unappressed Membrane
Domains in the Process of pD1 Synthesis and Maturation-Different
forms of chlorophyll protein
complexes related to RCII could be separated by nondenaturing gel electrophoresis as carried out in this work. To establish
their relative distribution, nondenaturing gels of the thylakoid
subfractions (grana, intermediate, and stroma) were run, and
the polypeptide compositions of the chlorophyll-protein complexes were resolved by denaturing electrophoresis in the
12566
Light
Regulation
of pD1
Synthesis
second dimension (Fig. 4). Three distinct forms of RCII were
resolved. These are denoted as a, a*, and b (Figs. 3 and 4,
CBB panels). In some experiments, complex a* could be
identified in two-dimensional gels of total thylakoids by immunoblotting as well as by staining (data not shown).
As can be seen, radioactively labeled pD1 identified as such
in Fig. 2. is present in complexes a and b in the stroma
membrane domain (Fig. 4, Stroma, Pulse panel, arrowhead).
The same is true for the intermediate fraction (Fig. 4, Intermediate, Pulse). The label marked by the arrowhead
(pD1)
also contains a small amount of mature Dl (as identified in
Fig. 2a). Comparison of Figs. 2, a and b, shows that at the end
of the pulse labeling most of the Dl is in the precursor form
(Fig. 2a, I, P), while in the immunoblot which shows the
steady state level of the protein, most of the Dl is in the
mature form (Fig. 2b, Z, P). Thus processing appears to take
place in this membrane fraction. The same complexes, however, contain processed Dl in the grana domains (Fig. 4,
Grana, Pulse, black arrow). After 30 min of chase no radioactive label is found in complex b in all membrane fractions.
Also, no radioactively labeled Dl is found in the complexes
present in the stromal membrane domains (Fig. 4, Chase,
Stroma panel). Following chase, labeled Dl is found in complex a in the intermediate and grana membranes and in
complex a* in the grana membrane domains (Fig. 4, Chase
panels,
black arrow).
The appearance of more than one type of RCII complex
can be attributed to: (i) changes in RCII organization during
Grana
Intermediate
1
1
t
7
t
-D
2
_Stroma
a=Q
t t
a b
t t
ab
a’
d
FIG. 4. Reaction center II containing
newly synthesized Dl
translocates from the unappressed to the appressed membrane
domains. Thylakoid membranes labeled and fractionated as in Fig.
2 were separated (3 pg of chlorophyll/fraction) into protein complex
polypeptide components as in Fig. 3: CBB, stained gel (representative
of both pulse and chase membrane fractions); P, pulse; C, chase.
Three forms of RCII are resolved in order of decreasingapparent
molecular mass:a*, a, and b. After pulse labeling, pD1 (arrowheads)
is found only in the a and b forms of RCII in unappressedmembranes.
These complexescontain a small amount of already processedDl in
the intermediate fraction. Dl (black arrow) is found in the a and b
forms of RCII and after chase, in a and in a and u* forms in the
intermediate and granal fraction, respectively.
Is Mediated
by RCII
the process of light-induced modification and Dl protein
exchange (see “Discussion”); (ii) the presence of other photosystem II polypeptides not resolved by this gel system, or
(iii) by the association of more than one RCII unit into
oligomeric complexes as has been described for the photosystern I reaction center (22).
DISCUSSION
Based on the data presented in this work we propose that
following its light-induced modification (6, 12), RCII dissociates in uiuo from the light-harvesting antennae in the granal
membrane domain (23) as an assembled complex and appears
to translocate to the unappressed membrane domains where
it serves as an acceptor for the newly synthesized pD1. Alternatively, one could consider that unstacking may occur where
modified RCII dissociates from the light-harvesting complex.
Unstacking, as examined by electron microscopy, does not
appear to occur in chloroplasts of cells exposed to high light
for up to 90 min under conditions enhancing Dl turnover
(11): Thus translocation of RCII seems to be the operative
control mechanism of Dl exchange.
The contention that light enhances translocation of RCII
to the unappressed membrane regions as an assembled complex is essential to this conclusion and is based on the fact
that neither pD1 nor the Dl, D2, or 47-kDa polypeptides of
RCII could be detected as free running polypeptides in the
second dimension electrophoresis.
The involvement of RCII components in the regulation of
Dl synthesis has been documented by results obtained with
C. reinhardtii
and cyanobacterial mutants. The Dl protein is
not detectable in mutants lacking D2 (9), the 47-kDa polypeptide (lo), or the cytochrome bss9 apoproteins (24, 25).
Conversely the latter polypeptides are synthesized but do not
accumulate in Dl-less mutants (26). Thus one would expect
that addition of chloramphenicol, preventing synthesis and
replacement of Dl protein, will promote the degradation of
all reaction center II proteins. This is, however, not the case
(ll), supporting the idea that RCII does not disassemble
during the process of Dl degradation and replacement. Thus
the increase in the amount of RCII polypeptides in the intermediate membrane fraction can be ascribed to translocation
of RCII complexes. The methodology used in this work did
not permit identification of Dl-less reaction centers in experiments in which cells were exposed to high light intensity in
the presence of chloramphenicol (data not shown). Whether
RCII directly regulates the translation of pD1 or stabilizes
pD1 after its synthesis, protecting it from immediate degradation, cannot yet be decided unequivocally. If pD1 is first
inserted into the membranes as a free polypeptide and RCII
only protects pD1 from proteolysis after its integration into
the complex, then the processes of integration and/or degradation of free pD1 are extremely fast since no free pD1 could
be detected at the end of the 2-min pulse. Translation control
by arrest of the elongation process in the absence of chlorophyll synthesis has been described for chlorophyll-binding
proteins (27). It is thus possible that pD1 chain elongation is
arrested unless the nascent chain is accepted by RCII. Binding
of newly synthesized chloroplast proteins to a specific acceptor protein (possibly the 47-kDa polypeptide) has been suggested before as a translation arrest mechanism (10).
The role of RCII as an acceptor of pD1 and the implication
that processing of pD1 occurs following integration into RCII
are supported by the previous observation that in a Scenedesmus mutant lacking the processing enzyme system, pD1 ap’ N. Adir, S. Shochat, and I. Ohad, unpublished data.
Light Regulation
of pD1 Synthesis Is Mediated
pears as a stoichiometric
stable component
of photochemitally active RCII (28, 29).
We have previously reported that both pD1 and Dl proteins
in situ are not susceptible to cross-linking
reagents unless
denatured by treatment of the membranes with certain detergents
and thus have proposed that they share a similar conformation
and/or environment
(19). The results presented
here are in agreement with these observations.
Three forms of RCII complexes of different apparent molecular mass could be resolved by the nondenaturing
electrophoresis technique used in this work. The a type complex
form appeared in all membrane
fractions. Considering
the
amount of grana membrane
domain relative to the total
membranes as estimated from the chlorophyll
amount in the
various fractions, the a form of RCII is the predominant
form
in C. reinhardtii
thylakoids.
The a* form which was found
only in the grana membranes could be an oligomeric form of
RCII. The b form of RCII was found mostly in the unappressed membrane
fractions
and thus amounts only to a
relatively
small fraction (about 10-E%)
of the total RCII
present in the thylakoids.
The presence of these RCII forms
is apparently
not an artifact of the detergent treatment. This
is strongly suggested by the specific and transient appearance
of pD1 in the a and b forms of RCII in the stroma membrane
fraction, by the transient appearance of the processed Dl in
the a and b forms of RCII in the intermediate
membrane
domain, and by the absence of labeled Dl in the a* RCII form
during pulse labeling and its appearance
during the chase
period.
One should stress here that the identification
of the radioactive spots corresponding
to the Dl protein in the various
forms of RCII as pD1 and Dl is based not only on the
denaturing
electrophoresis
carried in the two-dimensional
gels
but also on the data obtained by gradient SDS-polyacrylamide
gel electrophoresis
shown in Fig. 2. The second dimension
gels serve mostly the purpose of demonstrating
the presence
of pulse-labeled
precursor and mature Dl in reaction center
II complexes. The fact that pD1 appears transiently
in two
different
RCII complex forms precludes the possibility
that
pD1 is integrated in the membranes as a free protein and that
its location in the lanes corresponding
to RCII forms is an
experimental
artifact.
Based on these and previous results (6, 12), one could
consider that the modified forms of RCII characterized
by
impaired
electron
flow and charge recombination
(6, 12),
induced by exposure of chloroplasts
to light, may be related
to the a and b RCII forms found in the unappressed membrane
domains. Reactivation
of photoinhibited
irreversibly
damaged
RCII requires replacement
of the altered Dl protein by a
newly synthesized
molecule (3, 12). Thus such RCII forms
may dissociate from the light-harvesting
complex (23) and
migrate to the unappressed
membrane domains where they
could serve as an acceptor for the newly synthesized pD1.
The average distance from the center of a granum stack to
the adjacent unappressed membrane domain in C. reinhardtii
thylakoids
is in the range of 200 nm (20). Translocation
of
RCII complexes from the grana to the unappressed
domains
is thus compatible
with the observed results considering
that
the diffusion
coefficient
of chloroplast
membrane-protein
complexes within the membrane bilayer is estimated as 2 x
10-l’ cm2 s-’ at 25 “C (30).
Following integration
of pD1 the properties of RCII change
concomitantly
with the processing of pD1 to mature Dl. Thus
while pD1 may be initially
integrated
in the b form of RCII,
conversion of b to a will result in the presence of pD1 in both
forms of RCII. It also appears that conversion of b to a is not
by RCII
a consequence of the processing step. Following translocation
to the appressed membrane domain, mature Dl is found for
a short time in both these forms of RCII. The presence of Dl
in the b form of RCII in the grana fraction at the end of the
pulse could be attributed
to the presence of unappressed
surface-exposed
membranes.
However, after only a 30-min
chase period the b form converts to the a or a* forms. These
forms having been translocated
back to the grana appressed
membrane domains complete the cycle of light-induced
inactivation/reactivation
of RCII (6, 12) and Dl turnover.
Reaction centers unable to transfer electrons to the plastoquinone
pool were reported to occur preferentially
in unstacked thylakoids
(p centers (31)). The relative amount of @
centers increases in light-exposed
cells (32). Hence, p centers
may include the light-modified
RCII (b form of RCII?) described above. Migration
of RCII components
to the stroma
membrane domains in photoinhibited
thylakoids was reported
to occur in vitro (33).
The data reported so far are consistent with the hypothesis
that RCII modified by light exposure shuttles from granal to
stromal membrane domains where it serves to regulate pD1
synthesis, possibly by translational
control. This involvement
of RCII provides a coupling mechanism between light intensity-dependent
damage to RCII and Dl degradation
and light
intensity-dependent
pD1 synthesis.
Acknowledgment-Antisera
against the 47-kDa
apoprotein
of the photosystem
Dr. R. Nechushtai,
The Hebrew
protein
and the
reaction
center
were the kind gift of
University,
Jerusalem,
Israel.
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