Tài liệu Báo cáo khoa học: ATPase activity of magnesium chelatase subunit I is required to maintain subunit D in vivo ppt

ATPase activity of magnesium chelatase subunit I is required
to maintain subunit D
in vivo
Vanessa Lake
1,2
, Ulf Olsson
2
, Robert D. Willows
1
and Mats Hansson
2
1
Department of Biological Science, Macquarie University, North Ryde, Australia;
2
Department of Biochemistry, Lund University,
Sweden
During biosynthesis of chlorophyll, Mg
2+
is inserted into
protoporphyrin IX by magnesium chelatase. This enzyme
consists of three different subunits of  40, 70 and
140 kDa. Seven barley mutants deficient in the 40 kDa
magnesium chelatase subunit were analysed and it was
found that this subunit is essential for the maintenance of
the 70 kDa subunit, but not the 140 kDa subunit. The
40 kDa subunit has been shown to belong to the family
of proteins called ÔATPases associated with various cellu-
lar activitiesÕ, known to form ring-shaped oligomeric
complexes working as molecular chaperones. Three of the
seven barley mutants are semidominant mis-sense muta-
tions leading to changes of conserved amino acid residues

in the 40 kDa protein. Using the Rhodobacter capsulatus
40 and 70 kDa magnesium chelatase subunits we have
analysed the effect of these mutations. Although having
no ATPase activity, the deficient 40 kDa subunit could
still associate with the 70 kDa protein. The binding was
dependent on Mg
2+
and ATP or ADP. Our study dem-
onstrates that the 40 kDa subunit functions as a chaperon
that is essential for the survival of the 70 kDa subunit
in vivo. We conclude that the ATPase activity of the
40 kDa subunit is essential for this function and that
binding between the two subunits is not sufficient to
maintain the 70 kDa subunit in the cell. The ATPase
deficient 40 kDa proteins fail to participate in chelation in
a step after the association of the 40 and 70 kDa subunits.
This step presumably involves a conformational change of
the complex in response to ATP hydrolysis.
Keywords: AAA; barley; chlorophyll; magnesium chela-
tase; Rhodobacter capsulatus.
The first unique enzymatic reaction of the (bacterio)chloro-
phyll biosynthetic pathway is the insertion of Mg
2+
into
protoporphyrin IX. Three different polypeptides participate
in the catalytic reaction and these constitute the subunits of
magnesium chelatase (Fig. 1). The subunits are designated
BchI, BchD and BchH in bacteriochlorophyll-synthesizing
organisms such as Rhodobacter and Chlorobium, while in
plants, algae and cyanobacteria, the homologous proteins

are generally named ChlI, ChlD and ChlH [1]. The average
molecular masses of BchI/ChlI, BchD/ChlD and BchH/
ChlH are 40, 70 and 140 kDa, respectively. The largest
subunit is red upon purification due to bound protopor-
phyrin IX [2–4] and binding studies of deuteroporphyrin IX
to the H-subunit show a K
d
value of 0.53–1.2 l
M
[5]. The
large subunit has therefore been suggested to be the catalytic
subunit. The exact role of the other two subunits is not
understood. It is known that they form a complex in the
presence of Mg
2+
and ATP [2,3,6,7]. The complex forma-
tion does not require hydrolysis of ATP, as ADP and
nonhydrolysable ATP analogues (but not AMP) allowed
complex formation [8]. It is clear, however, that the overall
magnesium chelatase reaction requires ATP hydrolysis. The
observations are consistent with earlier observations with
pea magnesium chelatase where the magnesium chelatase
reaction was demonstrated to be a two-step reaction,
consisting of an activation step followed by the actual Mg
2+
insertion step [9]. The activation step could proceed with
ATP-c-S, whereas ATP was required for the chelation.
The three-dimensional structure of the Rhodobacter
capsulatus BchI has recently been determined and it was
found to belong to the large family of ÔATPases associated

with various cellular activitiesÕ, or AAA
+
proteins [10].
AAA
+
proteins are important mechanoenzymes that
transform chemical energy into biological events and they
are usually found in various multimeric states [11,12]. They
play essential roles in a broad range of cellular activities,
including DNA replication, membrane fusion, cytoskeletal
regulation, protein folding and proteolysis [11–13], and now
also in porphyrin metallation [10]. The N-terminal half
of the BchD subunit is homologous to BchI, while the
C-terminal half includes a metal ion coordination motif
characteristic for integrin I domains [10]. Integrins are
known to participate in cell–matrix and cell–cell interactions
[14,15]. They are involved in signalling to and from cells in
various physiological processes, including morphogenesis,
cell migration, immunity and wound healing [16,17]. The
proposed models for the magnesium chelatase reaction all
involve an Mg
2+
- and ATP-dependent complex formation
of the 40 and 70 kDa subunits. In a subsequent step the
complex triggers the Mg
2+
insertion into protoporphyrin
by the 140 kDa subunit [2,4,18–20]. Our present model for
Correspondence to M. Hansson, Department of Biochemistry, Lund
University, Box 124, S-22100 Lund, Sweden. Fax: + 46 46 2224534,

Tel.: + 46 46 2220105, E-mail:
Abbreviations: AAA
+
proteins, ATPases associated with various
cellular activities.
(Received 1 December 2003, revised 20 February 2004,
accepted 2 April 2004)
Eur. J. Biochem. 271, 2182–2188 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04143.x
the magnesium chelatase reaction mechanism also takes into
account the structural data of the R. capsulatus BchI and
general functional aspects of AAA
+
proteins [10]. In this
model not only the BchI proteins are organized in an AAA
+
hexamer, but also BchD as the amino acid sequence of the
BchD N-terminal half is homologous to BchI. The interac-
tions between the BchI and BchD proteins are suggested to
occur via three b-hairpin elements, which protrude from the
core of the BchI structure and which do not belong to the
traditional structure of an AAA
+
protein. In the double-
ringed BchI-BchD structure the ATPase activity of BchI is
blocked. A conformational transition upon binding to
BchH may bring the integrin I domain of BchD into contact
with the integrin-binding motif of BchH, simultaneously
triggering porphyrin metallation. This would also lead to a
release of the blockade of the ATP-binding site of BchI by
the integrin I domain, triggering ATP hydrolysis [10]. It is

still an open question which subunit provides the Mg
2+
to
be inserted into protoporphyrin, as all three subunits have
some relationship with Mg
2+
. Concerning the 40 kDa
subunit, it is well-known that Mg
2+
is required to perform
the ATP hydrolysis. In addition, kinetic analysis had shown
binding of Mg
2+
to this subunit [21]. An integrin I domain,
suggested to exist in the C-terminus of the 70 kDa subunit,
is a metal binding site (MIDAS motif) that can be expected
to bind Mg
2+
[10]. The 140 kDa subunit shows consider-
able sequence homology to the CobN subunit of the aerobic
cobaltochelatase. CobN binds both the Co
2+
and the
hydrogenobyrinic acid a,c-diamide substrate [22]. It can
therefore be expected that the 140 kDa magnesium chela-
tase subunit, in analogy with CobN, binds the two
substrates of the magnesium chelatase reaction.
Several barley (Hordeum vulgare L) mutants deficient in
magnesium chelatase activity were isolated during the 1950s
andreferredtoastheXantha-f, -g and -h loci [23]. It is now

known that the 40 kDa subunit is encoded by Xantha-h,the
70 kDa subunit by Xantha-g and the 140 kDa subunit
by Xantha-f [24]. The mutations are all lethal. Among
the known seven mutant alleles of the Xantha-h gene
encoding the smallest subunit of barley magnesium chelatase
(corresponding to R. capsulatus BchI), four are recessive
(xantha-h
30
, -h
38
, -h
56
and -h
57
) and three are semidominant
(Xantha-h
clo 125
, -h
clo 157
and -h
clo 161
). The homozygous
mutant plants are all yellow and lack chlorophyll. On the
other hand, the heterozygous mutants carrying the recessive
allele are all green and indistinguishable from the wild-type
plants. In contrast the heterozygous plants carrying the
semidominant allele are pale green. It has been shown that
the recessive mutations prevent transcription of the Xantha-h
gene [24], while the semidominant alleles are mis-sense
mutations leading to changes of single amino acid residues

[25]. The mis-sense mutations have previously been con-
structed in the corresponding gene, bchI,ofR. capsulatus
[26]. The amino acid exchanges in the three mutants are
D207N, R289K and L111F (numbered according to
R. capsulatus BchI). These three residues are close to the
ATP-binding site located at the interface between two BchI
subunits in a presumed oligomeric ring [10]. The mutations
D207N and R289K are located at one side of the ATPase
active site, while L111F is found at the opposite side at the
neighbouring subunit. The deficient BchI proteins also
showed a dominant effect in vitro with respect to magnesium
chelatase activity. In contrast, they were recessive with
respect to the ATPase activity, but could still associate in
oligomeric complexes with themselves as well as with wild-
type BchI [26]. It was concluded that an intact BchI oligomer
is required to support magnesium chelation, whereas ATP
hydrolysis is achieved by autonomously working BchI
subunit interfaces. In the present work we have expanded
the study of the BchI subunits with the exchanges D207N,
R289K and L111F, and analysed their ability to interact with
BchD. The ATPase-deficient BchI proteins provide a tool to
dissect the interaction between BchI and BchD and rule out
the importance of the ATPase activity in this process.
Although the ATPase-deficient 40 kDa BchI subunits can
bind the 70 kDa BchD protein in vitro, our in vivo analysis
show that the barley 70 kDa subunit is absent in homo-
zygous mutants of the Xantha-h gene encoding the 40 kDa
protein.
Materials and methods
Biological material

Barley wild-type (cv. Svalo
¨
f’s Bonus) and barley magnesium
chelatase mutants [23] were grown in moist vermiculite at
20 °C in 12 h dark/light cycles for 8 days. Lights were
turned on at 07:00 h. Yellow homozygous mutant leaves
were sorted from green wild-type leaves and put in liquid
nitrogen. Total barley protein was isolated from frozen
leaves according to [27].
Recombinant R. capsulatus BchI and BchD magnesium
chelatase subunits were used in the study. The BchD protein
was expressed as a His-tagged fusion protein. The BchI and
BchD proteins were produced and purified as described
previously [4].
Ni-affinity chromatography
The Ni-affinity chromatography system of Novagen was
used to immobilize the His-tagged BchD. Ni
2+
was bound
to 1 mL HiTrap Ni-affinity columns (Pharmacia). The
wash buffer contained 20 m
M
imidazole instead of the
recommended 60 m
M
. Four separate columns were used for
the interaction analysis of His-tagged BchD with the three
BchI mutant proteins and the BchI wild-type.
Fig. 1. The reaction catalyzed by magnesium chelatase. The insertion
of Mg

2+
into protoporphyrin IX is the first unique reaction of the
chlorophyll biosynthetic pathway. The reaction requires ATP hydro-
lysis and is catalyzed by magnesium chelatase, which consists of three
different subunits.
Ó FEBS 2004 ATPase activity is required to maintain subunit D (Eur. J. Biochem. 271) 2183
SDS/PAGE and Western blot analysis
SDS/PAGE [10% (w/v) acrylamide] was performed accord-
ingtoFlingandGregerson[28]withtheTris/Tricinebuffer
system of Scha
¨
gger and von Jagow [29]. SDS/PAGE
loading buffer consisted of 200 m
M
Tris/HCl pH 8.8,
20% (v/v) glycerol, 4% (w/v) SDS, 200 m
M
dithiothreitol
and 0.01% (w/v) Bromophenol blue. Proteins on SDS/
PAGE were visualized by staining with colloidal Coomassie
Brilliant Blue G-250 [30]. For Western blot analysis, 5 lgof
total protein was separated on SDS/PAGE and electro-
transferred to Immobilon P filters (Millipore) according to
Towbin et al. [31] using a semidry electroblotter. Polyclonal
antibodies against the three barley magnesium chelatase
subunits were from rabbit. Goat anti-rabbit IgG conjugated
to alkaline phosphatase was used as secondary antibody.
Antigens on filters were visualized using a chemilumines-
cence detection system (Clontech Laboratory Inc.).
Magnesium chelatase antisera

Antibodies were produced against truncated His-tagged
versions of the three barley magnesium chelatase subunits
expressed from derivatives of plasmid pET15b. The plas-
mids containing Xantha-f, -g and -h were named pAntF1:1,
pAntG1 and pAntH, respectively. Plasmid pAntF1:1 con-
tains 741 bp of the Xantha-f gene. The produced polypep-
tide corresponds to amino acid residues E541 to E781 of the
full length XAN-F polypeptide of 1381 amino acid residues
(numbered according to [24]). Plasmid pAntG1 has an insert
of 717 bp of genomic Xantha-g DNA and produces
54 residues of the C-terminal half of the XAN-G protein.
TheXAN-GspecificresiduesAVRVGLNAEKSGDVG
RIMIVAITDGRANVSLKKSNDPEAAAASDAPRPST
QELK follow after the His-tag. Plasmid pAntH contains
749 bp of Xantha-h,  70% of the gene. The XAN-H-
specific amino acid sequence of 239 residues starts with
EVMGP after the His-tag and ends with DISTV. The
fusion proteins were produced in Escherichia coli
BL21(DE3) using the inducible T7 RNA polymerase
system [32]. Cells from 1 L cultures were harvested and
lysed by sonication. His-tagged magnesium chelatase
polypeptides were purified from crude cell extracts accord-
ing to Novagen. All buffers used for the purification of the
XAN-G polypeptide had to contain 6
M
urea to prevent
the protein from precipitation. The proteins were desalted
into 10 m
M
Na-phosphate pH 7.4, 150 m

M
NaCl and
dispensedinto100lg aliquots of which four portions were
given to the rabbit. The desalted XAN-G also contained
1
M
urea.
mRNA analysis
The presence of Xantha-g mRNA was analysed by cDNA
synthesis from total RNA. First, 1 lgtotalRNA,1lL
dNTP (10 m
M
)and1lL oligo(dT)
15
(0.5 mgÆmL
)1
)were
mixed with water to a total volume of 10 lL followed by
heating to 65 °C for 5 min. Then 4 lL5· first strand buffer,
4 lLMgCl
2
(25 m
M
)and2lL dithiothreitol (0.1
M
)were
added. After 2 min at 42 °C, 0.5 lL Superscript II reverse
transcriptase (200 unitsÆlL
)1
; Life Technologies) was added

followed by incubation at 42 °C for 50 min and 70 °Cfor
15 min. One microlitre of RNaseH was added and incuba-
ted for 20 min at 37 °C. The synthesized first strand
cDNA was used as template in a PCR amplification, where
Xantha-g-specific primers were utilized. The primers
EXgLp67 (5¢-CGTAGATACAAACTTGTTCTCGGT
AT-3¢) and EXgUp70 (5¢-GCATTTATTCCCTTCCGTG
GAGACT-3¢) are separated by two introns in the chromo-
somal Xantha-g DNA. Therefore a DNA fragment ampli-
fied from genomic DNA is 566 bp, whereas a fragment
amplified from cDNA is 378 bp. The 50 lL reaction
contained 2 lL first strand cDNA, 5 lL10· reaction
buffer, 3 lLMgCl
2
(25 m
M
), 0.5 lLdNTP(20m
M
),
2 lL of each primer (10 l
M
)and0.5lL Taq DNA
polymerase (5 unitsÆlL
)1
). Thirty-five cycles were per-
formed: 94 °C, 30 s; 58 °C, 30 s; 68 °C, 40 s. After the
PCR was completed the DNA fragments were analysed
with agarose-gel electrophoresis and DNA sequencing.
Results
Presence of magnesium chelatase subunits

in barley
xantha-h
mutants
In a previous study of the 70 kDa barley XAN-G magnes-
ium chelatase subunit, it was found that the XAN-G protein
was missing in total cell extracts of the xantha-h
56
and
xantha-h
57
mutants [33]. The two mutant plants are
suggestedtolackexpressionoftheXAN-Hproteinasno
Xantha-h mRNA could be detected in these strains [24]. In
contrast, the 70 kDa ChlD protein of Arabidopsis thaliana
accumulates to wild-type levels under conditions where no
40 kDa ChlI protein could be detected [34]. Our analysis
was extended to all of the barley xantha-h mutants to
determine if the lack of XAN-G is a general feature in these
mutants. Crude cell extract was isolated from leaves of
mutants grown in 12 h dark/light cycles for 7 days and the
presence of XAN-G was analysed by Western blotting,
using antibodies raised against the C-terminal half of the
barley XAN-G protein. High amounts of XAN-G could
only be detected in the wild-type. The seven xantha-h
mutants probably lack XAN-G totally or contain only trace
amounts of the protein (Fig. 2A). The XAN-H protein was
found at wild-type level in the semidominant Xantha-
h
clo 125
,-h

clo 157
and -h
clo 161
mutants (Fig. 2B), which have
altered amino acid residues in their resulting 40 kDa
protein. No XAN-H could be found in the recessive
xantha-h
30
, -h
38
,-h
56
and -h
57
mutants (Fig. 2B). This is in
agreement with the lack of Xantha-h mRNA in these
mutants [24]. The large 140 kDa XAN-F protein was not
affected by the mutations and was detected in all of the
seven xantha-h mutants (Fig. 2C).
Binding of mutated BchI to BchD
The barley Xantha-h
clo 125
, -h
clo 157
and -h
clo 161
mutations
have been constructed in the orthologous R. capsulatus
40 kDa magnesium chelatase subunit, BchI, in a previous
study [26]. We analysed the ability of these BchI proteins,

with the exchanges D207N, R289K and L111F, to bind
tothe70kDaBchDproteinwithanN-terminalHis-tag.
His-tagged proteins have affinity to immobilized Ni
2+
and
usually remain bound to the column when it is washed
2184 V. Lake et al.(Eur. J. Biochem. 271) Ó FEBS 2004
with 60 m
M
imidazole-containing buffer. The His-tagged
BchD, however, eluted at 60 m
M
imidazole and 20 m
M
imidazole-containing buffers had to be used in the wash
steps (Fig. 3). In the experiment 50 lL of the BchI protein
(3 mgÆmL
)1
)in50m
M
Tricine/NaOH pH 8.0 was mixed
with 50 lLof50m
M
Tricine/NaOH pH 8.0, 8 m
M
ATP,
8m
M
dithiothreitol, 30 m
M

MgCl
2
.Themixturewas
addedto10lL His-tagged BchD (5 mgÆmL
)1
in 50 m
M
Tricine/NaOH pH 8.0, 4 m
M
ATP, 4 m
M
dithiothreitol,
15 m
M
MgCl
2
) and left on ice for 90 min. The resulting
110 lL were mixed with 4 mL binding-buffer (20 m
M
Tris/HCl pH 7.9, 0.5
M
NaCl, 4 m
M
ATP, 15 m
M
MgCl
2
)
and loaded on a Ni
2+

-containing HiTrap Ni-affinity
column equilibrated with binding-buffer. The column was
washed with 8 mL wash-buffer (binding-buffer containing
20 m
M
imidazole) before bound proteins were eluted with
4 mL elute-buffer (20 m
M
Tris/HCl pH 7.9, 0.5
M
NaCl,
1
M
imidazole, 6
M
urea). Two 2 mL fractions were
collected from the run-through, four 2 mL fractions
were collected from the wash and four 1 mL fractions
were collected from the elute. The collected proteins were
precipitated by addition of 100% (w/v) trichloroacetic
acid to a final concentration of 20% (w/v). Imidazole at
1
M
concentration in the elute-buffer inhibited trichloro-
acetic acid precipitation, but the problem was overcome
by including urea in the buffer. After a wash with acetone
the precipitated proteins were dried and resolved in
200 lL SDS/PAGE loading buffer. Ten microlitres were
analysed by SDS/PAGE followed by staining with
colloidal Coomassie Brilliant Blue. Pure His-tagged BchD

andpurewild-typeBchIwereloadedoneachgelto
identify the proteins in the run-through, wash and elute
fractions. The analysis showed that the His-tagged BchD
bound to the column, as His-tagged BchD was only
found in the elute fractions and not, or to very little
extent, in the run-through and wash fractions. The three
BchI proteins with the exchanges D207N, R289K and
L111F, as well as the wild-type BchI, were found in the
run-through fractions and the first wash fractions, but
also in the elute fraction (Fig. 4A). The experiment was
also performed without His-tagged BchD. The various
BchI proteins probably show some affinity to the HiTrap
Ni-affinity column (Fig. 4C). However, as the amount of
BchI in the elute fractions were much higher when His-
tagged BchD was present in the experiment we conclude
that the four different BchI proteins can all bind to His-
tagged BchD. Further experiments showed that the
binding of wild-type BchI to His-tagged BchD was
dependent on Mg
2+
and that ADP, but not AMP, could
be used instead of ATP. The three modified BchI proteins
could also bind to His-tagged BchD when ADP was used
instead of ATP (Fig. 4B).
Presence of
Xantha-g
mRNA in
xantha-h
mutants
A possible explanation for the absence of 70 kDa XAN-G

protein in the xantha-h mutants could be that the XAN-H
protein affects the level of Xantha-g mRNA. Therefore,
the presence of Xantha-g mRNA was analysed in one
semidominant and one recessive xantha-h mutant (Xantha-
h
clo 157
and xantha-h
57
, respectively). First strand cDNA
was synthesized from total RNA of the mutants. Total
RNA of a wild-type strain grown in parallel with the
mutants was used as a positive control. The first strand
Fig. 3. Coomassie-stained SDS/polyacrylamide gels. The strength of
His-tagged BchD binding to Ni
2+
immobilized on a HiTrap
Ni-affinity column was analysed. (A) The column was washed with
buffer containing 60 m
M
imidazole before being eluted with 1
M
imidazole. (B) The wash buffer contained only 20 m
M
imidazole. The
wash buffer containing 20 m
M
imidazole was used in the following
experimentsas60m
M
imidazole elutes the His-tagged BchD from the

column. W1, wash fraction 1; W2, wash fraction 2; W3, wash fraction
3; W4, wash fraction 4; E1, elute fraction 1; E2, elute fraction 2; E3,
elute fraction 3. The arrows indicate the His-tagged BchD.
Fig. 2. Western blot analysis. Analysis of magnesium chelatase sub-
units XAN-G (70 kDa; A), XAN-H (40 kDa; B) and XAN-F
(140 kDa; C) in barley wild-type (Wt) and mutants xantha-h
30
,-h
38
,
-h
56
,-h
57
,-h
clo 125
(DN), -h
clo 157
(RK) and -h
clo 161
(LF). The arrows
indicate the XAN-G, XAN-H and XAN-F antigens.
Ó FEBS 2004 ATPase activity is required to maintain subunit D (Eur. J. Biochem. 271) 2185
cDNAwasthenusedinanordinaryPCRamplification
and the resulting DNA fragments were isolated after
agarose gel electrophoresis and analysed by DNA
sequence analysis. The oligonucleotides used as primers
are separated by two introns in the genomic DNA
fragment. The expected size of a DNA fragment amplified
from the cDNA was 378 bp, whereas the size of a DNA

fragment amplified from genomic Xantha-g DNA was
566bp.DNAfragmentsof378bpcouldbeisolatedfrom
the wild-type as well as the two mutants, demonstrating
that the absence of XAN-G protein in the mutants cannot
be explained by abnormalities at the transcriptional level
(Fig. 5).
Discussion
The structural analysis of BchI clearly revealed it as an
AAA
+
protein [10]. It is therefore logical to search for the
function of BchI among the various functions of AAA
+
proteins. The AAA
+
proteins represent one type of
molecular chaperones and their function is to control the
fate of proteins or DNA. This is done by facilitating protein
folding and unfolding, assembly and disassembly of protein
complexes, degradation of protein, replication and tran-
scription of DNA, etc. [11–13]. Here we found that mRNA
encoding the 70 kDa XAN-G subunit is present in both a
recessive and a semidominant barley xantha-h mutant. This
is in accordance with an earlier study, where wild-type levels
of Xantha-g mRNA were detected by Northern blot analysis
in the four recessive xantha-h mutants[33].Itistherefore
likely that the lack of XAN-G protein in xantha-h mutants
is due to failure of the mutated 40 kDa XAN-H proteins
to interact with XAN-G in a normal way. This protective
interaction seems to be specific for the XAN-H protein

because wild-type levels of XAN-G are found in eight
available barley Xantha-f mutants deficient in the 140 kDa
XAN-F magnesium chelatase subunit [35]. In the xantha-
h
30
, -h
38
, -h
56
and -h
57
mutants the failure to maintain
XAN-G is easily explained by the absence of XAN-H
protein. In the Xantha-h
clo 125
, -h
clo 157
and -h
clo 161
mutants,
however, the lack of XAN-G has to be explained by an
inhibited activity of the deficient XAN-H proteins. Inter-
estingly, the recombinant R. capsulatus BchI proteins with
exchanged amino acid residues orthologous to the Xantha-
h
clo 125
, -h
clo 157
and -h
clo 161

mutations could still bind to the
His-tagged BchD protein. In addition, the binding of BchD
Fig. 4. Coomassie-stained SDS/polyacrylamide gels. The ability of wild-type BchI and BchI with modifications D207N, L111F and R289K to bind
His-tagged BchD was analysed in a so-called pull-down experiment. A new column was used for experiments with individual mutant samples. The
columns were stripped and recharged with Ni
2+
between each experiment. (A) Buffers contained ATP. (B) Buffers contained ADP. (C) Control
experiment without His-tagged BchD. Buffers contained ATP. W1, wash fraction 1; W2, wash fraction 2; W3, wash fraction 3; W4, wash fraction 4;
E1, elute fraction 1; E2, elute fraction 2; R, run-through fraction 2.
Fig. 5. Presence of Xantha-g mRNA in barley wild-type, the recessive
mutant xantha-h
57
and the semidominant mutant Xantha-h
clo 157
. First
strand cDNA synthesis was performed with total RNA, followed by
ordinary PCR. DNA fragments were separated with agarose gel
electrophoresis and stained with ethidium bromide. The amplified
378 bp fragment indicates the presence of Xantha-g mRNA in all three
strains tested. DNA fragments of 566 bp originate from the amplifi-
cation of contaminating genomic DNA.
2186 V. Lake et al.(Eur. J. Biochem. 271) Ó FEBS 2004
showed the same dependence on ATP or ADP as the wild-
type BchI. Previously, the mutant BchI proteins were found
to associate in oligomeric complexes with themselves as well
as with wild-type BchI [26]. Thus, the deficient BchI proteins
interact in a similar way to wild-type BchI although
they cannot contribute to magnesium chelation. The lack
of ATPase activity is their major divergence. Our study
demonstrates that the 40 kDa subunit is a chaperone that is

essential for the survival of the 70 kDa subunit in vivo.We
conclude that the ATPase activity of the 40 kDa subunit is
essential for the function of this subunit as a chaperone
and that binding of I to D is not enough to maintain
the D subunit in the cell. Our study suggests that ATP
hydrolysis is important for a mechanistic step after the
formation of an ID complex. This is supported by studies
performed with N-ethylmaleimide-treated 40 kDa ChlI
subunit of Synechocystis [19]. Similarly to the effects of
the barley Xantha-h
clo 125
, -h
clo 157
and -h
clo 161
mutations
studied here, N-ethylmaleimide treatment abolished ATP
hydrolysis and magnesium chelatase activity, but still
allowed complex formation between the 40 and the
70 kDa subunits. It should be noted that there are examples
of AAA
+
proteins that might function without ATP
hydrolysis [12]. On the other hand it has been shown for
several AAA
+
proteins that a significant change of
conformation occurs during the ATP hydrolysis cycle and
it has been suggested that this may be a general feature of
these proteins [12,36–41].

Further studies have to be performed to understand the
reason and a possible function of the instability of the
70 kDa subunit. Obviously, the D subunit is a substrate of
the I subunit, which led to questions concerning the
assembly of the I and D building blocks into the suggested
hexameric double-ring structure [10]. Several pathways are
possible, among which are that (a) the I and D subunits first
assemble into pure I and pure D hexamers, which then form
an ID complex; (b) an I-hexamer is first formed, which then
helps in the stepwise formation of a D-hexamer; (c) the
hexameric double-ring is assembled in total random order,
i.e. any combinations of pure I, pure D or mixed ID
complexes are likely to exist.
Acknowledgements
Dr Salam Al-Karadaghi is acknowledged for fruitful discussions.
This work was made possible thanks to generous support from the
Swedish Research Council, the Swedish Research Council for
Environment, Agricultural Sciences and Spatial Planning, and the
Magn. Bergvall Foundation to M. H., and the Australian Research
Council (grant no. A09905713) and an IREX award (grant no.
X00001636) to R. D. W.
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