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 t
Trang 1ATPase activity of magnesium chelatase subunit I is required
Vanessa Lake1,2, Ulf Olsson2, Robert D Willows1and Mats Hansson2
1
Department of Biological Science, Macquarie University, North Ryde, Australia;2Department of Biochemistry, Lund University, Sweden
During biosynthesis of chlorophyll, Mg2+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 ATPases associated with various
cellu-lar activities, 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 Mg2+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 Mg2+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 deuteroporprotopor-phyrin IX
to the H-subunit show a Kdvalue of 0.53–1.2 lM[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 Mg2+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 Mg2+ 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 capsulatusBchI has recently been determined and it was found to belong to the large family of ATPases associated with various cellular activities, 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 Mg2+- and ATP-dependent complex formation
of the 40 and 70 kDa subunits In a subsequent step the complex triggers the Mg2+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: mats.hansson@biokem.lu.se
Abbreviations: AAA + proteins, ATPases associated with various
cellular activities.
(Received 1 December 2003, revised 20 February 2004,
accepted 2 April 2004)
Trang 2the 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 Mg2+to
be inserted into protoporphyrin, as all three subunits have
some relationship with Mg2+ Concerning the 40 kDa
subunit, it is well-known that Mg2+is required to perform
the ATP hydrolysis In addition, kinetic analysis had shown
binding of Mg2+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 Mg2+[10] The 140 kDa subunit shows
consider-able sequence homology to the CobN subunit of the aerobic
cobaltochelatase CobN binds both the Co2+ 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
and referred to as the Xantha-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-h30, -h38, -h56and -h57) and three are semidominant
(Xantha-hclo 125, -hclo 157 and -hclo 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, of R 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
20C 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 Ni2+was bound
to 1 mL HiTrap Ni-affinity columns (Pharmacia) The wash buffer contained 20 mM imidazole instead of the recommended 60 mM 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.
Trang 3SDS/PAGE and Western blot analysis
SDS/PAGE [10% (w/v) acrylamide] was performed
accord-ing to Flaccord-ing and Gregerson [28] with the Tris/Tricine buffer
system of Scha¨gger and von Jagow [29] SDS/PAGE
loading buffer consisted of 200 mM Tris/HCl pH 8.8,
20% (v/v) glycerol, 4% (w/v) SDS, 200 mMdithiothreitol
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 lg of
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
54residues of the C-terminal half of the XAN-G protein
The XAN-G specific residues AVRVGLNAEKSGDVG
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 6Murea to prevent
the protein from precipitation The proteins were desalted
into 10 mM Na-phosphate pH 7.4, 150 mM NaCl and
dispensed into 100 lg aliquots of which four portions were
given to the rabbit The desalted XAN-G also contained
1Murea
mRNA analysis
The presence of Xantha-g mRNA was analysed by cDNA
synthesis from total RNA First, 1 lg total RNA, 1 lL
dNTP (10 mM) and 1 lL oligo(dT)15(0.5 mgÆmL)1) were
mixed with water to a total volume of 10 lL followed by
heating to 65C for 5 min Then 4 lL 5· first strand buffer,
4 lL MgCl2(25 mM) and 2 lL dithiothreitol (0.1M) were
added After 2 min at 42C, 0.5 lL Superscript II reverse
transcriptase (200 unitsÆlL)1; Life Technologies) was added
followed by incubation at 42C for 50 min and 70 C for
15 min One microlitre of RNaseH was added and incuba-ted for 20 min at 37C 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 lL 10· reaction buffer, 3 lL MgCl2 (25 mM), 0.5 lL dNTP (20 mM),
2 lL of each primer (10 lM) and 0.5 lL Taq DNA polymerase (5 unitsÆlL)1) Thirty-five cycles were per-formed: 94C, 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 barleyxantha-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-h56 and xantha-h57 mutants [33] The two mutant plants are suggested to lack expression of the XAN-H protein as no Xantha-hmRNA 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-hclo 125, -hclo 157and -hclo 161mutants (Fig 2B), which have altered amino acid residues in their resulting 40 kDa protein No XAN-H could be found in the recessive xantha-h30, -h38, -h56and -h57mutants (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-hclo 125, -hclo 157 and -hclo 161mutations 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
to the 70 kDa BchD protein with an N-terminal His-tag His-tagged proteins have affinity to immobilized Ni2+and usually remain bound to the column when it is washed
Trang 4with 60 mM imidazole-containing buffer The His-tagged
BchD, however, eluted at 60 mM imidazole and 20 mM
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) in 50 mM Tricine/NaOH pH 8.0 was mixed
with 50 lL of 50 mM Tricine/NaOH pH 8.0, 8 mM ATP,
8 mM dithiothreitol, 30 mM MgCl2 The mixture was
added to 10 lL His-tagged BchD (5 mgÆmL)1in 50 mM
Tricine/NaOH pH 8.0, 4mM ATP, 4mM dithiothreitol,
15 mM MgCl2) and left on ice for 90 min The resulting
110 lL were mixed with 4mL binding-buffer (20 mM
Tris/HCl pH 7.9, 0.5MNaCl, 4mMATP, 15 mMMgCl2)
and loaded on a Ni2+-containing HiTrap Ni-affinity
column equilibrated with binding-buffer The column was
washed with 8 mL wash-buffer (binding-buffer containing
20 mMimidazole) before bound proteins were eluted with
4mL elute-buffer (20 mM Tris/HCl pH 7.9, 0.5M NaCl,
1M imidazole, 6M 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
1M 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
and pure wild-type BchI were loaded on each gel to
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 Mg2+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 ofXantha-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-hclo 157 and xantha-h57, 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 Ni2+ 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 experiments as 60 m 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.
Trang 5cDNA was then used in an ordinary PCR amplification
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
566 bp DNA fragments of 378 bp could be isolated from
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] It is therefore 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-h30, -h38, -h56 and -h57 mutants the failure to maintain XAN-G is easily explained by the absence of XAN-H protein In the Xantha-hclo 125, -hclo 157and -hclo 161mutants, 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-hclo 125, -hclo 157and -hclo 161mutations 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 Ni2+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.
Trang 6showed 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-hclo 125, -hclo 157and -hclo 161mutations
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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