From the
The
The light reactions of photosynthesis generate reducing
equivalents in the form of NADPH and energy in the form of ATP. During
photosynthetic electron transfer, protons are unidirectionally
deposited into the lumen of the thylakoid membrane. The chloroplast ATP
synthase (CF
ATP synthases from all species are highly regulated
(4) . One
possible mode of regulation that appears to be unique for plant enzymes
is the redox state of the
In contrast to other coupling factor complexes, CF
DNA was isolated from
parental strain nit1-305, atpC1, and each of the
photoautotrophic colonies and subjected to Southern blot analysis with
PstI digestion. Photoautotrophic colonies were labeled as
putative cotransformants if they contained DNA fragments that
hybridized to
The
Previous studies have shown that a wild-type CF
There are already several examples of stable nuclear transformation
in C. reinhardtii (30, 31, 32, 33, 34, 35) .
However, we believe that this is the first example of stable nuclear
transformation in which gentically engineered genes have been used to
complement a known defect. That we were successful demonstrates the
utility of atpC1 as a model system to study the CF
A growth curve for wild type,
atpC1, and each of the three mutants was performed in order to
determine the effects, if any, of each of the
Examination of enzymatic
activities of each of the mutants using a partially purified, enriched
cell lysate
(18) revealed that in each case the mutant enzymes
behaved in similar fashion to parental nit1-305 with respect
to manifest activity and stimulation by 20% (v/v) ethanol. However,
nit1-305 showed an increase in ATPase activity when 20%
ethanol and 50 mM DTT were included in the reaction mixture
when compared with the same experiment minus DTT. The soluble CF
We believe that the data from the PMS-dependent photophosphorylation
experiments support the model of Nalin and McCarty
(5) , which
implicates the disulfide bond within the CF
The data from the growth curves may also suggest that the
in vivo form of coupling factor in the light is the reduced
form of the enzyme. The PMS-dependent photophosphorylation experiments
indicate that the mutant enzymes have activities that are virtually
identical to that of the reduced form of the wild-type enzyme. In wild
type, the reduced form of CF
In the work presented in
this report it was important for us to first demonstrate that we could
obtain viable transformants and functional CF
The
coding strand primer for all reactions was
5`-GGAGGAAGGGGGTGTTGCGGGGAGGTCCTG-3`.
Listed reactions contained 20% (v/v) ethanol, 50 mM DTT, and 330 µg of chicken egg yolk antibodies prepared either
prior to (preimmune) or after (immune) immunization of a chicken with
purified CF
We thank Dr. Pete Lefebvre for plasmid PMN24, helpful
hints about Chlamydomonas transformation, and continued
interest and support. We are also grateful to Susanne Selman-Reimer for
assistance with the ATPase assays, to Tracy Baas for synthesizing
oligonucleotides, to Adam Steinberg and Laura Vanderploeg for artwork,
and to Drs. Milo Aukerman and Michael Sussman for helpful suggestions
about the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-subunit of coupling factor 1 (CF
) contains
a cysteine bridge that is thought to be involved in the redox control
of enzymatic activity. In order to test the regulatory significance of
this disulfide bond, genetic transformation experiments with
Chlamydomonas reinhardtii were performed. C. reinhardtii strain atpC1 ( nit1-305, cw 15,
mt
), which is null for the
-subunit, was
transformed and complemented with
-subunit constructs containing
amino acid substitutions localized to the cysteine bridge between
Cys
and Cys
. Successful complementation was
confirmed by phenotypic selection, Northern blot analysis, reverse
transcription polymerase chain reaction, and cDNA sequencing. CF
ATPase activities of the soluble enzymes were measured in the
presence and absence of dithiothreitol (DTT). Mutant CF
enzymes showed no effect of DTT although increased activity was
observed for the wild-type enzyme. In vitro, phenazine
methosulfate-dependent photophosphorylation assays revealed that
wild-type CF
exhibits a 2-fold stimulation in the presence
of 25 mM DTT, whereas each of the mutant enzymes has
activities that are DTT-independent. Growth measurements indicated that
despite the absence of a regulatory disulfide/dithiol, the mutant
strains grew with the same kinetics as wild type. This study provides
evidence to illustrate the involvement of the
-subunit in the
redox regulation of ATP synthesis in vivo. This work is also
the first demonstration in C. reinhardtii of stable nuclear
transformation using mutated genes to complement a known defect.
CF
)
(
)
couples
the chemical potential energy of the protonmotive force to the
synthesis of ATP
(1) . ATP synthase contains two functionally
distinct domains, designated CF
and CF
.
CF
is an integral thylakoid membrane protein complex that
functions as a proton channel. CF
is a peripheral membrane
complex that is located on the stromal side of the thylakoid; it
contains the catalytic sites for ATP synthesis
(2, 3) .
-subunit
(5, 6, 7, 8, 9, 10, 11, 12, 13) .
The Chlamydomonas reinhardtii CF
-subunit,
like other plant
-subunits, contains an intrapeptide disulfide
bond between positions 198 and 204 of the mature polypeptide. This is
the only disulfide bond within the entire multisubunit CF
complex
(5) . Protein sequence comparisons between the
known plant CF
-subunits and their F
mitochondrial and bacterial counterparts reveal that this
disulfide bond is a unique feature of the plant enzymes
(14) .
Several in vitro chemical modification studies have indicated
that the disulfide bond may be important for regulatory control of ATP
synthesis. In the presence of reducing agents, such as DTT, CF
exhibits elevated ATPase activity
(5, 6, 7, 8, 9, 10, 11, 12, 13) .
has
not been fully reconstituted from its component subunits
(4) .
Furthermore, in plants, it is difficult to perform genetic
manipulations that affect the photosynthetic apparatus without causing
lethal effects on the plants. However, Ort and colleagues
(15) have reported experiments where they screened a large
population of Arabidopsis thaliana to look for mutant
phenotypes affiliated with the redox state of coupling factor. A few
candidate strains have been obtained, and the characterization of these
mutant strains is currently under way
. (
)In this
study we utilized the green algae C. reinhardtii, which can be
grown photoautotrophically or heterotrophically on acetate, thereby
rendering the photosynthetic apparatus dispensable. Recently, a strain
of C. reinhardtii designated atpC1, which is null for
CF
-subunit, was isolated and characterized
(17) . Here we describe nuclear transformation of atpC1
with mutated CF
-subunit genes. We believe that this
is the first demonstration of stable nuclear transformation and
complementation of a defective nuclear gene in C. reinhardtii using mutant genes. With this model system, we were able to test,
by site-directed mutagenesis of the
-subunit, its putative role in
redox control of coupling factor 1.
Generation of CF
Fig. 1
shows the cloning strategy utilized.
Site-directed mutageneses of the cysteine bridge region were achieved
by four subcloning steps. The genomic CF
-Subunit
Mutations
-subunit
clone, P
, was utilized. This clone, a SalI restriction
fragment isolated from a C. reinhardtii EMBL-3
phage
library, contains the transcription unit plus 762 bp of upstream
promoter sequence of the CF
-subunit gene cloned into
the SalI site of pBluescript SK
(Stratagene,
La Jolla, CA)
(18) . Three mutations were generated within the
168-bp BglII region located between positions 1811 and 1979 of
the
-subunit transcription unit. Due to the presence of a
BglII site within the promoter region at position
122,
P
was subcloned by digestion with SacI into two halves,
designated plasmids P
1 and P
2, with the latter containing the
BglII region of interest. Using PCR mutagenesis, we generated
three different mutations within the BglII cassette, all of
which are localized to the cysteine bridge between positions 198 and
204 of the
-polypeptide. The PCR products were restricted with
BglII and inserted into P
2 to yield plasmid P
3.
Proper sequence and orientation were confirmed by dideoxy sequencing
(19) and restriction digestion analyses. The P
3 inserts
were restricted with SacI and subcloned into P
1 to yield
P
4 plasmids. In total, three mutations were generated at each or
both of the cysteine residues at positions 198 and 204 of the CF
-subunit polypeptide. They are designated C198S, C204S, and
C198S/C204S to indicate that a serine residue has replaced the cysteine
at position 198 or 204, or both 198 and 204. In generating
site-directed mutants by PCR, the 168-bp region in P
, which is
flanked by BglII restriction sites, served as the template.
The noncoding strand primers were designed to introduce the mutations
into the targeted region. They are listed in .
Figure 1:
Cloning
strategy used to make CF
-subunit mutants for
cotransformation into atpC1 cells. P
, the plasmid
containing the wild-type atpC gene, was subcloned by
SacI digestion into two segments designated P
1 and
P
2. PCR primers were used to generate each of the three mutants by
amplifying a 168-bp fragment that was restricted with BglII
and inserted into P
2 to yield P
3 constructs. P
3
constructs were restricted with SacI and inserted into
P
1, which yielded P
4 constructs.
Introduction of Plasmids C198S, C204S, and C198S/C204S
into atpC 1 Cells by Cotransformation
Because we could not
a priori predict the phenotypes of atpC1
cells complemented with mutant CF-subunit genes, we
employed a cotransformation protocol. This approach takes advantage of
the fact that strain atpC1 is also null for nitrate reductase.
For the first selection, the atpC gene constructs were
cotransformed along with plasmid PMN24
(20) , which encodes
C. reinhardtii nitrate reductase, into nit1-305,
cw-15, mt
, atpC1 cells. Cells were grown in
liquid Sager-Granick II-NH
(SGII-NH
) medium
(21, 22) under continuous light to midlogarithmic phase (1-2
10
cells/ml) and concentrated to 3
10
cells/cotransformation experiment by centrifugation at 4,000
g for 5 min. The cells were washed two times and
finally suspended in 0.5 ml of SGII-NO
.
atpC1 cells were cotransformed according to the glass bead
protocol described by Kindle
(23) . The cells were uniformly
plated onto 150
15-mm Petri plates containing
SGII-NO
, 1% agar (previously washed six
times with double distilled water) and cultured under continuous
illumination of approximately 120 µmol/m
s from
cool white fluorescent bulbs. Nit
colonies appeared in
about 2 weeks. They were reselected two times in 96-well microtiter
plates in liquid SGII-NO
medium.
Surviving colonies were replica plated two times into liquid
SGII-NH
acetate minus medium and selected
for photoautotrophic growth, again under low light of
120
µmol/m
s. In each case about 15% of the
nit
colonies were able to grow photoautotrophically,
and they were thus labeled as putative cotransformants.
Photoautotrophic colonies were grown in
SGII-NH
medium prior to isolation of DNA,
RNA, or protein.
DNA and RNA Isolation, Electrophoresis, and
Blotting
Cells were grown in
SGII-NH media to midlogarithmic phase and
pelleted by centrifugation at 4,000
g for 5 min. DNA
isolation, RNA isolation, gel electrophoresis, and Southern and
Northern blotting were all performed as described previously
(17, 24) . Blots were probed with CF
-cDNA (or
-subunit cDNA) excised from host plasmid by
EcoRI digestion
(25) , gel-purified, and randomly
primed with [
P]dCTP. All blots were exposed to
x-ray film with an intensifying screen.
Reverse Transcription PCR
Five µg of total RNA
were reverse transcribed with 50 ng of random hexamers by following the
manufacturer's protocol (Life Technologies, Inc.). The cDNA
products were phenol/chloroform-extracted, precipitated, and
resuspended in 80 µl of Tris/EDTA (pH 8.0). Two µl were used
for each PCR reaction. PCR reactions were carried out as described
previously
(17) . The reaction conditions were 1 cycle at 94
°C for 5 min, 35 cycles at 94 °C for 2 min, 57 °C for 1
min, and 72 °C for 2 min.
Preparation of Thylakoid Particles
1 liter of
cells were grown to midlogarithmic phase, collected by centrifugation
(5 min at 200 g), washed in 50 mM
Tricine-NaOH (pH 8.0) buffer, and resuspended in 50 mM
Tricine-NaOH (pH 8.0) to an equivalent concentration of 1 mg of
chlorophyll/ml. This suspension was sonicated in 3-ml batches at room
temperature in a water bath-type sonicator (Laboratory Supplies Co.,
Inc., Hicksville, NY; power output, 80 kHz, 80 watts) for 20 s. The
resulting suspension was centrifuged at 200
g for 1
min in order to pellet unbroken cells. Thylakoid particles were
obtained by centrifugation of the supernatant at 30,000
g for 10 min. When used to prepare ATPase, the particles were
resuspended (at
0.5 mg of chlorophyll/ml), washed three times in
10 mM NaPP
(pH 7.8), and centrifuged at 30,000
g for 10 min. When used for photophosphorylation
experiments, the thylakoid particles were resuspended in a buffer that
contained 20 mM Tricine-NaOH (pH 8.0), 0.5 mM
MgCl
, 0.3 M sucrose, and 10 mM NaCl and
maintained at 4 °C until they were used.
Growth Curves
About 1 10
cells
for each strain were placed into 250 ml of
SGII-NH
acetate minus medium and were
cultured in an 8-h light/16-h dark photoperiod for several days in a
growth chamber kept at 18 °C. The light intensity during the 8-h
light period was
120 µmol/m
s. At the
indicated times duplicate 1 ml samples of cells were harvested,
immobilized by the addition of 50 µl of a solution containing 1
mM iodine dissolved in 95% ethanol, and counted with a
hemacytometer.
ATPase Assays
Cells were grown to midlogarithmic
phase. Isolation of enriched fractions of CFwas performed
as described previously
(18, 26) . The ATPase reaction
mixture, in a total volume of 0.1 ml, contained the following: 20
mM Tricine-NaOH (pH 8.0), 1 mM EDTA, 5 mM
MgCl
, 10 mM [
-
P]ATP
(
2
10
cpm), 50 mM dithiothreitol, and
0.5 µg of protein. Reaction mixtures were incubated for 6 min at 37
°C. The reactions were terminated as described previously
(26) , and 1 ml of released [
P]phosphate
was counted in a Packard 460C liquid scintillation counter.
Phenazine Methosulfate (PMS)-dependent
Photophosphorylation
Reaction mixtures for PMS-dependent
photophosphorylation experiments contained, in a total volume of 0.1
ml, 50 mM Tricine-NaOH (pH 7.8), 10 mM NaCl, 0.5
mM MgCl, 3 mM ADP, 2 mM
[
P]phosphate (
2
10
cpm), and 0.08 mM PMS. Thylakoid particles (3-10
mg of chlorophyll/ml) were added to the reaction mixture, and the
reactions were initiated by the addition of DTT to a final
concentration of 25 mM or ddH
O and illuminated at
20 °C under white light isolated from a 150-watt halogen projection
light pipe (Ealing Optics, South Natick, MA; 2.4
10
µmol/m
s) for 0, 15, 30, 45, 60, and 90 s.
Esterified phosphate was extracted as described previously
(27) and counted in a Packard model 460 C liquid scintillation
counter.
Introduction of Nitrate Reductase and Mutant
CF
Introduction of mutant
CF
-Subunit Genes into atpC1 Cells and
Identification of Cotransformed Strains
-subunit genes into atpC1, which is null
for both
-subunit and nitrate reductase, was accomplished by
cotransformation using the glass bead protocol
(23) . After
about 10 days small, green nit
colonies appeared on
SGII-NO
plates. The nit
transformation efficiency was about 25 cells/2 µg of
circularized DNA for each experiment. The nit
colonies
were reselected two times by transferring them into a 96-well
microtiter plate, which contained liquid
SGII-NO
medium. Surviving colonies were
then replica-plated two times and selected for photoautotrophic growth
in SGII-NH
minus acetate medium. In each
case about three to five colonies were positive for photoautotrophic
growth. Therefore, about 15-20% of the nit
transformants were cotransformants.
P-labeled CF
-cDNA that
were in addition to the endogenous atpC PstI gene fragments.
All putative cotransformants displayed additional bands, and in no case
was there any indication of a reversion event (data not shown).
Northern Blot Analysis of atpC mRNA from Cotransformed
Cells
To determine whether the transformed atpC mutant
genes were being expressed, total, steady-state RNA was isolated from
parental nit1-305, atpC1, and each of the
cotransformed strains and subjected to Northern blot analysis. All of
the photoautotrophic colonies appear to have accumulated atpC mRNA to a level similar to that of the parental nit1-305
strain (Fig. 2). As previously demonstrated
(17) , strain
atpC1 does not accumulate a -subunit message. In
contrast, all the cell strains accumulated mRNA for the CF
-subunit to levels similar to nit1-305 (wild type).
Reverse Transcription PCR, Cloning, and Sequencing of nit1-305,
atpC1, and Cotransformant cDNAs-In order to determine whether the
expressed mRNAs in the putative cotransformants contained the sequences
we engineered and introduced into atpC1, we utilized a reverse
transcription PCR technique. Samples containing 5 µg of RNA from
nit1-305, atpC1, and each of the cotransformants were
reverse transcribed in order to generate cDNA. The cDNA from each
reaction was then subjected to PCR amplification. In order to ensure
that the PCR products arose from cDNA and not from the inherent, albeit
nonfunctional,
-subunit gene within strain atpC1, we
chose to perform PCR using two primers that flank intron 4 of the
genomic
-subunit gene. The presence of contaminating DNA could
then be detected by the presence of a 1015-bp band that would
co-migrate with a control PCR product amplified from P
, the
CF
-subunit genomic clone. As a size control we
amplified the
-subunit cDNA using the same two primers.
Fig. 3B shows that the parental nit1-305,
C198S, C204S, and C198S/C204S strains each amplify a 308-bp fragment
that co-migrates with the positive control PCR product from cloned
-cDNA. Since this 308-bp band contains the 168-bp BglII
region of interest, each band was gel-purified, digested with
BglII, inserted into the BamHI site of pBluescript
SK
, and screened by blue/white detection. Plasmid DNA
from white colonies was isolated and sequenced for each cell line. The
RT-PCR reactions yielded the expected 0.3-kilobase product from
nit1-305 and each of the cotransformant lines but not from
strain atpC1. The lack of an RT-PCR product from
atpC1 is consistent with the Northern blot results for this
cell strain. The dideoxy cytosine track of each construct is shown in
Fig. 3C, and the arrows indicate where each of
the base pair changes has been engineered. Thus, the data from RT-PCR
clearly demonstrate that each of the engineered CF
-subunit constructs is expressed in its respective cell
line.
Figure 2:
Northern blot analysis of total RNA from
nit1-305, atpC1, and putative cotransformants.
Samples of (5 µg) RNA were separated in a 1% denaturing agarose
gel, blotted onto a nylon membrane, and probed with randomly primed
P-labeled CF
-subunit cDNA ( lower panel) and
-subunit cDNA ( upper panel). Lane 1, nit1-305; lane 2, atpC1; lane 3, C198S;
lane 4, C204S; lane 5,
C198S/C204S.
Figure 3:
Reverse transcription PCR and cDNA
sequencing of nit1-305, atpC1, and atpC1
cotransformants. Samples (5 µg) of each RNA were reverse
transcribed by following the Life Technologies, Inc. protocol and
suspended in 80 µl of Tris/EDTA, pH 8.0, and 2 µl were used for
each PCR reaction. A, map depicting the area of the CF-subunit cDNA subjected to PCR. Primers A and B were
designed to amplify through intron 4 of the genomic
-subunit gene.
The black box represents the 168-bp BglII
region, which contains the cysteine bridge. B, ethidium
bromide-stained 1% agarose gel, which shows RT-PCR results. For
nit1-305 and each of the putative cotransformants there is a
308-bp product that comigrates with the cDNA control. atpC1
does not yield a cDNA product. The map shows both of the PCR primers
relative to intron 4 of the
-subunit genomic clone. kb,
kilobase. Lane 1, nit1-305; lane 2, atpC1; lane
3, C198S; lane 4, C204S; lane 5, C198S/C204S;
lane 6,
-cDNA; lane 7,
-genomic clone. C,
partial cDNA sequence that shows only the dideoxycytidine track. The
arrows indicate the engineered sequences that were introduced
into atpC1. Lane 1, nit1-305; lane 2, C198S;
lane 3, C204S; lane 4,
C198S/C204S.
Photoautotrophic Growth in an 8-h Light/16-h Dark
Photoperiod
In order to examine the effects, if any, that the
mutations within the CF-subunit disulfide bond might
have on photoautotrophic growth, strains nit1-305,
atpC1, and each of the cotransformants were subjected to an
alternating light/dark growth regime under conditions that demanded
photoautotrophic growth (that is without acetate in the growth medium).
At specific time points 1 ml of each cell type was counted in duplicate
using a hemacytometer. Fig. 4demonstrates that each of the
cotransformant strains grows with kinetics similar to the wild-type
nit1-305 cells and that each reaches the same cell density. In
contrast, strain atpC1 dies within 12 h after plating into
medium without acetate, presumably because it fails to accumulate the
CF
-subunit polypeptide, which apparently prevents the
assembly of CF
(18) .
Figure 4:
Growth curves of nit1-305, atpC1,
C198S, C204S, and C198S/C204S grown in an 8-h light/16-h dark period in
the absence of acetate. At the indicated times, 1 ml of cells were
harvested and counted using a hemacytometer. The data points represent
the average of the duplicates where the standard deviation was less
than 10% of the average.
Soluble ATPase Assays
Thylakoid-associated
proteins were obtained from chloroform-extracted membranes, and this
``enriched'' fraction was utilized for ATPase studies. The
catalytic activities of parental nit1-305, atpC1, and
mutant enzymes were measured as Mg-dependent ATPases
(26) . The enzyme from the parental strain, nit1-305,
has manifest activity that is stimulated about 5-fold by the addition
of 20% ethanol to the reaction mixture ()
(18, 26) . demonstrates that each of the
mutant CF
enzymes behaves in a similar fashion. It should
be noted, however, that the ethanol-induced nit1-305 ATPase
activity is further increased by 50 mM DTT. In comparison, the
ATPase activities of the mutant enzymes are DTT-independent, and
furthermore, the rates are similar to the rate of ATP hydrolysis of the
wild-type enzyme in the presence of both ethanol and DTT. The presence
of a D. salina anti-CF
antibody
(18, 28) completely inhibits ATPase activity with all of the
coupling factors.
PMS-dependent Photophosphorylation
Fig. 5
shows the results of photophosphorylation measurements for
parental nit1-305 and each of the three mutant CFenzymes. Panel A demonstrates that the rate of
photophosphorylation with nit1-305 is stimulated approximately
2-fold by 25 mM DTT. In contrast, DTT has no effect on the
rate of PMS-dependent photophosphorylation with thylakoids isolated
from any of the three mutants ( Panels B-D).
Moreover, the rate of phosphorylation activity with all of the mutant
thylakoids is the same as the rate of phosphorylation with wild-type
thylakoids assayed in the presence of DTT. Thus, it appears that the
site-directed mutants of CF
-subunit have been
rendered DTT-independent.
Figure 5:
PMS-dependent photophosphorylation of
nit1-305 and atpC1 cotransformants. Dark-relaxed
thylakoids (3-10 µg of chlorophyll ( chl.)) were
assayed in the presence or absence of 25 mM DTT for ATP
synthesis from ADP and P-labeled orthophosphoric acid for
0, 15, 30, 45, 60, and 90 s. The data points are the means of the
duplicate experiments, each with a standard deviation not greater than
10%.
-subunit of CF
is believed to play an
important role in the regulation of ATP synthase
(4, 29) . Using thiol reducing agents such as DTT, the
redox state of the
-subunit disulfide bond has been correlated
with the activation of soluble ATPase activity
(5, 6, 7, 8, 9, 10, 11, 12, 13) .
Sequence comparisons between F
-subunits from various
sources illustrate that the cysteine bridge domain is unique to the
enzyme in Chlamydomonas and other plants
(12) . Recent
developments in C. reinhardtii genetics have made it possible
to address the functional significance of the CF
-subunit disulfide bond in vivo in this model plant
system.
-subunit gene can complement the defect in C.
reinhardtii mutant strain atpC1
(18) . However, in
order for strain atpC1 to be useful for these studies, we had
to demonstrate that we could successfully perform transformations with
mutant CF
-subunit genes, which would be inserted into
the nuclear genome and be expressed in a stable fashion. In this study
we have succeeded in doing this by using a cotransformation protocol
that takes advantage of the fact that the atpC1 cell line is
also null for nitrate reductase
(17, 30) . This
procedure was advantageous, because if the mutant
-subunit genes
failed to complement the defect in atpC1 cells, potential
cotransformants could still have been screened by Southern blot
analysis. In the three cases studied, we were able to demonstrate that
each of the mutant CF
-subunit genes was stably
expressed and assembled into apparently functional CF
complexes. Using RT-PCR, we were also able to demonstrate that
the expressed
-subunit mRNAs each contained mutant sequences that
we engineered and cotransformed into atpC1. Furthermore,
Southern blot analysis indicated that complementation and not a
reversion event restored photoautotrophic growth to atpC1.
Indeed, to date we have never observed a reversion event in
atpC1 in transformed or untransformed cells.
(
)
The results from the reverse transcription PCR studies
coupled with the photoautotrophic phenotypes strongly suggest that each
strain analyzed expressed mutant CF
-subunits.
-subunit. This genetic system allows us to alter almost any
amino acid within the
-subunit and study its possible effect(s) on
CF
assembly, function, and regulation. In addition, this
work with an enzyme involved in photosynthesis provides a strategy for
site-directed mutagenesis studies of other nuclear encoded genes
involved in other cellular processes.
-subunit mutations
on photoautotrophic growth. McCarty and colleagues
(6) have
shown that reduced CF
can catalyze ATP synthesis in the
light and ATP hydrolysis in the dark. We therefore reasoned that
because the mutant enzymes may be locked in a ``reduced''
form that they too might catalyze ATP hydrolysis in the dark. If this
were the case, then one might expect that the cells would become sick
or even die since ATP synthesis activity would be balanced by ATP
hydrolysis activity in a futile cycle. The data from the
photoautotrophic light-dark growth curves argue against ATP hydrolysis
in the dark because the mutant cells grew with the same kinetics and
reached the same cell density as wild type. Therefore, deactivation of
CF
in vivo probably involves an alternative form
of regulation such as the binding of ADP to the
-subunit, which
has previously been shown to deactivate CF
enzymatic
activity in the dark
(16) .
enzyme from C. reinhardtii does not exhibit the marked
DTT-stimulatory effects associated with the spinach enzyme
(5, 6, 7, 8, 9, 10, 11, 12, 13, 26) .
Nevertheless, we believe the increase in nit1-305 ATPase
activity in the presence of DTT to be noteworthy because this effect is
not seen in any of the mutants. In comparison, there is no detectable
ATPase activity in partially purified atpC1 cell extracts,
which is in agreement with previously published results
(18) .
-subunit
in the redox control of ATP synthesis. In the nit1-305 (wild
type) assay the amount of P
esterified is increased about
2-fold by 25 mM DTT. The DTT effect was abolished in each of
the three CF
mutants assayed. We believe that the effect of
DTT is on the CF
complex because Mills and Mitchell
(7) have previously shown that DTT has no effect on
photosynthetic electron transfer. The three mutant enzymes in this
study involve changes in the cysteine bridge by replacement of each or
both of the cysteine residues with serines. In these mutants the
CF
enzymes have no redox state by way of a disulfide bond,
and therefore, if the model is correct, they would not be affected by
DTT reduction. Indeed this is the case, since C. reinhardtii strains C198S, C204S, and C198S/C204S all exhibited high rates of
PMS-dependent photophosphorylation independent of DTT. Furthermore, the
rates of ATP synthesis for the mutant enzymes, regardless of the
presence of DTT, were identical to that of wild type in the presence of
DTT.
catalyzes rates of ATP
synthesis that are about two times as fast as the oxidized form of the
enzyme. Because the wild-type nit1-305 cells grow with the
same kinetics as the cells harboring mutations in the
-subunit,
which effectively ``reduce'' the
-subunit disulfide
bond, this implies that the in vivo form of the wild-type
enzyme is in fact the reduced conformation.
enzymatic
activity from our mutant enzymes before proceeding to further
biochemical characterization. The molecular data strongly indicate that
the restoration of photoautotrophic growth to cotransformed
atpC1 cells was accomplished by complementation with mutant
-subunit genes. The data from the soluble ATPase assays and
PMS-dependent photophosphorylation experiments suggest that the mutant
C. reinhardtii strains harbor CF
enzymes, which
behave differently from wild type in that they are not affected by DTT
reduction and have activities identical to wild type in the presence of
DTT. Furthermore, growth curves may imply that the in vivo form of the wild-type enzyme is the reduced form but that
reoxidation of the CF
-subunit disulfide bond may not
be required to shut down the enzyme in vivo. This work
represents the first complementation of a known nuclear defect with
mutated genes. It also provides data that support the redox model for
control of ATP synthesis
(5) as mediated by the CF
-subunit.
Table: Noncoding strand PCR primers
Table: C. reinhardtii CF ATPase assay with an enriched protein fraction isolated from
parental nit1-305, atpC1, and atpC1 cotransformants
from Dunaliella salina. This antibody
has been shown to cross-react with C. reinhardtii CF
.
, coupling factor o;
CF
, coupling factor 1; DTT, dithiothreitol; Tricine,
N-tris(hydroxymethyl)methylglycine; PMS, phenazine
methosulfate; PCR, polymerase chain reaction; RT-PCR, reverse
transcriptase-PCR; bp, base pair(s); SGII, Sager-Granick II.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.