Download The 18-kD Protein That Binds to the Chloroplast DNA

The Plant Cell, Vol. 1, 551-557, May 1989 0 1989 American Society of Plant Physiologists
The 18-kD Protein That Binds to the Chloroplast DNA
Replicative Origin 1s an Iron-Sulfur Protein Related to a
Subunit of NADH Dehydrogenase
Madeline Wu,' Z.Q. Nie, and Junming Yang
Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, Maryland 21228
From a high-salt extract of the purified thylakoid membrane, an 18-kD protein was detected. This protein was
translated by the chloroplast ribosomes and could form a stable DNA-protein complex with a cloned chloroplast
DNA replicative origin [Nie, Z.Q., Chang, D.Y., and Wu, M. (1987) MOI. Gen. Genet. 209, 265-2691. In this paper, the
18-kD protein is linked to frxB, a chloroplast-encoded, ferredoxin-type, iron-sulfur protein, by N-terminal microsequencing of the purified protein and computer analysis. The identification is further supported empirically by the
fact that the electron paramagnetic resonance spectra of the protein indicate the presence of iron-sulfur clusters.
A polyclonal antibody raised against a synthetic pentadecameric peptide with amino acid sequence corresponds to
the highly conserved region of the frxB protein and reacts strongly and specifically with the 18-kD protein band in
protein gel blot analyses. The 18-kD iron-sulfur protein is found to be related to a subunit of the respiratory chain
NADH dehydrogenase by its cross-reaction with a polyclonal antibody raised against highly purified NADHubiquinone oxidoreductase, a key enzyme of the respiratory chain. These data are consistent with chlororespiration,
and, thus, possible implication of chlororespiration in regulating the initiation of chloroplast DNA replication is
discussed.
INTRODUCTION
All photosynthetic plant cells contain chloroplasts that
belong to a family of closely related organelles, the plastids.
Plastids are present in various forms in all living plant cells,
each form having a distinctive morphology and performing
a different function. For example, chloroplasts are found
in cells of green leaves, chromoplasts in flower petals, and
leucoplasts in interna1 tissue. All forms develop from proplastids, small organelles in meristematic cells. Multiple
copies of the plastid genome occur in all plastids, but the
number may vary from a few copies in a proplastid to a
few hundred copies in a chloroplast (Aguettaz et al., 1987).
We were interested in investigating the mechanism that
regulates the replication of the plastid genome, using the
chloroplast DNA of Chlamydomonas reinhardtii as a model
system.
A chloroplast DNA replicative origin of C. reinhardtii has
been mapped by using electron microscopy (Waddell,
Wang, and Wu, 1984), and the DNA sequence of the
cloned origin determined (Wu et al., 1986). We established
a crude in vitro DNA replication system for functional
assays and investigated the DNA-binding properties of
' To whom correspondence should be addressed.
various protein components of the in vitro system (Nie,
Chang, and Wu, 1987). From a high-salt extract of the
thylakoid membrane, we identified an 18-kD protein that
was translated by the chloroplast ribosomes and strongly
bound to the cloned DNA replicative origin (Nie et al.,
1987). We used the complete DNA sequences of the
chloroplast genomes from liverwort (Ohyama et al., 1986)
and tobacco (Shinozaki et al., 1986) in conjunction with
Severa1 conventional techniques to determine the putative
coding sequence and function of the 18-kD protein.
RESULTS
N-Terminal Amino Acid Sequence of 18-kD Protein
Matches That of the ORF 167 Protein of Tobacco Chloroplast Genome
Our previous studies using two-dimensional gel electrophoresis demonstrated that the high-salt extract of the
thylakoid membrane contains a single protein in the 18-kD
band (Nie et al., 1987). That band was recovered from
severa1 large preparative gels and concentrated by ammonium sulfate precipitation. Figure 1 shows the band
552
The Plant Cell
I
2
M
**••.
68.0
43.0
(ORF) determined by Shinozaki et al. (1986) in the complete nucleotide sequence of the tobacco chloroplast genome. Ten of 14 residues of the 18-kD protein matched
the ORF 167 protein of the tobacco chloroplast genome,
which has a projected molecular mass of 19.53 kD. The
gene in liverwort chloroplast DMA corresponding to ORF
167 was termed frxB, a cysteine-rich ORF. The periodicity
of the cysteines in the frxB gene was typical of that in a
4Fe-4S ferredoxin (Ohyama et al., 1986). The equivalent
gene in wheat chloroplast DNA has also been sequenced
(Dunn and Gray, 1988). Figure 2 shows the amino acid
sequences of all the above-mentioned frxB genes, which
are aligned for detection of maximal amino acid sequence
conservation. The periodicity of cysteines and the sequences adjacent to the cysteines are highly conserved.
The 18-kD Protein Is an Iron-Sulfur Protein
25.7
I8.4
The most sensitive physical method for detecting ironsulfur protein is electron paramagnetic resonance (EPR)
spectroscopy. Reduced iron-sulfur centers display characteristic EPR signals caused by molecular antiferromagnetism between the high-spin irons. This technique
was used to investigate whether the purified 18-kD protein
contains a functional iron-sulfur cluster for electron transfer. Figure 3a shows the EPR spectra of the oxidized 18kD protein; a sharp peak was detected at g = 2.02. After
partial reduction with sodium dithionite, the g = 2.02 peak
decreased and an additional small new peak was detected
at g = 2.08. These spectral characteristics resemble that
4.3
Figure 1. Electrophoretic Protein Gel Pattern of Gel-Purified 18kD Protein and the High-Salt Extract of the Purified Thylakoid
Membrane Used for the Isolation of 18-kD Protein.
M F P A H T E F K N Y G O O - - - - - - - - - I8KD. protein (Chlomydomonas )
b)
d)
In lane 1, approximately 0.1 ng of gel-purified 18-kD protein was
loaded. In lane 2, high-salt extract containing 1 ^g of protein was
loaded. Proteins were resolved in a 15% polyacrylamide gel
containing 1% SDS, and were visualized after silver staining.
Marker proteins in lane M are lysozyme (14.3 kD), /Mactoglobulin
(18.4 kD), o-chymotrypsinogen (25.7 kD), ovalbumin (43 kD), and
BSA (68 kD). Molecular masses (kD) of the marker proteins are
indicated at the corresponding bands.
M L P M I T E F I N Y G O O T I R A A R Y I G Q G F M I T L S H A
M F S I I N G L K N Y N O Q A I Q A A R Y I G O G F L V T L O H M
M N M F P M V T G F M S Y G Q Q T I R A T R Y I GOSF I T T L S H T
N R L P V T I Q Y P Y E K L I T S E R F R G R I H F E F D K C
N R L P T T I Q Y P Y E K L I P S E R F R G R I H F E F D K C
N R L P I T I H Y P Y E K S I T P E R F R G R I H F E F D K C
C E V C V R V C P I D L P V V D W K L E T D I R K K R L L N Y S I
C E V C V R V C P I N L P V V D W E L K K T I K K K Q L K N Y S I
C E V C V G V C P I D L P V V D W R F E K D I K K K Q L L N YS I
DFG
I C I F C G N C V E Y C P T N C L S M T E E Y E L S T Y D R
D F G V C I F C G N C V Q Y C P T N C L S M T E E Y E L S T Y N R
D F G V C I F C G N C V E Y C P T S C L S M T E E Y E L S T Y D R
H E L N Y N O I A L G R L P M S
H E L N Y O Q I A L G R L P I S
H E L N Y N O I A L S R L P I S
I D D Y T
I E D S T
M G D Y T
R T I S N L P Q I K
E N I F N L T C L P
OT I R N S S E S K
e ) - » - H E L N Y N Q I A L G R L P M - (BSA)
patterns of the gel-purified 18-kD protein (lane 1) and the
clean high-salt extract used for the isolation of an 18-kD
protein (lane 2), which was a dominant band in that preparation. The gel-purified protein was used for subsequent
analyses.
We determined the N-terminal amino acid sequence of
the purified 18-kD protein. This sequence was compared
with the amino acid sequences of all open reading frames
K G K I E G H I Y S R N I T N I V N *
I N K E K S S N S
o r f ! 6 7 (t.obocco)
f r x B ( liverwort 1
frxB (wheat)
Figure 2. Amino Acid Sequences of frxB Proteins.
(a) N-terminal of the gel-purified 18-kD protein from C. reinhardtii;
(b) ORF 167 of tobacco chloroplast DNA; (c) frxB of liverwort
chloroplast genome; (d) frxB of wheat chloroplast genome; (e) the
synthetic pentadecamer that was linked to BSA at the C-terminal
end. The conserved regions are shaded, and the periodic cysteines are marked by dots.
Iron-Sulfur Protein Binds to DNA Replicative Origin
553
end of the peptide was conjugated to BSA as described
in Methods. The conjugate proved to be a good antigen
for the production of polyclonal antibodies in mice.
I
Protein Gel Blot Analyses
3000
3200
3400
Gauss
Figure 3. EPR Spectra of the Purified 18-kD Protein.
Protein concentrationwas approximately 1OY5 M. Temperatureof
measurement was 1O0 K at the microwave power of 1 mW. The
spectra were recorded at a microwave frequency of 9.1 18 GHz
and a magneticfield modulationamplitudeof 1O G.(a) No dithionite
was added; high-spin iron species were observed at g = 2.02. (b)
The protein was reduced with 4 mM dithionite; some high-spin
iron species were shifted from g = 2.02 to g = 2.08.
of the 2 [4Fe-4S] ferredoxin isolated from Clostridium
acidi-urici (Orme-Johnson and Sands, 1973). Therefore,
the 18-kD protein is probably an iron-sulfur protein, which
can function as an electron carrier by undergoing reversible
Fe[ll]-Fe[lll] transitions.
Preparation of frxB Protein-Specific Antibody
The complete amino acid sequence and an electron density
map at 2.8-A resolution for Pepfococcus aerogenes ferredoxin containing 2 [4Fe-4S] clusters are available from
Adman, Sieker, and Jenson (1973). That protein contains
54 amino acids, and cysteine is located at positions 8, 11,
14, and 18, and at 35, 38, 41, and 45. In frxB proteins,
which contain 167 to 183 amino acids, cysteine is located
at positions 64, 67, 70, and 74, and at 104, 107, 11O, 114,
and 118. Periodicities of cysteine in both proteins were
similar. These facts suggest that a similar type of folding
might be used to form the iron-sulfur complexes in both
types of protein. The detailed structural information on P.
aerogenes ferredoxin was used for selecting a pentadecameric peptide region of frxB protein for the production
of an frxB protein-specific probe. The amino acid sequence
of the synthetic peptide is shown in Figure 2e. The carboxyl
The polyclonal antibody raised against the pentadecamerBSA conjugate reacted with the pentadecameric peptide,
BSA, and the purified 18-kD protein. Figure 4 (lane 2)
shows that, in a protein blot prepared from thylakoid
membrane extract, this antibody reacted specifically with
the 18-kD band. This result supports the conclusion that
the 18-kD DNA-binding protein is the gene product of frxB.
The following information led us to investigate whether
the 18-kD protein is related to a subunit of NADH
dehydrogenase.
In the chloroplast genome of both tobacco and liverwort,
the frxB gene is flanked by ndhA on one side and ndhE,
D, F on the other. These ndh genes have been identified
by their DNA sequence homology with human mitochondrial URF1,4,4L, and 5, respectively (Ohyamaet al., 1986;
Shinozaki et al., 1986). The protein products of these
human mitochondrial URFs have been identified as components of the respiratory chain NADH dehydrogenase by
their reaction with antibodies raised against highly purified
native beef heart NADH dehydrogenase (Chomyn et al.,
1985). And recently it was demonstrated in tobacco chloroplast that all ndh genes are actively expressed (Matsubayashi et al., 1987).
NADH dehydrogenase isolated from purified mitochondria is the most complicated enzyme of the respiratory
chain. The purified form is generally known as Complex I
or NADH-ubiquinone oxidoreductase. It has the ability to
reduce ubiquinone analogs in a rotenone-sensitive manner.
All purified preparations of Complex I contain flavin, nonheme iron, and acid-labile sulfide (Ragan, 1980). When
polypeptide components of Complex I were analyzed by
two-dimensional gel electrophoresis, 26 subunits were
detected. The EPR spectra of purified Complex I indicated
the presence of five distinct iron-sulfur paramagnetic centers. The iron-sulfur protein fraction contained severa1 subunits with the molecular masses of 75 kD, 49 kD, 30 kD,
and 18 kD, and three polypeptides with molecular masses
close to 15.5 kD. Based on extensive structural studies, a
model was proposed to show the organization of NADH
dehydrogenase in the membrane. In this model, the ironprotein fragments are exposed to both sides of the membrane. Antisera raised against either Complex I or the ironsulfur protein fragment coordinately precipitate all the subunits of the enzyme (Ragan, 1980).
A thylakoid membrane-bound NADH-plastoquinone oxidoreductase has been enriched from C. reinhardfii. Spectral properties indicate that the enzyme is a flavoprotein
containing an iron-sulfur group. It oxidizes NADH and
NADPH, with plastoquinone acting as an effective electron
554
The Plant Cell
M 12 3
4 5
H
68.0-r
43.0-
25.7-
18.4-II
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14.3 -1
Figure 4. Protein Gel Blot Analysis Using High-Salt Extract of the
Purified Thylakoid Membrane.
Lane 1, the band pattern of the protein blot used for reaction with
antibodies. Approximately 12 Mg of protein was loaded in lane 1.
After electrophoretic separation in a 10% polyacrylamide gel
containing 1% SDS and electrophoretic transfer, protein bands
on the nitrocellulose sheet were visualized by staining with India
ink. Lane 2, an identical protein blot after protein gel blot analysis
using the frxB protein-specific antibody raised against the synthetic pentadecameric peptide-BSA conjugate. Lane 3, an identical protein blot after reaction with NADH dehydrogenase antibody.
(The position of the 18-kD band is indicated by an arrow.) Lanes
4 and 5, controls for lanes 2 and 3, respectively; identical protein
blots were reacted with preimmune antisera. Lane M, the positions
of proteins used as size markers.
acceptor. The bound enzyme is sensitive to rotenone and
inhibitors of photosynthetic electron flow (Godde, 1982).
In chloroplasts, the plastoquinone pool is reduced by Photosystem II and is oxidized by Photosystem I. Using a
mutant that is devoid of a Photosystem I reaction center,
Bennoun (1982) presented strong kinetic evidence for the
presence of a respiratory chain in the thylakoid membrane
of C. reinhardtii. He named the process chlororespiration,
which is an O2 uptake process distinct from photorespiration and Mehler reaction. He proposed that these mechanisms ensure recycling of the ATP and NADPH generated
by the glycolytic pathway converting starch into triose
phosphate (Bennoun, 1982; Lemaire, Wollman, and Bennoun, 1988).
On the basis of this information, we used immunological
cross-reaction to determine whether the 18-kD iron-sulfur
protein is a subunit of the NADH dehydrogenase located
in the thylakoid membrane. A rabbit polyclonal antibody
raised against highly purified native beef heart NADHubiquinone oxidoreductase was provided for this purpose.
As shown in Figure 4 (lane 3), this antibody reacted with
the 18-kD band as well as with several other bands of
higher molecular weight on a protein blot prepared from a
high-salt extract of an extensively purified thylakoid membrane of C. reinhardtii. It is known that NADH dehydrogenase may be dissociated into subunits by a wide variety
of treatments (Ragan, 1980). Therefore, it is conceivable
that other subunits of the membrane-bound enzyme could
be dislodged during the extraction procedure and react
with this polyclonal antibody.
In eukaryotic cells, electron transport pathways to oxygen are located in the inner membranes of mitochondria
and in the endoplasmic reticulum (Sato and Omura, 1978).
These different respiratory processes are referred to as
mitorespiration and cytorespiration, respectively (Bennoun, 1982). In this study, the thylakoid membrane used
for the preparation of high-salt extract was purified by two
rounds of sucrose floatation. Loosely attached components were removed by extensive extractions with 0.15 M
NaCI buffer. Examination by electron microscopy showed
no contamination of the purified thylakoid membrane fraction by intact mitochondria. The amount of thylakoid membrane used for each extraction was estimated by its chlorophyll content (Arnon, 1949). The yield of high-salt extract
per milligram of chlorophyll was quite consistent. Therefore, we believe that proteins bound tightly to the thylakoid
membrane are responsible for these cross-reactions. Additional evidence supports the conclusion that the 18-kD
protein is a thylakoid membrane-bound protein: It is translated inside the chloroplast (Nie et al., 1987), and its coding
sequence is located inside the chloroplast genome
(Ohyama et al., 1986; Shinozaki et al., 1986). Therefore,
these data support our hypothesis that the 18-kD ironsulfur protein is related to a subunit of the NADH dehydrogenase located in the thylakoid membrane. We also
noticed that, when the antibody against the synthesized
probe was used, two adjacent bands in the 18-kD region
were detected (Figure 4, lane 2). When the antibody
against the purified Complex I was used, a broad diffused
band in the 18-kD region was detected (Figure 4, lane 3).
Iron-SulfurProtein Binds to DNA ReplicativeOrigin
Whether these patterns reflect the existence of this ironsulfur protein in two forms with slightly different mobilities
is unknown.
DISCUSSION
Reduced forms of ferredoxin-type proteins provide electron sources for a variety of reductive reactions such as
sulfite or nitrite reduction. The strongly reducing electrons
can also be shunted toward the reduction of pyridine
nucleotides for biosynthetic reactions (Yasunobu and Tanaka, 1980). A subunit of bacteriophage T7 DNA polymerase has been identified as thioredoxin (Mark and Richardson, 1976), an fscherichia coli protein involved in the
synthesis of deoxyribonucleotides (Reichard, 1967). Thioredoxin can provide the reducing power in the form of 2
cysteine residues for the reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates in a reaction catalyzed by ribonucleoside diphosphate reductase.
In the process, the 2 cysteine residues of thioredoxin are
oxidized to cystine, and the reduced form of thioredoxin is
regenerated by the action of thioredoxin reductase at the
expense of one molecule of NADPH. Mark and Richardson
(1976) suggested that the reduction of nucleotides at the
polymerization site could enhance the efficiency of reduction and polymerization, or it could function as a control
mechanism. Another possibility is that i 7 DNA polymerase
could play a role in a hypothetical in situ reduction of
ribonucleotide primers at the moment of initiation of DNA
replication (Mark and Richardson, 1976). We speculate
that similar results could be achieved by a close association of the replication origin with a redox subunit of the
thylakoid membrane-bound NADH dehydrogenase. This
arrangement could also serve as a device to coordinate
the initiation of DNA replication with the available reducing
power in situ. Our future study will be directed toward the
detection of this association in vivo.
METHODS
Purification of 18-kD Protein
Cultivation of the alga1 cells and the isolation of thylakoid membrane have been described previously (Chua and Bennoun, 1975;
Nie et al., 1987). The purified thylakoid membrane was first
extracted extensively with 0.15 M NaCl buffer to removeali loosely
attached proteins. To isolate the high-salt extract, the membrane
was then extracted with 1 M NaCl buffer. In addition to NaCI, the
extraction buffer consisted of 20 mM Hepes-KOH (pH 7.3, 5 mM
MgCI,, 1 mM DTT, 15% glycerol, 0.1 mM phenylmethylsulfonyl
fluoride,0.1 mM benzamidineHCI, 0.5 mM 6-amino-N-caproic acid.
Protein gel electrophoresis was performed on 15% or 10%
555
polyacrylamidegel containing 1% SDS; the stacking gel was 5%
polyacrylamide.The electrophoresis buffer contained 25 mM Trisglycine (pH 8.3). Electrophoresis was performed at a constant
current of 30 mA for 3 to 4 hr or until the tracking dye was 1.O
cm from the bottom (Laemmli, 1970). Proteins used as molecular
weight standards were purchasedfrom Bethesda Research Laboratories. Peptide bands were visualized after silver staining on
the gei (Giulian, Moss, and Greaser, 1983) or staining with lndia
ink after being transferred to nitrocellulose filter. Recovery of the
18-kD protein band from the SDS gel or the nondenaturing gel
was performed according to the method of Hager and Burgess
(1980).
N-TerminalMicrosequencing,Computer Analyses, EPR
Spectral Analyses, and Preparation of Peptide
The 18-kD protein band, which was electroblotted onto activated
glass (Aebersoldet al., 1986), was analyzed in an Applied Biosystems model 470A gas-phase protein sequenator (Hewick et al.,
1981). Computer analyses were performed with a Tandy 1200
computer.
The EPR spectra were obtained with a Varian Century-100 Xband spectrometer. The sample was cooled with liquid nitrogen.
and temperature for each experiment was determined by using a
germanium resistor. The magnetic field intensity was monitored
by counting the frequency of the proton nuclear magnetic resonance, and the microwave frequency was calibrated with a cavity
wavemeter.
Peptides were synthesized by using a Biosearch peptide synthesizer, model 9500. Since the C-terminal of the designated
peptide probe was a methionine, an extra glycine was added for
cyanogen bromide cleavage to generate a lactone. The lactone
was coupled to the 6-NH2 group of lysine on BSA. The peptideBSA conjugate was used to generate polyclonal antibodies in
mice.
Preparation of Polyclonal Antibodies and Protein Gel Blot
Analyses
Balb/cj mice were immunized by one initial peritoneal injection
with 5 pg of peptide-BSAconjugate mixed with Freund'scomplete
adjuvant (Sigma) and two booster injections, each with 5 pg of
peptide-BSAconjugate mixed with Freund's incomplete adjuvant
(Sigma). lnduction of immune polyclonal ascites fluid in the immunized mice was performed according to the method of Lacy
and Voss (1986).
Electrophoretic transfer of proteins from polyacrylamidegels to
nitrocellulose sheets was performed according to the method of
Towbin, Staehelin, and Gordon (1979). Hybridization of the nitrocellulose sheets with mouse antibody was performed according
to the method of Johnson et al. (1984), except that the concentration of nonfat dry milk (Carnation)was reduced to 0.5% (w/v).
For the color reaction, the blots were incubated with Protein Aalkaline phosphatase conjugate (Sigma), washed, and then developed with 5-bromo-4-chloro-3-indoyl phosphate and nitroblue
tetrazolium (Sigma) color reagent.
Rabbit polyclonal antibody against purified native beef heart
NADH-ubiquinone oxidoreductase (Complex I) was provided by
Drs. A. Chomyn and G. Attardi.
556
The Plant Cell
ACKNOWLEDGMENTS
We are grateful to Dr. James S. Vincent for EPR spectroscopic
analyses, to Dr. Tomas Kempe for N-terminal microsequencing
and the synthesis of peptide, and to Drs. A. Chomyn and G.
Attardi for the gift of NADH dehydrogenaseantibody. This work
has been supported by the National Science Foundation Grant
DCB-8609764 and a grant from the Center of Agricultura1 Biotechno!ogy of the University of Maryland.
Received January 4, 1989; revised March 1O, 1989.
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Plant Cell 1989;1;551-557
DOI 10.1105/tpc.1.5.551
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