From the
We identified two neighboring missense mutations in the
chloroplast atpA gene which are responsible for the defect of
ATP synthase assembly in the FUD16 mutant from Chlamydomonas
reinhardtii. The two corresponding amino acid substitutions,
Ile
The H
Our primary interest has been to
elucidate the assembly process of the chloroplast ATP synthase in C. reinhardtii by utilizing the numerous mutants available
that are defective in correct ATP synthase assembly. Assembly mutants
are useful for the dissection of the steps involved in the proper
interaction of the nuclear and cytoplasmic derived components of the
complex. The extent to which the assembly process can proceed when one
of the ATP synthase subunits is not synthesized or is synthesized in an
altered form can provide valuable information concerning the assembly
process as well as the cellular functions which are necessary to cope
with defective complex formation. In most cases of ATP synthase
assembly mutants, improper assembly of the ATP synthase results in an
instability of the unassembled or partially assembled complexes and a
loss of subunits normally found in ATP synthase (Lemaire and Wollman,
1989b). An exception to this case is the mutant FUD16, defective in ATP
synthase, which was previously characterized by Woessner et
al.(1984) as bearing a chloroplast mutation, which belongs to
complementation group II when compared with a series of ATP synthase
mutants of chloroplast origin. In a subsequent biochemical analysis,
Lemaire and Wollman (1989b) showed that the FUD16 mutant displayed
improper assembly of CF1 on the thylakoid membranes together with
increased intracellular levels of the
The FUD16 mutant has a
2-fold interest. It provides a means to further understand a process of
protein assembly but it should also help to determine the outcome of
overexpressed proteins in organellar compartments. Quite often the
overexpression of proteins in procaryotes and eucaryotes results in the
formation of substructures, termed inclusion bodies (Mitraki and King,
1989). These structures have been found in the cytosol and are composed
of high molecular weight aggregates of the overexpressed protein.
Whether this process occurs in organellar compartments has not been
observed until now. The expression of organelle-encoded proteins is
blocked or down-regulated in a number of mutants from yeast or Chlamydomonas (see reviews of Tzagoloff and Dieckmann(1990)
and Rochaix(1992)). In contrast, isolation of mutants overexpressing an
organelle-encoded protein is difficult to achieve. Several
nuclear-encoded proteins, which are targetted to the mitochondria, have
been overexpressed in yeast and in some cases displayed an increased
steady-state concentration in the mitochondria but their state of
assembly or self-aggregation was not characterized (van Loon et
al., 1983a, 1983b).
In this study we have identified the
mutation in FUD16 as a 2-base pair substitution in the atpA gene coding for the
Induction of gametes, crosses,
maturation of zygotes, and dissection of tetrads were carried out
according to Levine and Ebersold(1960). Chloroplast recombination tests
were achieved according to Girard-Bascou(1987).
The thm24.FUD16
double mutant was selected from tetrads obtained after FUD16.mt+
Anti-
Pulse labeling
experiments were carried out according to Delepelaire (1983). Cells
were labeled for 5 min with 5 µCi/ml
[
For the immunocytochemistry analysis, cells
were fixed in 2% paraformaldehyde and 1% glutaraldehyde in 4
mM potassium phosphate buffer plus 10 mM
MgCl
A
restriction map of the Chlamydomonas chloroplast DNA region
containing the atpA gene is presented in Fig. 1A. Transformation rescue experiments were
performed on the FUD16 mutant, with various restriction fragments from
WT atpA gene region. After particle bombardment, transformants
with a WT phenotype were selected by their ability to grow in
phototrophic conditions. A restriction fragment containing the atpB gene was used in control transformations. As shown on Fig. 1B, all fragments which restored phototrophic
growth mapped in the coding region of the atpA gene. The
smallest fragment of this series was a 940-base pair EcoRI-PstI fragment which mapped within the first
half of the atpA coding region.
We also examined the steady-state concentrations of
the
In the FUD16 profile, most of the
We
then attempted to disrupt the
We characterized these double mutants for their
rates of synthesis of the
The FUD16 phenotype is thus remarkable since it
demonstrates that the chloroplast compartment can accommodate inclusion
bodies which, up to now, were observed only in the cytosol upon
expression of abnormal proteins or overexpression of endogeneous or
foreign proteins, in bacteria (Nicolas et al., 1993; Marston,
1986) or in Saccharomyces cerevisiae (Binder et al.,
1991).
In a thorough
survey of the various reports on inclusion body formation, Mitraki and
King(1989) concluded that inclusion bodies result from the aggregation
of partially folded intermediates. This view is consistent with our
observation that some CF
We observed that the rate of
synthesis of the
At the moment we cannot discriminate between
co-translational interactions and early post-translational
interactions. The nucleotide fold of newly synthesized
Polypeptides were separated by urea/SDS-PAGE, transferred to
nitrocellulose, detected with antibodies against
We thank R. Kuras for numerous suggestions in the
course of this work, D. Picot for comments on the F
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Asn and Asn
Tyr, occurred
at strictly conserved sites among the
and
subunits of
(C)F
complexes from bacteria, mitochondria, and
chloroplasts. The altered region in the
polypeptide chain is
located 7 amino acids downstream of the P-loop, which forms most of the
conserved nucleotide binding site. Although the resulting chloroplast
mutant fails to accumulate most of the ATP synthase subunits, it
displays an increased intracellular content in both the
and
subunits. We demonstrate that the two subunits do not bind to the
thylakoid membranes but associate and overaccumulate in the chloroplast
stroma as inclusion bodies. Increased rates of synthesis of the two
subunits in the mutant point to an early interaction between the two
subunits during their biogenesis.
-ATP synthase, located on bacterial,
mitochondrial, and chloroplast inner membranes, is highly conserved
from procaryotes to eucaryotes (Nelson, 1992). It consists of two
functionally independent components, the extrinsic soluble portion,
(C)F
, which is the site of nucleotide binding and ATP
catalysis, and the intrinsic membrane sector, (C)F
, which
is responsible for the proton flux across the membrane. The (C)F
sector consists of three copies each of two large subunits,
and
and single copies of three smaller subunits,
,
,
and
. The crystal structure of the F
-ATPase from
bovine heart mitochondria has been recently solved at atomic resolution
(Abrahams et al., 1994). Three
heterodimers are
arranged around a central
-helix formed by the C-terminal domain
of the
subunit. The (C)F
sector forms a pore with
1-2 copies of subunit I, 1 copy of subunits II and IV, and
10-12 copies of subunit III. In the chloroplast, both CF
and CF
sectors are comprised of nuclear- and
chloroplast-encoded subunits. In Chlamydomonas reinhardtii, as
in higher plants, the CF
subunits
,
,
, and
the CF
subunits I, III, and IV are chloroplast-encoded
while CF
subunits
and
, and CF
subunit II are nuclear-encoded (Lemaire and Wollman, 1989a;
Herrmann et al., 1993). Therefore the two compartments of the
cell must cooperate to regulate the proper assembly of the CF
and CF
complexes.
and
subunits. The
complementation group II was then tentatively ascribed to the atpA locus (Lemaire and Wollman, 1989b).
subunit, which is located immediately
downstream from the conserved region involved in nucleotide-binding
(Walker et al., 1982). We show that the overaccumulation of
the mutated
subunit is accompanied by the formation of inclusion
bodies in the chloroplast. This process requires the interaction of the
mutated
subunit with the
subunit which also accumulates in
the inclusion bodies. We discuss the molecular basis for the altered
interactions between the two subunits.
Growth Conditions, Strains, and Genetic
Characterization
Wild-type (WT)(
)and
mutant strains were grown at 300 lux (5.86 µE/m
s PAR)
in Tris acetate-phosphate (TAP) medium. The WT strain is derived from
strain 137c. The nuclear mutants F54, thm24, and ncc1 and the
chloroplast mutant FUD16, have been previously described (Lemaire and
Wollman, 1989b; Drapier et al., 1992). The chloroplast mutant
10-6C has been shown to bear a mutation in the rbcL gene
by Dron et al.(1983).
thm24.mt- crosses. Two clones, out of four per tetrad,
were selected as double mutants based on the absence of atpB transcripts as determined by mRNA hybridization analysis.
FUD16.mt+
ncc1.mt- crosses yielded two double mutant
clones per tetrad. These were selected as affected in their atpA transcript accumulation upon mRNA analysis.
Protein Analysis
Thylakoid membranes were isolated
as described by Chua and Bennoun(1975). Protein content was analyzed
after urea/SDS-polyacrylamide gel electrophoresis according to Piccioni et al.(1981). Gels were either silver-stained as in Rabilloud et al.(1988) or used for immunoblotting experiments as in de
Vitry et al.(1989). Antibody labeling was detected with I-protein A (Amersham, France) or the enhanced
chemiluminescence method (ECL, Amersham, France).
,
anti-
, and anti-
were kindly provided by C. Lemaire (Centre
de Genetique Moleculaire, Gif sur Yvette, France) and anti-CF
subunit III by O. Vallon (Institut Jacques Monod, Paris, France)
and B. Lagoutte (CEN, Saclay, France). Anti-P10 was kindly provided by
R. Bassi (Universita di Verona, Verona, Italy) and anti-LHC
(light-harvesting complex) II was prepared in the laboratory as
described in de Vitry et al.(1989).
C]acetate (56 mCi/mM, Amersham,
France) in the presence of 6.6 µg/ml cycloheximide which inhibits
translation in the cytoplasm. Quantification of the
C
labeling or
I labeling was performed using a
PhosphorImager (Molecular Dynamics).
Separation of Protein Complexes on Sucrose
Gradient
16 10
cells, resuspended in 4 ml of
buffer (25 mM HEPES, pH 7.5, 10 mM EDTA, 0.3 M sucrose) were broken in a French press, directly loaded on a
continuous 20-80% (w/v) sucrose gradient, containing 10 mM HEPES, pH 7.5, and centrifuged at 38,000 rpm for 60 or 1 h in a
Beckman SW-41 Ti rotor. Fractions of 0.5 ml were collected and analyzed
by immunoblotting as described above.
Assays of Dissociation of the Inclusion Bodies in FUD16
Strain
Fractions from the sucrose gradient containing the
inclusion bodies were diluted four times with 10 mM HEPES, pH
7.5, and centrifuged at 50,000 rpm for 15 min in a Beckman TLA-100.3
rotor. The pellet was resuspended in 25 mM HEPES, pH 7.5, 10
mM EDTA, 0.3 M sucrose, then incubated, at 4 °C
for 30 min, with either 2% Triton X-100 or 8 M urea with
gentle stirring. Samples were loaded on a continuous 20-80% (w/v)
sucrose gradient centrifuged at 54,000 rpm for 40 min in a Beckman
TLS-55 rotor. Fractions of 300 µl were collected and analyzed on
urea/SDS-acrylamide gels which were silver-stained.
CF1 Analysis
Chloroform-release
extracts of purified thylakoid membranes were prepared and ATPase
activity tested as in Piccioni et al.(1981). Sedimentation of
WT CF, prepared by the chloroform release procedure was
carried out on a continuous sucrose gradient as described above.
DNA Analysis
Chloroplast DNA was isolated
according to Rochaix et al.(1988). For isolation of the R7 and
R15 EcoRI fragments (nomenclature according to Rochaix, 1978),
chloroplast DNA was digested with the restriction enzyme EcoRI
and subcloned in the pUC21 vector by standard protocols (Sambrook et al., 1989). After transformation into Escherichia
coli, tetracycline-resistant colonies were hybridized against WT C. reinhardtii atpA R7 and R15 gene fragments. Plasmid DNA
from the selected colonies was isolated and analyzed by DNA filter
hybridization with WT fragments. Fragments were subcloned in
pBSKS- (Stratagene) for DNA sequence analysis. Double-stranded
plasmids were sequenced using Sequenase dideoxy nucleotide sequencing
(U. S. Biochemical Corp.) according to the manufacturer's
instructions. Sequence from both strands was obtained and compared with
the atpA sequence obtained from Leu et al.(1991) and
Dron et al.(1982).
Transformation Protocols and Plasmids
Chloroplast
transformation was carried out according to Kuras and Wollman(1994).
Cells were grown up to 2.5 10
cells/ml in liquid
TAP medium after treatment for about 6 generations (48 h) with
3-fluorodeoxyuridine. Cells were then plated on TAP medium containing
500 units/ml penicillin at a density of 10
cells/plate for
transformation. Cells were bombarded with 1.2-µm tungsten particles
containing appropriate DNA. Cells were then plated on TAP medium for 5
days under dim light (300 lux), then transferred to high light (4000
lux) on minimal medium for recovery of phototrophic transformants.
Electron Microscopy
Thin sections of intact cells
were performed on samples fixed in 4% paraformaldehyde and 2%
glutaraldehyde, postfixed in 1% osmium tetroxyde and embedded in
Epon-Araldite resin.
, rinsed in the same buffer plus 0.1 M glycine
and embedded in Lowicryl K4M resin. Lowicryl thin sections were labeled
with antisera (1/200 dilution) as described in Vallon et
al.(1985), using colloidal gold-labeled protein A to reveal
binding of primary antibodies.
FUD16 Mutant Strain Bears Two Base Substitutions in the
atpA Gene
The atpA gene is located between the atpH and rbcL genes on the C. reinhardtii chloroplast
genome, with the rbcL gene being transcribed in the direction
opposite to atpA. The genetic lesion in FUD16 mapped next to
the rbcL gene, yielding 1-2% recombinants upon crosses
with the rbcL mutant 10-6C (experiments not shown). This
result strongly suggested that the mutation was in atpA.
Figure 1:
Determination of the 2-bp substitutions
conferring the FUD16 phenotype. A, schematic restriction map
of the atpA gene region in the chloroplast DNA. The
nomenclature of the chloroplast fragments (R15,R7) is from Rochaix
(1978); arrows point to directions of transcription for rbcL and atpA. The adenine additions between the two
start sites of the transcripts are indicated by , whereas the
two substitutions responsible for FUD16 phenotype are indicated by *. H3, HindIII; P, PstI; RI, EcoRI; X, XbaI. B, results from the
transformation of the FUD16 strain with wild-type chloroplast DNA
fragments. The fragments listed, with the exception of Bam5 which
contains the atpB gene, are shown diagramatically in A. Selection was based on recovery of phototrophic
transformants as discussed in the text. C, location of the two
base substitutions in the atpA gene with the two corresponding
amino acid changes in the polypeptide sequence. Nucleotide numbers
start by +1 at the first A in the AUG start codon of atpA.
We then cloned the
choroplast DNA fragments comprising the atpA gene from the
FUD16 strain (see ``Materials and Methods''). A comparison of
the atpA gene sequences in WT (as determined by Leu et
al.(1991) and Dron et al.(1982)) and FUD16 strains,
revealed two base changes at positions 551 and 556 (with nucleotide
number 1 being the A of the AUG start codon for atpA) in the
coding region of the atpA gene (Fig. 1C). The
two base changes are located 1930 and 1935 base pairs upstream from the rbcL mutation 10-6C, at the distance we expected for the
1-2% recombination frequency (Girard-Bascou et al.,
1987). The observed T551A and A556T substitutions resulted in amino
acid changes Ile
Asn and Asn
Tyr. We also found a few adenine additions at positions -453,
-524, and -553 in the extragenic region between the atpA and rbcL genes. As mentioned above, FUD16
transformation with the EcoRI-PstI fragment, which
restored only the two substituted bases from the coding region of atpA, proved sufficient to restore a WT phenotype. We thus
conclude that the chloroplast mutation responsible for the FUD16
phenotype corresponds to two amino acid substitutions located within a
tripeptide segment of the
polypeptide chain.
Rates of Synthesis of the
Lemaire and Wollman (1989b) previously
reported that the FUD16 mutant displayed an increased synthesis of the
and
Subunits Are
Increased in FUD16
subunit (hereafter referred to as
). This
conclusion was drawn from a comparison of pulse-labeled FUD16 cells
with a control strain which was subsequently demonstrated to bear a
nuclear mutation (ncc1) altering the rates of synthesis of the
and
subunits (Drapier et al., 1992). We therefore
re-examined, by [
C]acetate pulse labeling
experiments, the rates of synthesis of these two subunits in
exponentially growing cells of the FUD16 and WT strains. Labeled
proteins (equivalent loads) from cell extracts were separated on
urea/SDS-PAGE and dried gels were exposed to autoradiography for
1-4 weeks. As shown in Fig. 2, the resulting labeling of
both the
and
subunits was higher in the FUD16
strain as compared to that in the WT strain. Quantification of the
respective labeling in the two strains by phosphoimaging showed that
FUD16 displayed a 4- and 3-fold increase in the rates of synthesis for
the
and
subunits, respectively (mean value of
three distinct experiments). We detected no parallel changes in the
amount of atpA and atpB transcripts in FUD16 cells
compared to WT cells (results not shown).
Figure 2:
Chloroplast translates in the 50-60
kDa region from WT and FUD16 cells, pulse-labeled with
[C]acetate for 5 min. Cells were labeled in the
presence of cycloheximide to inhibit synthesis of proteins in the
cytoplasm. Polypeptides were separated by urea/SDS-polyacrylamide gel
electrophoresis and viewed by autoradiography. LS, the large
subunit of ribulose-P
carboxylase, migrates in the same
region of the gel as the
and
subunits.
The relative amounts of
and
Subunits
Overaccumulate in FUD16
and
subunits, present in exponentially
growing cells or purified thylakoid membranes from FUD16, were
estimated by immunoblotting using specific antibodies conjugated with
iodinated protein A (Fig. 3A). We used combined antisera
for both the
and
subunits, which show a higher titer for
antibodies against the
subunit (Lemaire and Wollman, 1989b).
Therefore the
:
stoichiometric ratio of 1:1 in WT samples
corresponds to a stronger labeling of the
subunit.
Figure 3:
Accumulation of and
subunits
in whole cells or thylakoid membranes from WT strain and mutant FUD16.
Polypeptides were separated by urea/SDS-PAGE, transferred to
nitrocellulose, and detected with specific antibodies. A,
immunoblots of whole cells (grown at 2
10
cells/ml)
and thylakoid membranes from WT strain and mutant FUD16 treated with
antibodies against
,
, or
subunits of chloroplast ATP
synthase and antibody against P10, an antenna polypeptide (loading
control). Binding of the antibodies was detected using radioiodinated
protein A. B, immunoblot reacted with antibody against subunit
III (suIII) of CF
; a diluted sample of WT membranes was
used to estimate the sensitivity of the method. Binding of the antibody
was detected by ECL, using a goat anti-mouse horseradish
peroxidase-conjugated antibody.
Most
strikingly, the FUD16 mutant showed an increased intracellular
accumulation in both the and
subunits whereas
purified thylakoid membranes displayed only trace amounts of these
subunits: FUD16 membranes displayed one-tenth of the
subunits and 1/35 of the
subunits present in WT membranes (). In contrast, FUD16 intracellular contents in
and
subunits were, respectively, three and
two times higher than that of
and
subunits in the WT. We
note that these values depended on the state of the cell culture for
FUD16: the content in
subunits increased in the
late phase of growth, whereas that in
subunits decreased ().
subunit of CF
(Fig. 3A) and
subunit III of CF
(Fig. 3B) with specific
antibodies. These two subunits remained below detection in the FUD16
samples. Thus the mutation in FUD16 strain prevents the intracellular
accumulation of both CF
and CF
.
FUD16 Thylakoid Membranes Still Accommodate Minor Amounts
of CF
Chloroform
extraction is a selective tool to extract CF-like Complexes
bound to
biological membranes (Beechey et al., 1975). Although the
rationale for this purification procedure is poorly understood, its use
with the WT of C. reinhardtii proved to be selective for both
chloroplast CF
(Piccioni et al., 1981) and
mitochondrial F
(Atteia, 1994). Although the FUD16 mutant
failed to accumulate significant amounts of CF
, we applied
the chloroform treatment to purified FUD16 thylakoid membranes in an
attempt to increase the sensitivity of our immunological detection. We
used a large amount of highly concentrated membranes purified from
cultures at 2
10
cells/ml. The chloroform-release
extract contained a low level of
and
subunits (Fig. 4A). Surprisingly, the two subunits now displayed
the same stochiometric ratio as in the WT extract, although the
starting membranes did not (). Moreover, the
chloroform-release extract from FUD16 contained detectable amounts of
subunit (Fig. 4B). These two criteria suggested
that chloroform extracted CF
-like complexes from FUD16
thylakoid membranes. Based on the respective amounts of WT and FUD16
membranes extracted with chloroform, we could estimate that assembled
CF
-like complexes in FUD16 represent approximately 3% of
the genuine CF
bound to WT thylakoids.
Figure 4:
Analysis of CF from WT and
CF
-like complexes from FUD16 by immunoblotting.
Polypeptides recovered in the aqueous phase after chloroform treatment
of thylakoid membranes were separated by urea/SDS-PAGE, transferred to
nitrocellulose, and probed with antibodies. A, immunoblot of
CF
from the two strains. Anti-
and anti-
binding
was detected with radioiodinated protein A. Samples corresponding to 50
µg of chlorophyll for WT and 500 µg of chlorophyll for FUD16
were loaded. B, subunit
was detected using a specific
antibody on the same immunoblot as in A revealed by the ECL
method.
CF released by chloroform extraction of WT thylakoid membranes still
showed some Ca
-ATPase activity as measured by
CaCl
precipitation on native gels (Piccioni et
al., 1981). Such an ATPase activity was also observed with the
FUD16 sample at the same migration position as that of the WT sample
(results not shown). No CaCl
precipitate was observed in
the control FUD50 strain, totally devoid of CF
complexes
(Lemaire and Wollman, 1989b), which supports the assignment of this
ATPase activity to assembled CF
-like protein complexes in
mutant FUD16.
Overaccumulated
Because of the discrepancy between the amount of
assembled CFand
Subunits Form Aggregates of High Molecular Weight in FUD16
Cells
-like complexes bound to FUD16 thylakoid
membranes, and the amount of
and
subunits
present in total cell extracts, we expected to find either soluble
-
complexes with abnormal stochiometry, or at
least individual
and
subunits in the
chloroplast stroma of FUD16. Therefore we separated cellular extracts
of the WT and FUD16 strains by differential centrifugation on
20-80% (w/v) sucrose gradients. We first chose a centrifugation
time of 60 h which was long enough for both membrane vesicles and
soluble oligomeric proteins to reach their equilibrium density. The
fractions were separated by urea/SDS-PAGE and analyzed by Western
blotting. Combined antisera against
and
subunits were used
to determine their location in the gradient and antibodies against
LHCII proteins were used to monitor the location of thylakoid
membranes. The data presented in Fig. 5result from
phosphoimaging analysis of the antibody labeling patterns. Antibodies
against
subunit were also used to determine the possible state of
assembly of the CF
(data not shown).
Figure 5:
Distribution profiles of and
subunits, released from WT and FUD16 cells, upon sucrose gradient
centrifugation. Cells were broken with a French press, then directly
loaded on a 20-80% (w/v) sucrose gradient and centrifuged at
240,000
g for 60 h. Fractions were collected from
bottom (fraction 1) to top (fraction 25) of the gradient. Graphs were
constructed using the
and
immunolabeling quantification of
the fractions by phosphoimaging. Also shown is the labeling of LHCII as
a marker of thylakoid membrane distribution in each gradient. All
profiles were normalized to the fraction showing maximal LHCII content
(fractions 9 or 10). A correction factor was applied to the
subunit labeling in order to provide a 1:1 ratio between the
and
subunits in CF1 from WT thylakoid membranes fractions. +,
subunit;
,
subunit; &cjs0800;,
LHCII.
In the WT profile,
the and
subunits co-localized with the
subunit, in
the same fractions as the LHCII (fractions 8-11). This reflects
the association of CF
with the thylakoid membranes.
CF
subunits were also detected in a minor peak (fraction
13), slightly above the bulk of the thylakoid membranes, with an
/
subunit ratio similar to that in fractions 8-11.
Fraction 13 most likely corresponds to a subset of thylakoid membranes
rather than to free CF
since (i) it displayed the same
amount of LHCII relative to that of cytochrome f as in
fractions 8-12 (data not shown) and (ii) chloroform-extracted
CF
from WT thylakoid membranes was not recovered at the
level of fraction 13 but in fraction 10, i.e. at 40% sucrose
density (results not shown).
and
subunits migrated to a higher density
(fractions 3-5) than that of the LHC-containing thylakoid
membranes (fractions 8-11 as in WT). The
:
ratio in these higher density fractions was
2.3, as calculated by phosphoimaging. These fractions contained neither
the
subunit nor any other protein components (see the
silver-stained gel on Fig. 6A). Some
and
subunits still sedimented to fractions 13-15 (Fig. 5), which corresponds to the minor
and
peak in
the WT gradient. It is of note that, in spite of the higher
accumulation of
than
subunits in FUD16, all
the fractions displaying a significant content in the
subunit also showed the presence of
subunit. Extracts from
older FUD16 cells displayed the same profile as those from younger
cells, although in an
:
ratio of 5, instead of
2.3, in fractions 3-5 (results not shown), which was consistent
with the changes in their relative accumulation described in .
Figure 6:
Identification of the inclusion bodies in
FUD16. A, fractions 3 and 4 from the sucrose gradient (Fig. 5)
were pooled, diluted in buffer, and centrifuged at 100,000 g. The pellet was resuspended and analyzed by silver staining
after urea/SDS-gel electrophoresis; B, after treatment with 8 M urea, the
and
subunits from A are
recovered at the top of a 20-80% (w/v) sucrose gradient. Note
that the faint high molecular weight band (*), detected in fractions
3-4 in A, remains close to the bottom of the gradient in B.
Thus, the experiment of Fig. 5clearly points to
the presence, in the FUD16 mutant, of a membrane-free protein complex
made of and
subunits. This
-
complex equilibrated to a 55% sucrose
concentration which corresponds to a density of 1.26. Surprisingly, in
a similar experiment in which the time of centrifugation was shortened
to 1 h, the same distribution of the major
-
peak was observed, peaking at 55% sucrose concentration (result not
shown). This latter observation further suggested that the
-
oligomer was of larger molecular mass than
expected from a regular oligomeric protein complex, well above 1000
kDa. Indeed, when we diluted the
-
-containing
fractions to a sucrose concentration lower than 0.3 M, we
recovered the
-
oligomers in the pellet, after
low speed centrifugation at 5000
g for 15 min.
-
association by
various treatments. Whereas 2% Triton X-100 had no effect, incubation
of
-
oligomers from fraction 3 with 8 M urea (see ``Materials and Methods'') totally destroyed
their association: when loaded on a second sucrose gradient, the
subunits were recovered in the two fractions close to the top of the
gradient (Fig. 6B). These observations strongly
suggested that
-
oligomers behaved as an
inclusion body, a subcellular structure observed, for example, when
foreign proteins are overexpressed in E. coli (Williams et
al., 1982).
Direct Observation of an
We looked at the ultrastructure of FUD16 cells
by electron microscopy. Fig. 7shows a thin section across a cell
region where the cytosol can be distinguished from the stroma of the
chloroplast by its granular and darker aspect. A striking
ultrastructural characteristic of the chloroplast in FUD16 cells was
the presence of a round-shaped amorphous and dark mass which is typical
of an inclusion body (labeled ib on Fig. 7). It was
unambiguously localized in the stroma next to the thylakoid membranes (arrows on Fig. 7) but not enclosed within a membrane
vesicle. Its average diameter was 1 µm, as measured on a
preparation of inclusion bodies purified from FUD16 broken cells by
sucrose gradient centrifugation (experiment not shown). It was similar
to that of the majority of the inclusion bodies recovered after
purification (Taylor et al., 1986).
-
Inclusion Body in FUD16
Chloroplasts
Figure 7:
Thin section of a FUD16 cell. The cell
contains a round-shaped and amorphous electron dense mass corresponding
to an inclusion body (ib). Note the presence of thylakoid
membranes (arrows) in the vicinity of the inclusion body
pointing to its chloroplast localization.
An immunocytochemical
characterization of the composition of these inclusion bodies was
undertaken, in situ, using various antibodies conjugated with
gold-labeled protein A (Fig. 8). The inclusion bodies (white
arrows), but not the thylakoid membranes (black arrows),
were heavily labeled with antibodies against the or
subunits (Fig. 8, a and b). We observed no
labeling of the inclusion bodies with antibodies against the
subunit of CF
(Fig. 8c) or against LHCII
subunits (Fig. 8d). The LHCII antibody, but not the
antibodies against the various ATP synthase subunits, heavily labeled
the thylakoid membranes in FUD16 (black arrows). This labeling
pattern of the membranes is consistent with their unaltered content in
peripheral antenna proteins but with extensive deficiency in ATP
synthase.
Figure 8:
Immunocytochemical characterization of the
inclusion bodies in the FUD16 mutant. Inclusion bodies were densely
labeled with antibodies directed against (a) and
(b)
subunits (white arrows) but not with antibodies directed
against the
subunit (c). Thylakoid membranes (black
arrows) were labeled only with anti-LHCII (d).
Nuclear Mutations, Which Block Synthesis of the
That the overexpressed
Subunit or Alter the Rates of Synthesis of the
Subunit, Prevent Inclusion Body
Formation
subunit in FUD16 was found in association with the
subunit
in inclusion bodies led us to suspect that an early recognition of the
mutated
subunit by a neighboring
subunit may be required
for its stable accumulation. In addition, this early recognition may
critically depend on the respective rates of synthesis of the two
subunits. Therefore we looked at the synthesis and accumulation of
and
subunits in nuclear contexts which
markedly altered their synthesis. Two nuclear mutants were used in this
study: the thm24 mutant which is unable to synthesize the
subunit, because of a destabilization of the atpB transcript,
and the ncc1 mutant which shows a decrease in the rate of synthesis of
the
subunit because of a decreased stability of the atpA transcript (Drapier et al., 1992). We placed the FUD16
chloroplast mutation in each of these nuclear backgrounds by selecting
double mutants from FUD16.mt+
thm24.mt- or
FUD16.mt+
ncc1.mt- crosses (see ``Materials and
Methods'').
and
subunits by
pulse labeling (Fig. 9A) and for the extent of
intracellular accumulation of the two subunits by Western blotting (Fig. 9B). The double mutant FUD16.thm24 had
characteristics similar to that of the thm24 single mutant. In vivo pulse labeling of chloroplast translates showed the absence of
synthesis and no overexpression of the
subunit (Fig. 9A). Pulse labeling of chloroplast
translates in the double mutant FUD16.ncc1 showed no differences with
that in the single mutant ncc1. In each case, the rate of synthesis of
the
(respectively
) subunit was severely
decreased as indicated by its much lower labeling than that of the
subunit (Fig. 9A) at variance with the situation
observed in the WT or FUD16 cells. The immunoblots on Fig. 9B show that neither the FUD16.thm24 nor the FUD16.ncc1 accumulated
the
subunit. The FUD16.ncc1 strain thus behaves
very differently from the single mutant ncc1 which accumulates
and
subunits in CF
complexes. The trace amount of
subunit visible in whole cells of FUD16.ncc1 is similar to the
phenotype of the F54 strain which totally lacks synthesis of the
subunit but accumulates small amounts of the
subunit (Drapier et al., 1992). Thus, the absence of inclusion bodies in the
two double mutants suggests that their formation critically depends on
the rates of association between
-
oligomers.
Figure 9:
Characterization of the double mutants
FUD16.thm24 and FUD16.ncc1. A, 50-60-kDa region of a
urea/SDS-acrylamide gel loaded with mutant cells pulse-labeled for 5
min. Same experimental conditions as described in the legend to Fig. 2.
The WT and single nuclear mutants thm24 and ncc1 are shown for
comparison with the double mutants. B, immunoblots of whole
cells from the same mutant strains as in A. and
subunits were detected with specific antibodies and radioiodinated
protein A, as in Fig. 3. The ncc1 accumulates the two subunits in the
same ratio as the wild type (Drapier et al.,
1992).
The Substitutions in FUD16 Occur at Strictly Conserved
Sites in
In the present
study we have identified the mutational event which prevents ATP
synthase assembly in the chloroplast mutant FUD16 of C.
reinhardtii. Two neighboring point mutations in the atpA gene result in the expression of an Subunits from (C)F
subunit
with two amino acid substitutions: the Ile
Leu
Asn
tripeptide segment from the
WT
subunit is converted into Asn
Leu
Tyr
in the FUD16
. The
IXN motif found in the WT
subunit is strictly conserved
among the 40 sequences of
subunits from bacterial, mitochondrial,
and chloroplast (C)F
available in the NBRF data bank. This
motif is also present in
subunits from bacterial, beef heart, and
yeast mitochondrial F
and higher plants and C.
reinhardtii CF
(Walker et al., 1985). These
two amino acid changes in
are located 7 and 9 amino
acids downstream the conserved nucleotide-binding fold
GXXXXGKT (Walker et al., 1982), a protein motif also
present in all
and
subunits of (C)F
-ATP
synthase. According to the three-dimensional structure of the
F
-ATPase from bovine heart mitochondria, which is now
resolved at 2.8-Å resolution (Abrahams et al., 1994),
the nucleotide-binding motif forms the ``P-loop'' between
strand 3 and helix B in the nucleotide-binding domain of either the
or
subunit. The two amino acid substitutions in
are thus located next to the end of helix B, toward
the periphery of the complex, and opposite the region facing the
internal cavity where the helical domains of the
subunit are
located.
Misfolded
Most strikingly, the conversion of the Ile-
Complexes Accumulate in the Chloroplast as Inclusion
Bodies
Leu
Asn
tripeptide
segment from the WT
subunit into Asn
Leu
Tyr
in FUD16 results in the
overproduction of the
subunit. The intracellular
concentration of the
subunit is also increased in the mutant but
to a more limited extent than that of the
subunit.
In contrast, the other subunits of the chloroplast ATP synthase, such
as the
subunit of CF
or subunit III of
CF
, were hardly detectable in this mutant strain. The bulk
of the
and
subunits in FUD16 copurified in
membrane-free, high molecular weight aggregates which sedimented upon
low speed centrifugation. These urea-sensitive aggregates had
morphological features and a density of 1.26 on sucrose gradients,
which are typical of previously characterized inclusion bodies from E. coli (Taylor et al., 1986). Although chloroplast
chaperonins can associate with
subunits in some instances (Lubben et al., 1989; Chen and Jagendorf, 1994), we found no evidence
for their presence in the inclusion bodies: silver-stained gels showed
that they contained no other proteins than the
and
subunits.
Inclusion Bodies Are Made of Partially Folded
Intermediates
It is of note that the mutation in FUD16 preserved
-
interactions but almost totally prevented
further interactions with the
subunit. Although the two point
mutations in
alter amino acids located toward the
periphery of CF
complexes, as viewed from the
three-dimensional structure of F
(Abrahams et al.,
1994), one could consider that these mutations should interfere with
the folding of the whole P-loop region thereby preventing interaction
with the
subunit. However, the study of Smart and Selman(1993)
showed that the mere absence of interactions between the
subunit
and
-
oligomers does not lead the latter to aggregate in
inclusion bodies. Instead the
and
subunits were rapidly
degraded in a nuclear mutant of C. reinhardtii disrupted in
the atpC gene encoding the
subunit.
, although in marginal amounts,
were properly assembled in the FUD16 mutant and displayed some ATPase
activity in nondenaturing gels. In addition, as we previously reported
(Lemaire and Wollman, 1989b), the FUD16 strain displays some ATP
synthase activity in vivo, as deduced from the increase in the
decay rates of the flash-induced transmembrane potential upon
preillumination of dark-adapted cells. This treatment is known to
activate the membrane-bound ATP synthase (Joliot and Delosme, 1974).
Thus the two point mutations in the FUD16 strain, instead of destroying
a site required for proper folding of the
subunit, would rather
alter its folding pattern during CF
biogenesis. Newly
synthesized
subunits still interact with
subunits and are thus protected from proteolytic degradation as
demonstrated by their destruction in the FUD16.thm24 mutant in which
the
subunit is not synthesized. The subsequent assembly pathway
in FUD16 would drive most, but not all, of the
-
folding
intermediates toward inclusion body formation rather than toward
CF
assembly. This explains why the inclusion bodies in
FUD16 comprise two components, the
and
subunits, instead of one only as usually observed (Mitraki and King,
1989). Our study is consistent with the folding pathway recently
proposed by Chen and Jagendorf(1994) based on a chaperonin-assisted in vitro system for reconstitution of
,
, and
subunits from CF
: several
-
dimers would assemble
up to an hexamer complex able to interact with the
subunit. The
FUD16 mutation would prevent formation of most of the hexamers
competent for
binding. A similar situation most likely prevails
after import of the
and
subunits of F
in the
mitochondria: Ackerman and Tzagoloff(1990) showed that the proper
folding pathway of
-
oligomers in the organelle requires
assistance of at least two nuclear-encoded proteins which are not part
of mitochondrial F
. Yeast mutants defective in either of
these two proteins displayed a phenotype which strikingly resembles
that of FUD16. They accumulated
-
aggregates of high
molecular weight in the mitochondria. We believe that further analysis
of these mutants would show genuine intramitochondrial inclusion bodies
made of
-
aggregates.
Increased Rates of Synthesis of the
Five-min pulse labeling
experiments showed that there was a 4-fold increase in the rate of
synthesis of and
Subunits in FUD16 Point to an Early
Interaction during their Biogenesis
in FUD16 as compared to that of
in the WT. As we discussed elsewhere (Kuras and Wollman 1994), these
rates of synthesis do not reflect the subsequent state of accumulation
of a given polypeptide and result from a combination of the rates of
translation with the rates of possible co-translational or early
post-translational modifications. According to the codon usage in the
chloroplast of C. reinhardtii (Rochaix, 1987) we could exclude
that either of the 2-base pair substitutions corresponded to a
conversion of a rare codon in a more frequently used codon, a situation
which may have accounted for an increased rate of translation of
. We can also exclude an increased translation due
to an increased availability in atpA transcripts since mRNA
hybridization experiments showed no changes in their accumulation
between the WT and FUD16 strains.
subunit was also increased in FUD16. It suggests
that the biogenesis of the two subunits are intimately related.
Aschenbrenner et al.(1993) working with mitochondrial yeast
F
had observed an increased synthesis of a mutated
subunit with a concomitant increase in synthesis of the
subunit.
Other evidence for an early interaction in the synthesis of the two
subunits came from our previous study of several nuclear mutants of C. reinhardtii (Drapier et al., 1992). These mutants
displayed changes in the rates of
synthesis associated with
changes in the rates of synthesis of the
subunit. Here, we
observed that FUD16.thm24 and FUD16.ncc1 double mutants no longer
displayed an increased rate of synthesis of the
subunit. In the two cases, the translated
subunits were rapidly degraded. This suggests that the increased
synthesis of
subunit observed in the single mutant
FUD16 is controlled by an early interaction with neighboring
subunits.
or
subunits could act as a translational regulator on the atpA or atpB transcripts as recently suggested for other
(di)nucleotide binding proteins (Hentze, 1994). Changes in ribosome
pausing during translation, at positions where proper folding
intermediates would otherwise accommodate cofactor-binding or further
interactions between nascent subunits and chaperonins (Kim et
al., 1991; Stollar et al., 1994), could also account for
our observations.
Table: Comparative accumulation of the and
subunits in cells and membranes preparations from WT and FUD16 strains
and
subunits, and radioiodinated protein A; quantification was performed by
phosphoimaging. Values in mutant FUD16 are given relative to those in
WT.
0
structure, F. Lacqueriere and M. Recouvreur for expert technical
assistance, and the members of the Service de Photosynthèse for
critical reading of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.