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Drapier et al, Supplemental Data
expression of a truncated subunit .
We generated a chloroplast mutant expressing a truncated subunit , by introducing a stop
codon within the atpA coding sequence at position 300, associated with the recycling aadA cassette for
selection of transformants (fig. S1A).
In pulse-labelling experiments, the resulting Tr strain (table I), showed no neosynthesised
subunit , but a new translation product (truncated subunit ) with an apparent molecular mass of ~30
kDa (fig. S1B). This truncated polypeptide is stable and accumulates to significant amount (fig. S1C).
In absence of an assembly- competent subunit , this truncated polypeptide behaves as full-length
subunit it shows a much reduced synthesis and does not accumulate (fig. S1B). We note also that
the synthesis of subunit  remains high in the absence of subunit  when subunit  is expressed in a
truncated form, indicating that  heteromers can not form in this latter strain.
atpA
rbcL
St
psbI
A)
Tr
filled-in
SexA1
B
cemA
K
WT
{ Tr, Tr}
C)
Tr
Tr


WT
B)
{ Tr, Tr}
Recycling cassette

Tr
Tr
*
OEE2
fig. S1: expression of a truncated subunit 
A) Strategy used to introduce a mutation (St) in the atpA gene, associated with the recycling
spectinomycin resistance cassette, schematically depicted by >> K >> (not to scale). B, BseRI. Arrow
indicates transcription start site. Coding sequences are depicted in grey. Because the mutation leads to
a premature stop codon (St), about one third of atpA coding sequence is not translated in the mutant
(white rectangle).
Synthesis (B) and accumulation (C) of subunits  and  in the wild type and in strains Tr
and {Tr,  Tr}.
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Drapier et al, Supplemental Data
Subunit  translated from an unrelated 5’UTR can be overexpressed in the absence of
subunit .
The chimeric gene 5’psaA-atpA contains the atpA coding region translated under the control of
the psaA 5’UTR (psaA encodes one of the major subunit of PSI, the expression of which is
independent of ATP synthase biogenesis). We replaced the atpA gene from the wild-type strain by this
5’psaA-atpA chimera (fig. S2A), associated with the aadA cassette. Spectinomycin-resistant
transformants aA  because they express a 5’psaA driven-subunit  - were able of phototrophic
growth. Chimeric subunit  is translated and accumulated to levels similar to the regular  subunit in
the wild type (fig. S2B). This construct was also transformed in strain Tr. In transformed strains {Tr,
aA}, translation of chimeric subunit  is similar to that observed in strain aA (fig. S2B), in contrast
to the endogenous subunit, which is poorly synthesised in the presence of a truncated  subunit (fig.
3). Subunit  is fairly well accumulated in strain {Tr, aA} (about 60% of the level found in strain
aA), indicating that it is quite stable, even in absence of subunit  (fig. S2B).
As we expected, translation of subunit  is no longer regulated by protein assembly when not
B
A N*
5’psaA
aA
rbcL
cemA
cemA


atpA
S
bAK
psbI
rbcL
psbI
atpA
WT
aA
B)
WT
A)
{ Tr, aA}
expressed from the atpA 5’UTR;
500 bp
Tr
Fig. S2: The atpA 5’UTR is required for the -dependent
control of subunit  expression.
A) Map of the atpA gene in wild-type and aA strains.
Relevant restriction sites are indicated: B, BseRI; N*, an NcoI
site engineered around the atpA initiation codon for cloning
purposes; A, AvrII. Arrows indicate transcription start sites.
bAK stands for the 5’psbA-aadA spectinomycin resistance
cassette (Minai et al, 2006), in opposite orientation with respect
to atpA.
B) Translation (upper panel) and accumulation (lower
panel) of the  and  subunits in the wild type and in strains
aA and { Tr, aA}. OEE2 provides a loading control.


OEE2
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Drapier et al, Supplemental Data
Cycloheximide pre-treatments modify the translation pattern of chloroplast genes.
For specific visualisation of chloroplast translation products, cycloheximide, an inhibitor of
cytosolic translation is added to cell suspensions prior of pulse-labelling experiments. However,
chloroplast gene expression is heavily dependent on nuclear gene products, which suggests that a
cycloheximide treatment could affect the rate of synthesis of chloroplast-encoded polypeptides.
Therefore we pulse-labelled wild-type cells, pre-treated with cycloheximide for increasing times prior
to the addition of
14
C-acetate. The general incorporation of
14
C within chloroplasts drops with
increasing time of cycloheximide pre-incubations, reflecting the decreased cellular metabolism when
cytosolic translation is impaired (Table SI). After correction for changes in
14
C incorporation, most
polypeptides showed unaltered rates of synthesis (see fig. S3 for polypeptides in the 30-60 kDa range
and Table SI). Subunit  from ATP synthase showed a limited decrease of its rate of translation upon
long time of preincubation with cycloheximide. By contrast, synthesis of subunit  drastically
decreased upon cycloheximide treatment and became hardly detectable after a 30 min preincubation.
Consequently, the relative synthesis of these two subunits, initially close to stoichiometric amounts,
became rapidly unbalanced (table S1).
From this experiment, we conclude that the unbalanced rate of synthesis of subunits  and 
observed in previous studies (Lemaire and Wollman, 1989)(Drapier et al., 1992) is likely an artefact
caused by the 10 min cycloheximide pretreatment. Thus, pulse labelling experiments performed in that
work, were done by adding 14C Acetate and cycloheximide simultaneously.
time of cycloheximide
preincubation (min.)


0’
10’ 30 ’
apoCP47
apoCP43
cyt. f
Fig. S3: Pulse-labelling of exponentially growing wild-type cells, pretreated with cycloheximide
(10 µgml-1) for the indicated time.
Position of neo-synthesized subunits  and  of ATP synthase, cytochrome f and PSII core subunits
are indicated.
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Drapier et al, Supplemental Data
Table SI:
Effect of cycloheximide pre-treatment on 14C incorporation and on the relative rates of synthesis
of ATP synthase subunits  and .
Cycloheximide
preincubation
(min)
14
0a
10
30
100 %
30 %
20 %
apo-CP47c
1.0
1.3
1.4
c
1.0
1.4
1.3
c
1.0
0.7
0.25
c
1.0
1.2
0.86
d
1.1
0.6
0.38
C incorporationb
apo-CP46
a
: cycloheximide was added simultaneously with 14C Acetate (experimental conditions chosen for this
study).
b 14
: C incorporation (measured as the total amount of radioactivity over the whole length of the lane) in
response to increasing time of cycloheximide pre-treatments; value for time 0 is taken as 100%.
c
: rate of synthesis corrected for variations in 14C incorporation.
d
: ratio between 14C incorporation into polypeptides  and  for each time point.
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Drapier et al, Supplemental Data
Materials and Methods
DNA Constructs
1- Construction of atpA deletion.
Plasmid patpA2 (Ketchner et al., 1995), encompassing the atpA gene was digested by BseRI
and PacI and ligated with the 2.95 kbp SacI-KpnI fragment from plasmid pKS-aadA-483 (Fischer et
al., 1996), both fragments being treated with T4 DNA polymerase to generate blunt ends. In the
resulting plasmid patpA::Kr, the atpA gene was replaced by the recycling aadA cassette, in a direct
orientation with respect to atpA.
2- Construction of chimeric genes.
i)
atpA
To create a chimeric atpA gene, a 245 bps DNA fragment containing the psaA promoter and
5’untranslated
regions
was
PCR-amplified
GACGTCCCCGGGTTTTACGAATACACATATGGT
with
3’)
and
primers
PsaA
(5’
PsaAPromB
(5’
GCG-
CGCGGATCCATGGTCA-
TGGATTTCTCCTTA 3’) from template plasmid ps1A1 (Kuck et al., 1987). This amplicon was
digested with AvrII and NcoI, two restriction sites (underlined) introduced in the sequence of primers,
respectively upstream of the 5’UTR and downstream of the psaA initiation codon and cloned into the
pFAAA plasmid (Drapier et al., 2002), digested with the same enzymes, to yield plasmid paA. The
1.7 kb 5’psbA-aadA spectinomycine resistance cassette was removed from plasmid pbAK (Minai et
al., 2006) by digestion with SmaI and EcoRV and inserted into the unique SmaI site (a site introduced
when designing primer PsaAPromB, written in bold) of plasmid paA, in direct orientation with respect
to the atpA gene, to create plasmid pKaA.
ii)
atpB
A DNA fragment containing the atpB promoter and 5’untranslated regions was removed from
plasmid pKS-atpB ATG 123 (Rimbault et al., 2000) by digestion with ClaI and NcoI and ligated into
vector pAFFF, digested with the same enzymes to yield plasmid pdBTf. The EcoRV-SmaI 1.9 kb aadA
cassette (Goldschmidt-Clermont, 1991) was then inserted, in reverse orientation with respect to the
petA gene, into this later plasmid digested with the unique cutter HincII to create plasmid pKdBTf.
3- Generation of truncated alleles
i) atpB gene.
Plasmid
P-112,
obtained
from
the
Chlamydomonas
Genetic
Center
(www.biology.duke.edu/chlamy/), contains a 5.3 kbp BamHI-EcoRI fragment from the chloroplast
genome encompassing the atpB gene (Woessner et al., 1986), subcloned into pUC8 vector. Selection
of transformed cells expressing a truncated version of the  subunit required the insertion of a
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Drapier et al, Supplemental Data
spectinomycin resistance cassette downstream of the atpB gene, in a region that belongs to the
inverted repeat. Gene conversion between the two copies of the inverted repeat could thus lead to the
duplication of the resistance cassette. The new copy of the cassette, not linked to the truncated atpB
allele, could hamper the selection of mutants. Thus, to prevent gene conversion and expedite the
process of homoplasmisation, the inverted repeat sequences downstream of the atpB gene were
removed. To do this, a 767 bp DNA fragment that contains the C-terminal part of the atpB coding
sequence and downstream regions, but no inverted repeat sequences, was PCR-amplified from
template P-112 with primers atpBcod (5’ AGGTCGTATGCCATCAGCTGTAGGTTACCAACCT 3’)
and atpBRev (5’ CGCGGTACCATA-CACGTTTAACGGCGTCCT 3’: KpnI). It was was digested by
ClaI (that cuts plasmid P-112 only once, within the amplified region) and KpnI (introduced when
designing primer atpBRev) and ligated into the vector P-112, digested with the same enzymes to yield
plasmid P-112IR. This later was then digested with ClaI, treated with Klenow enzyme and religated
on itself to form plasmid patpB335St. This treatment created a new NruI restriction site and caused a +2
frameshift in the coding sequence of atpB, leading to a premature stop codon 335 residues after the
initiation codon. The recycling aadA cassette was then introduced into the KpnI site of vector
patpB335St, in the same orientation as atpB, to create plasmid patpB335StKr.
ii) atpA gene.
Plasmid patpA2 was digested with SexAI, filled-in with Klenow and relegated on itself to
create plasmid patpA2300St. This generated a five bases frame-shift, causing the premature termination
of translation 300 residues after the initiation codon. The recycling aadA cassette was then introduced
into the BseRI site of vector patpA2300St, in the same orientation as atpA, to create plasmid
pKratpA300St.
Transformation experiments
Proper insertion of transforming DNA and homoplasmy were checked by RFLP analysis of
specific PCR amplification products, since all chimeric genes used in that study led, upon PCR
amplification of the region surrounding the 5’UTR (i.e. with a forward primer upstream of the petA
gene PetAprom2 –5’ GCGAATTCGCAGGCAGTGGCGGTACC 3’-and a reverse primer within the
coding sequence petArevA -5’ ACAGCTTGTGGTACTTCGATTTC-AACTGCT 3’-), to amplicons
of different size whether the template was the wild-type or the chimeric petA genes. For deletion or
truncated strains, homoplasmy was first deduced from the absence of growth of the transformed
strains on minimum medium, indicative of the loss of the wild-type version of the gene of interest and
further confirmed by size and/or RFLP analysis of specific PCR products encompassing the modified
region.
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Drapier et al, Supplemental Data
References
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Chlamydomonas identifies a factor controlling chloroplast mRNA stability by acting on the
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