From the Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078
Received for publication, August 7, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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Mature core I and core II proteins of the bovine
heart mitochondrial cytochrome bc1 complex were
individually overexpressed in Escherichia coli as soluble
proteins using the expression vector pET-I and pET-II, respectively.
Purified recombinant core I and core II alone show no mitochondrial
processing peptidase (MPP) activity. When these two proteins are mixed
together, MPP activity is observed. Maximum activity is obtained when
the molar ratio of these two core proteins reaches 1. This indicates
that only the two core subunits of thebc1
complex are needed for MPP activity. The properties of reconstituted
MPP are similar to those of Triton X-100-activated MPP in the bovine
bc1 complex. When Rieske iron-sulfur protein
precursor is used as substrate for reconstituted MPP, the processing
activity stops when the amount of product formation (subunit IX) equals
the amount of reconstituted MPP used in the system. Addition of Triton
X-100 to the product-inhibited reaction mixture restores MPP activity,
indicating that Triton X-100 dissociates bound subunit IX from the
active site of reconstituted MPP. The aromatic group, rather than the
hydroxyl group, at Tyr57 of core I is essential for
reconstitutive activity.
Most nuclear-encoded mitochondrial proteins are synthesized on
cytoplasmic ribosomes as larger precursors with presequences for
targeting into mitochondria (1). These presequences are proteolytically
removed during or after import of the precursors into mitochondria.
Three types of processing peptidases are involved in removal of the
presequence from precursors: mitochondrial processing peptidase
(MPP)1 (2), mitochondrial
intermediate peptidase (3), and inner membrane protease I (4, 5).
MPP cleaves all or part of the presequence as the initial processing
step. Many proteins are mature after a one-step cleavage by MPP.
Mitochondrial intermediate peptidase catalyzes a second-step cleavage
in the two-step processing of some precursor proteins. The inner
membrane protease I cleaves intermediate forms of proteins routed to
the intermembrane space. The last two peptidases act sequentially after
cleavage of the matrix targeting sequences by MPP. Thus, MPP plays an
important role in the proteolytic processing of precursor proteins in
the mitochondria.
MPP is located in the matrix of fungal and mammalian mitochondria and
in the inner membrane of plant mitochondria (2). Matrix-localized MPP
has been studied extensively and purified to homogeneity from
Neurospora crassa (6), Saccharomyces cerevisiae (7), and rat liver (8, 9). Purified, matrix-localized MPP contains two
nonidentical subunits, Although a wealth of information has been generated from the study of
matrix-localized MPP, less is known about membrane-localized MPP. MPP
activity associated with the inner mitochondrial membrane was first
observed by Braun et al. (22) in potato tuber and by
Eriksson and Glaser (23) in spinach leaf in 1992. Purification of plant
MPP revealed that the enzyme constitutes an integral part of the
bc1 complex of the respiratory chain (22-24).
Because of the sequence homology (17, 24) and immunological similarity between the subunits of matrix-localized MPP and the core subunits of
the plant bc1 complex (17, 25, 26), plant MPP
activity is thought to be associated with the core proteins of the
bc1 complex. The core I subunit corresponds to
Recently, MPP activity was detected in the bovine heart mitochondrial
bc1 complex after Triton X-100 treatment (28).
Based on the three-dimensional structure of this complex (29, 30), the
lack of MPP activity in the crystalline bovine complex was thought to
be due to the binding of an inhibitor polypeptide (subunit IX) to the
active site of MPP, which is located at the interface of core I and
core II (28). Triton X-100, at concentrations that disrupt the
structural integrity of the bc1 complex as
indicated by the loss of its electron transfer activity, weakens the
binding of inhibitor polypeptide (subunit IX) to the active site of MPP in core subunits and thus restores MPP activity (28).
Subunit IX of the bovine bc1 complex is the
73-amino acid presequence of ISP (31, 32). This presequence is removed
in one-step and remains as a subunit (subunit IX) in the
bc1 complex (32). This is different from the
process in S. cerevisiae (33) and N. crassa (34),
where the ISP presequence is removed in two steps and then degraded
in vivo by another, as yet unidentified, protease.
The Triton X-100-activated MPP in the bovine complex
(MPP/bc1) has properties similar to MPP detected
in the plant bc1 complex (28). They are
completely inhibited by metal ion chelators such as EDTA and the
EDTA-inhibited activity can be partially restored by addition of
divalent cations. The cleavage site specificity of activated MPP/bovine
bc1 depends more on the length of the amino acid
sequence in the mature protein portion than on that in the presequence
portion, when a synthetic peptide composed of N-terminal residues of
mature protein and C-terminal residues of its presequence is used as a
substrate (28). This finding is inconsistent with the present popular
speculation that substrate recognition by MPP requires only structural
elements in the presequence (35-41).
Our structure-function study of the MPP/bc1
complex requires reconstitutively active core I and core II subunits of
the bovine complex. There are two ways to obtain purified core
subunits: by biochemical resolution of the bc1
complex or by gene expression to generate recombinant core I and core
II. The availability of the cDNA sequences of bovine core I and
core II (Ref. 42 and GenBankTM accession number X59692)
together with our past experience in overexpressing soluble active
mitochondrial electron transfer proteins in E. coli (44-46)
encouraged us to generate core I and core II by gene expression. The
pET expression system was used because it introduces a His6
tag upstream from the N terminus of the expressed protein. This allows
a one-step purification of recombinant protein with Ni-NTA gel. Herein
we report the construction of the expression vectors, pET-1, pET-II,
and pET-pISP for core I, core II, and Rieske iron-sulfur protein
precursor (pISP), respectively; growth conditions for overexpression of
the active soluble form of core I, core II, and pISP in E. coli; reconstitution of MPP/bc1 with
purified recombinant core I and core II; and properties of reconstituted MPP. The inhibitory effect of subunit IX on reconstituted MPP using pISP as substrate is examined. The structural importance of
Tyr57 of core I in reconstitution is investigated.
Materials--
TA Cloning kit was from Invitrogen.
Taq DNA polymerase, T4 DNA ligase, and restriction
endonucleases were obtained from either Promega or Life Technologies,
Inc. Ampicillin, tetracycline, cytochrome c,
isopropyl- Bacterial Strains and Plasmids--
E. coli INV DNA Manipulation and DNA Sequencing--
General molecular
genetic techniques were performed according to procedures described in
Sambrook et al. (47). DNA sequencing was performed with an
Applied Biosystems model 373 automatic DNA sequencer at the Recombinant
DNA/Protein Resources Facility at Oklahoma State University.
Polypeptide Substrates--
Polypeptides composed of
varying lengths of C-terminal regions of presequences and varying
lengths of N-terminal regions (as indicated by bold type) of mature
subunit V were synthesized by the Recombinant DNA/Protein Resource Core
Facility at Oklahoma State University. These synthetic polypeptides
are:
VPASVRYSHTDIK( Construction of E. coli Strains Expressing Core I, Core II, and
the pISP--
The 1341-base pair NcoI-HindIII
cDNA fragment encoding mature core I and the 1320-base pair
BamHI-HindIII fragment encoding mature core II
were amplified from a bovine heart cDNA library by polymerase chain
reaction (PCR) using two synthetic primers: 5'-CAATGGATACCGCCACCGCCACCTACGCC-3' (the sense primer) and
5'-AAGCTTGAAGCGCAGCCA-3' (the antisense primer) for core I;
and 5'-GAATCCGTCCCTCAAAGTTGCT-3' (the sense primer) and
5'-AAGCTTCAACTCATCAATGAA-3' (the antisense primer) for core
II. The 830-base pair BamHI-HindIII cDNA
fragment encoding pISP was amplified from the pGEM/ISP plasmid (31) by PCR using two synthetic primers:
5'-TTCGGATCCATGTTGTCGGTTGCC (the sense primer) and
5'-GCGCAAGCTTACCAACAATCACCATATCATC (the antisense primer).
PCR amplification was performed in a minicycler from M. J. Research. The thermal cycle was set as follows: step 1, 95 °C for 1 min for initial denaturation; step 2, 94 °C for 1 min for
denaturation; step 3, 50 °C for 2 min for annealing; and step 4, 70 °C for 2 min for extension. A total of 30 cycles were performed
with a final extension step of 5 min. PCR products were confirmed by agarose gel electrophoresis and cloned into pCR2.1 cloning vectors from
Invitrogen to generate pCR/I, pCR/II, and pCR/pISP, respectively. The
inserts in these plasmids were confirmed by DNA sequencing.
The NcoI-HindIII fragment from pCR/I, the
BamHI-HindIII fragment from pCR/II, and the
BamHI-HindIII fragment from pCR/pISP were ligated
into their respective sites in pET-30a(+) vector to generate pET-I,
pET-II, and pET-pISP plasmids, respectively, which were transformed
into BL21(DE3) cells by electroporation. E. coli
transformants producing core I, core II, or pISP were identified by
immunological screening of colonies with antibodies against core I,
core II, or ISP, respectively.
Isolation of Recombinant His6-tagged Core I, Core II,
and pISP--
25 ml of overnight culture of E. coli
BL21(DE3)/pET-I, E. coli BL21(DE3)/pET-II, or E. coli BL21(DE3)/pET-pISP was used to inoculate 500 ml of LB medium
containing 2.5 mM betaine, 400 mM sorbitol, and
50 µg/ml kanamycin, and incubated at 37 °C with vigorous shaking
until OD660 nm reached 0.7. The culture was cooled to
about 27 °C, and IPTG was added to a final concentration of 0.5 mM to induce synthesis of recombinant proteins. Cells were grown at 27 °C for 3 h before being harvested by centrifugation at 8,000 × g for 30 min. About 1.8 g of cell
paste/500-ml culture was obtained and stored at
1.8 g of cell paste was suspended in 20 ml of 50 mM
sodium/potassium phosphate buffer, pH 8.0, containing 300 mM NaCl (NaCl-Pi buffer, pH 8.0). Cells were
broken with a Fisher Ultrasonics cell disrupter and treated with
aliquots of phenylmethylsulfonyl fluoride (100 mM in
absolute alcohol) to a final concentration of 1 mM. The
suspension was gently stirred for 30 min on ice and centrifuged at
30,000 × g for 25 min. About 50% of the expressed
core I, core II, or pISP in E. coli was recovered in the
supernatant, which was mixed with 4 ml of Ni-NTA-agarose slurry
equilibrated with NaCl-Pi buffer, pH 8.0. The slurry
mixture was slowly shaken for 1 h at 4 °C and packed into a
column (1.5 × 20 cm) that was washed, in sequence, with the
NaCl-Pi buffer, pH 8.0, until the
A280 nm in the column effluent dropped to less
than 0.01, NaCl-Pi buffer, pH 6.0, containing 10% glycerol
(10 column volumes), and NaCl-Pi buffer, pH 8.0, containing
80 mM imidazol (10 column volumes). Recombinant core
protein was eluted from the column with 20 ml of NaCl-Pi
buffer, pH 8.0, containing 250 mM imidazol and dialyzed against 50 mM Tris-HCl, pH 8.0, containing 0.25 M sucrose, overnight, to remove NaCl and imidazol. The
dialyzed sample was concentrated by centricon-10 and frozen at
Removal of the His6 Tag Containing Fragment from
Recombinant pISP, Core I, and Core II--
10 µg of recombinant
His6-tagged pISP was mixed with 0.5 unit of recombinant
enterokinase in 50 µl of cleavage buffer (20 mM Tris-HCl,
pH 7.4, containing 50 mM NaCl and 2 mM
CaCl2). After the mixture was incubated at 20 °C for 30 min, 50 µl of enterokinase capture agarose slurry equilibrated with
cleavage buffer was added. The mixture was incubated for 5 min and
centrifuged to remove enterokinase captured agarose gel. The cleavage
products, untagged pISP and the His6-tagged fragment
containing 44 amino acid residues, and uncleaved
His6-tagged pISP was recovered in the supernatant fraction.
When necessary, untagged pISP was separated from the His6
tag containing fragment and uncleaved His6-tagged pISP by Ni-NTA gel. Removal of the His6 tag-containing fragment
from recombinant core I and core II was accomplished in the same way as
described for obtaining untagged pISP.
Construction of E. coli Strains Expressing Core I
Mutants--
Core I DNA mutations were generated by site-directed
mutagenesis using the Altered SitesTM Mutagenesis system
from Promega. A 1362-base pair KpnI-HindIII fragment was excised from pET/I plasmid and cloned into the
KpnI and HindIII sites of pSELECT-1 vector to
generate pSELECT/I. The single-stranded pSELECT/I was used as the
template in the mutagenesis reactions. The mutagenic oligonucleotides
used were as follows: Y57T, AACGGGGCTGGCACATTTGTGGAGCATCTG;
Y57H, GGGGCTGGCCACTTTGTGGAG; Y57F,
GGGGCTGGCTTCTTTGTGGAG; and Y57W,
GGGGCTGGCTGGTTTGTGGAG. Each of these oligonucleotides was
used in combination with an ampicillin repair oligonucleotide and
annealed to the single-stranded pSELECT/I.
A 1362-base pair KpnI and HindIII fragment
containing mutated core I was excised from PSELECT/Im and
cloned into KpnI and HindIII sites of pET vector
to generate pET/Im, which was then transformed into BL21
cells. Mutations were confirmed by DNA sequencing of both
pSELECT/Im and pET/Im. Transformants expressing
the core Im protein were identified by immunological
screening of colonies with antibodies against the bovine cytochrome
bc1 complex.
Enzyme Preparations and General Biochemical
Techniques--
Bovine heart mitochondrial cytochrome
bc1 complex was prepared and assayed as
previously reported (48). MPP/bc1 activity was
measured with two substrates: synthetic peptides and pISP. When peptide
substrate was used, the substrate disappearance and product formation
was determined by HPLC (28). When recombinant pISP was used, the
production formation and substrate disappearance was measured by
SDS-PAGE. Protein concentration was routinely determined by the
Bradford assay (49) using a kit from Bio-Rad; for more accurate
determination of protein, the Biuret method was used (50). SDS-PAGE was
done according to the method of Laemmli (51) and, for high resolution,
according to Schägger et al. (52).
Effect of Induction Growth Conditions on Production of Recombinant
Core I, Core II, and pISP--
To facilitate the study of
MPP/bc1, recombinant core I, core II, and pISP
were generated. Core I and core II are for reconstitution studies and
pISP is for studies of MPP/bc1 inhibition by
subunit IX, because pISP is the natural substrate for
MPP/bc1 and the cleaved product is subunit IX.
Production of core I, core II, and pISP by E. coli was first
attempted with the pGEX system because this system was successfully
used to overexpress functionally active QPc-9.5 kDa of the beef
bc1 complex (44), QPs1 (45) and QPs3 (46) of
beef succinate-Q reductase, and subunit IV of the
Rhodobacter sphaeroides bc1 complex (53)
in E. coli. Unfortunately, the expression levels of core I,
core II, and pISP were low in this system. Taking the advantage of the
commercially available His6-tagged polypeptide expression
system, we used the pET system to express beef heart mitochondrial core
I, core II, and pISP in E. coli and isolated the expressed
proteins by a one-step purification with Ni-NTA gel.
Production of recombinant His-tagged core I, core II, or pISP depends
on IPTG concentration, induction growth time, medium, and temperature.
The yield increases as the IPTG concentration and induction growth time
are increased, reaching a maximum at 0.5 mM IPTG and 3 h post-induction growth. When cells are grown for more than 3 h,
the total yield decreases and degradative products increase, as
determined by Western blotting using antibodies against their
respective proteins.
Although the expression level for these three proteins in E. coli is high using LB medium at 37 °C (accounts for 30% of the total cellular protein), about 95% of the expressed protein is in
inclusion body precipitate. Solubilization of inclusion bodies of
recombinant His6-tagged proteins with 8 M urea
followed by dialysis and Ni-NTA column chromatography yielded only
small amounts of inactive soluble proteins. Because it has been
reported that including betaine and sorbitol in the induction growth
medium and lowering the induction growth temperature greatly increases the yield of soluble expressed protein in E. coli (44, 46), these induction conditions were adopted. About 40% of the expressed core I, core II, and pISP is in the soluble form when IPTG induction growth is in LB medium, containing 0.44 M sorbitol and 2.5 mM betaine at 27 °C for 3 h. About 6 mg of purified
recombinant His6-tagged core I, 5 mg of core II, and 6 mg
of pISP were obtained from 500 ml of their respective cell cultures.
The purified, recombinant His6-tagged core I, core II, and
pISP show single protein bands with apparent molecular masses of 55, 51, and 40 kDa, respectively (Fig. 1).
These are larger than the protein masses of core I and core II in
mitochondrial bc1 complex and of pISP calculated
from its protein sequence because of the addition of 44 amino acid
residues to the N termini of mature core I and core II and of pISP
during genetic manipulation. These extra residues are, in sequence, a
His6 tag, thrombin recognition sequence, S tag, and
enterokinase recognition sequence. Removal of these extra residues from
recombinant His6-tagged core I, core II, and pISP to
generate their respective untagged proteins can be achieved by
enterokinase digestion followed by Ni-NTA column chromatography. The
enterokinase used is removed from the digestion mixture with an
enterokinase-capture gel. The uncleaved, His6-tagged core
I, core II, or pISP and cleaved residues are separated from the
untagged proteins on a Ni-NTA column. However, the yields of untagged,
mature core I and core II and pISP obtained by this method are low,
mainly because of the low efficiency of enterokinase digestion (about
30%) and nonspecific binding of untagged proteins to the Ni-NTA
column.
Reconstitution of MPP from Purified Recombinant Core I and Core
II--
Purified recombinant core I and core II alone have no MPP
activity. When they are mixed together MPP activity is reconstituted. The efficiency of reconstitution increases by 20% if the mixture is
subjected to a freezing (
The reported failure of reconstitution of MPP from potato core proteins
produced in E. coli (24) may have resulted from improper
unfolding-refolding of the expressed protein produced as inclusion body
precipitate rather than a need for the structural integrity of the
complex for MPP/bc1 activity. In fact, when
recombinant beef heart core I and core II are purified from their
inclusion body precipitates, the resulting proteins are
reconstitutively inactive. The freeze-thaw step included in
reconstitution process may convert some inactive recombinant core I or
core II to an active form or may enhance interaction between core I and
core II. Because His6-tagged recombinant core I and core II
have the same reconstitutive activity as untagged proteins, a
structural requirement for amino acid residues near the N terminus of
these two protein is not critical. Addition of 44 amino acid residues at the N termini of both proteins has no ill effect on reconstitution. This is in line with the structural arrangement of the N termini of
these two core proteins in the bc1 complex
revealed from the x-ray crystallography (29).
Properties of Reconstituted MPP--
Fig.
3A compares the time course of
product peptide generation by reconstituted MPP from recombinant core I
and core II and Triton X-100-activated MPP in the bovine complex. When
a substrate peptide made of 5 residues from the C-terminal end of the
presequence and 20 residues from the N-terminal end of mature ISP
(
Fig. 3B shows the substrate peptide
concentration-dependent processing activity of
reconstituted MPP. When reconstituted MPP activity is measured with
increasing concentrations of substrate peptide
Like Triton X-100-activated MPP in the bc1
complex (28), the processing activity of reconstituted MPP is inhibited
by the metal ion chelator, EDTA. The EDTA-inhibited activity can be
partially restored by the addition of divalent cations. Of the divalent cations tested, Zn2+ is most effective (68%; Table
I), suggesting that
MPP/bc1 is a Zn metallopeptidase. Yeast MPP has
been shown to be a zinc metallopeptidase (43) by metal ion-protein
interaction studies using recombinant enzyme. Although both
MPP/bc1 and yeast (or other matrix) MPP are
probably zinc metallopeptidases, the role of Zn2+ in these
two classes of MPPs should be different. The activity of
MPP/bc1 is stimulated by (25%) but not totally
dependent on the addition of Zn2+, whereas yeast MPP has an
absolute requirement for Zn2+.
The substrate specificity of reconstituted MPP was tested with three
synthetic peptides composed of various lengths of C-terminal presequence and N-terminal sequence of mature subunit V (the
iron-sulfur protein):
The optimal assay conditions for reconstituted MPP are 15 mM Tris-Cl buffer, pH 8.0, at 37 °C, similar to those
described for Triton X-100-activated MPP/bovine
bc1. In 15 mM Tris-Cl buffer, the
activity of reconstituted MPP is not affected by addition of KCl up to
100 mM but decreases at higher salt concentrations.
The Effect of Subunit IX on Processing Activity of Reconstituted
MPP--
Based on the structure of the bovine heart mitochondrial
bc1 complex, it was suggested (28) that binding
of subunit IX to the active site of MPP, located in the interface of
core I and core II, explains the lack of MPP activity in this complex.
If this suggestion is correct one should observe end product inhibition of reconstituted MPP when ISP precursor protein is used as substrate, because subunit IX is the presequence of ISP. Alternatively, the processing activity of reconstituted MPP should be inhibited by the
addition of purified or recombinant subunit IX when substrate peptide
is used. The successful overexpression of pISP in E. coli, using the pET system, enables us to use recombinant pISP as substrate to examine the processing kinetics of reconstituted MPP.
When His-tagged pISP (Fig. 4, lanes
2 and 2') is used as substrate for reconstituted MPP,
no cleavage is observed (Fig. 4, lanes 3 and 3').
However, when enterokinase-free, untagged pISP (Fig. 4, lanes
4 and 4'), obtained by enterokinase digestion of His6-tagged pISP followed by treatment with
enterokinase-capture gel, is used as substrate, processing activity is
observed (Fig. 4, lanes 5 and 5'). These results
indicate that the N-terminal sequence of pISP is important for
processing by reconstituted MPP. This is in line with the report that
the structural elements at the N-terminal region of the presequence are
important for processing by MPP (37-41).
When a constant amount of untagged pISP is used as substrate for
varying amounts of reconstituted MPP, the amount of product formed,
subunits V and IX, increases as the amount of reconstituted MPP in the
system is increased (Fig. 4, lanes 5-7 and
5'-7'). However, the reaction stops when the amount of
product formed equals the amount of reconstituted MPP used, suggesting
that the reconstituted MPP catalyzes only one turnover.
To further confirm that reconstituted MPP shows end product inhibition,
the processing reaction was carried out in the presence and absence of
0.1% Triton X-100 (Fig. 5). In the
presence of Triton X-100, the amount of product formed increases as the
reaction time increases until the substrate is exhausted (Fig. 5,
lanes 3-5). In the absence of Triton X-100, the reaction
stops when the amount of product formed equals the amount of
reconstituted MPP used (Fig. 5, lanes 6-8). These results
are those predicted for end product inhibition and for Triton X-100
prevention subunit IX binding to the active site of MPP (28).
In the absence of Triton X-100 one of the cleaved products, subunit IX,
remains bound to the active site of MPP, rendering the enzyme inactive.
Because reconstituted MPP exhibits only one turnover, the amount of
production formation should equal the amount of reconstituted MPP used
in the system. In the presence of Triton X-100, subunit IX cannot bind
to the active site, no inhibition is observed, and the reaction
proceeds until pISP is exhausted. Triton X-100 not only prevents
binding of subunit IX to the active site of reconstituted MPP but also
dissociates bound subunit IX from the active site. Addition of Triton
X-100 to a subunit IX inhibited system (as in lane 8 of Fig.
5) restores the processing until the substrate disappears (Fig. 5,
lanes 9 and 10).
To further confirm that the observed resumption of processing activity
is due to dissociation of bound subunit IX from the active site of
reconstituted MPP by Triton X-100, the enzyme kinetics of inhibited
reconstituted MPP treated with Triton X-100 was examined with
The Effect of Mutation at Tyr57 of Core I on
Reconstituted MPP Activity--
Unlike MPPs from other sources, which
contain an inverted zinc-binding motif,
HXXEHX76E, in the
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-MPP and
-MPP, each with molecular mass of
around 50 kDa. The cDNAs encoding
- and
-MPP from these three
sources have been cloned, sequenced (6, 7, 9-13), and overexpressed in
Escherichia coli cells (13-15). MPP is classified in the
pitrilysin family (16) of zinc metalloproteases because of the presence
of an inverted zinc binding motif, HXXEH76H, in
the
-MPP (17). Processing activity requires both subunits because
recombinant
-MPP and
-MPP alone show no activity and activity is
restored upon mixing the two subunits (13-15). Although the role of
each subunit is largely unknown, accumulating evidence suggests that
-MPP is the catalytic subunit (18, 19) and that both
and
-MPP
are involved in substrate binding (20, 21).
-MPP and core II subunit to
-MPP. Traditionally the term core
protein has been used to describe the two high molecular mass protein
subunits of the bc1 complex. In contrast to
matrix-localized MPP, recombinant plant core I and core II produced in
E. coli are reconstitutively inactive (24, 27). This failure
limits the study of membrane-associated MPP at the
bc1 complex level.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-thiogalactopyranoside (IPTG), Triton X-100,
betaine, and sorbitol were from Sigma. Centricon-30 and centriprep-30
were from Amicon. LB agar, LB broth base, yeast extract, and selected peptone were from Life Technologies, Inc. Nitrocellulose membranes were
from Schleicher & Schuell. Oligonucleotides were synthesized by the
DNA/Protein Core Facility at Oklahoma State University. Antibodies
against bovine bc1 complex and ISP were
generated in rabbits and purified by a previously reported method (31).
Plasmid mini-prep kit, gel extraction kit, and Ni-NTA gel were
purchased from Qiagen. Recombinant enterokinase cleavage kit was from
Novagen. Other chemicals were obtained commercially in the highest
purity available.
F'
[F'endA, recA, hsdR17
(rk
mk
), supE44,
thi
1, gyr96, relA1,
80
litersacZ
m15,
(lac
ZYA-argF)U169
] and E. coli BL21(DE3) [F
ompT
hsdSb(rB
mB
)
gal dcm(DE3)] were used as hosts for plasmids of pCR2.1
(Invitrogen) and pET 30a(+) (Novagen), respectively.
7V+6); ASVRYSHTDIKVPDFSDYRRPEVLD(
5V+20);
and RPLVASVSLNVPASVRYSHTDIKVPDF(
17V+10).
20 °C until use.
80 °C until use.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
SDS-PAGE of recombinant
His6-tagged core I, core II, and pISP. Lanes
1, 6, and 10 were total cellular proteins;
lanes 2, 7, and 11 were insoluble
fractions; lanes 3, 8, and 12 were
soluble fractions; and lanes 4, 9, and
13 were purified recombinant His6-tagged
proteins, for core I, core II, and pISP, respectively. Lane
5 shows the molecular mass standards: phosphorylase B, 107 kDa;
bovine serum albumin, 74 kDa; ovalbumin, 49.3 kDa; carbonic anhydrase,
36.4 kDa; soybean trypsin inhibitor, 29.5 kDa; and lysozyme, 20.9 kDa.
80 °C for more than 30 min) and thawing process. The reconstituted MPP activity increases as the molar ratio of
core I/core II increases (Fig. 2).
Maximum activity is obtained when the molar ratio reaches 1, the same
as that in bovine bc1 complex activated by
Triton X-100, when calculation are based on the core I and core II
content of the complex. This result indicates that recombinant core I
and core II are active, MPP activity detected in Triton X-100-treated
complex is associated with core I and core II, and the structural
integrity of the bc1 complex is not required for
MPP/bc1 activity.
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Fig. 2.
Reconstitution of MPP with varying molar
ratios of recombinant core I and core II. The
His6-tagged recombinant core I (2 mg/ml) and core II (2 mg/ml) in 15 mM Tris-HCl buffer, pH 8.0, containing 20 µM of Zn + were mixed to give indicated molar
ratios between core I and core II in a final volume of 20 µl. After
these reconstituting mixtures were subjected to the freeze-thaw
procedure, 4 µl of substrate peptide 5V+20
(10 mg/ml) was added. After 12 h of incubation at 37 °C, MPP
product was analyzed by HPLC. The percentage of MPP activity was
calculated using the highest product peak as 100%.
5V+20) is incubated with reconstituted MPP
at 37 °C, product peptide formation increases with reaction time,
but at a decreasing rate and reaching a maximum after 12 h. Under
identical conditions, product peptide formation by Triton
X-100-activated MPP in the bc1 complex reaches a
maximum after 24 h. The specific activity of reconstituted MPP,
calculated based on product peptide generation in the first 2 h of
reaction time, is approximately twice that of Triton X-100-activated
MPP in core proteins of the bc1 complex.
View larger version (12K):
[in a new window]
Fig. 3.
Time and substrate concentration dependent of
product generation by MPP reconstituted from core I and core II and
Triton X-100 activated in bovine bc1
complex. For preparation of reconstituted MPP ( ): 60 µl of recombinant core I, 2 mg/ml, in 15 mM Tris-Cl
buffer, pH 8.0, containing 20 µM of Zn 2+ was
mixed with 60 µl of core II, 2 mg/ml, in the same buffer. The mixture
was subjected to the freeze-thaw process as described under
"Experimental Procedures." For preparation of Triton X-100-treated
cytochrome bc1 (
): 400 µg of the cytochrome
bc1 complex in 200 µl of 15 mM
Tris-Cl, pH 8.0, containing 1.5 mM Triton X-100 and 20 µM of Zn2+ was incubated at 37 °C for
1 h. A, reconstituted MPP and Triton X-100-treated
bc1 complex were added to 24 and 40 µl of
substrate peptide
5V+20 (10 mg/ml),
respectively, and incubated at 37 °C. For time course study, 10- and
24-µl aliquots were withdrawn from reconstituted MPP and Triton
X-100-treated complex, respectively, and assayed for product peptide
generation by HPLC at indicated time intervals. B, for the
substrate peptide concentration-dependent study of the
reconstituted and Triton X-100-activated MPP activity, various
substrate peptide
5V+20 concentrations were
used, and products formed in a 12-h incubation were determined.
5V+20, the activity increases with substrate peptide concentration. If one takes the concentration of substrate peptide that gives half of maximum activity as the apparent
Km, then the Km for
5V+20 for reconstituted MPP is 33.7 µM. Under identical assay conditions, the apparent
Km for
5V+20 for Triton
X-100-activated MPP in the bovine bc1 complex is
236 µM. Because reconstituted MPP has a
Km lower than the Triton X-100-activated MPP, the
substrate binding affinity of the former is probably higher than that
of the latter. This increase in the apparent Km for
Triton X-100-activated MPP probably results from a decrease in
substrate accessibility because of incomplete dissociation of inhibitor
peptide (subunit IX) from core I and core II in the detergent-treated complex.
Effect of EDTA and Zn2+ on reconstituted MPP
7V+6,
5V+20, and
17V+10. Only when
5V+20 is used as substrate is a
product peptide obtained that has an N terminus corresponding to that of mature subunit V. This result agrees with those obtained with Triton
X-100-activated MPP/bovine bc1, which showed
that cleavage site specificity of MPP depends more on the length of the
amino acid sequence from the mature protein portion and less on the presequence portion (28).
View larger version (68K):
[in a new window]
Fig. 4.
Assay of reconstituted MPP using
His6-tagged and untagged pISP as substrate.
A, lane 1, molecular mass standards
(phosphorylase B, 107 kDa; bovine serine albumin, 74 kDa; ovalbumin,
49.3 kDa; carbonic anhydrase, 36.4 kDa; soybean trypsin inhibitor, 29.5 kDa; and lysozyme, 20.9 kDa); lane 2, 5 µg of
His6-tagged pISP; lane 3, 5 µg of
His6-tagged pISP incubated with 4 µg of reconstituted
MPP; lane 4, 25 µl of enterokinase-free pISP; lanes
5-7, enterokinase-free pISP was incubated with 1, 2, and 4 µg
of reconstituted MPP; lane 8, 2 µg of mitochondrial
bc1 complex. B, Western blot of the
samples in A. Antibodies against bovine ISP were used as
first antibody and protein A-horseradish peroxidase conjugate as second
antibody.
View larger version (59K):
[in a new window]
Fig. 5.
Time course of product generation by
reconstituted MPP using pISP as substrate in the presence and absence
of Triton X-100. Lane 1, molecular mass standards
(phosphorylase B, 107 kDa; bovine serine albumin, 74 kDa; ovalbumin,
49.3 kDa; carbonic anhydrase, 36.4 kDa; soybean trypsin inhibitor, 29.5 kDa; and lysozyme, 20.9 kDa); lane 2, 25 µl of
enterokinase-free pISP preparation; lanes 3-5, 25-µl of
enterokinase-free pISP incubated with reconstituted MPP in the presence
of 0.1% Triton X-100 at 30 °C for 30, 60, and 120 min,
respectively; lanes 6-8, 25 µl of enterokinase-free pISP
incubated with reconstituted MPP in the absence of 0.1% Triton X-100
at 30 °C for 30, 60, and 120 min, respectively; lanes 9 and 10, samples from lane 8 were added (Triton
X-100 to 0.1%) and incubated for 1 and 2 h, respectively. All
samples were subjected to SDS-PAGE.
5V+20 as substrate. The amount of product
peptide produced by inhibited reconstituted MPP treated with Triton
X-100 increases with reaction time until substrate peptide is
exhausted. As expected, no product peptide generation is observed with
inhibited MPP without Triton X-100 treatment. These results confirm
that Triton X-100 dissociates bound subunit IX from the active site of
reconstituted MPP.
-subunit, beef
heart mitochondrial MPP has the
Y57XXEHX76E
sequence motif in the core I subunit. Mutational analysis indicate that
both histidines and the distal glutamate in the HXXEHX76E motif of
-subunit are
zinc binding residues and constitute the active site of MPP (18, 19).
Because in the beef core I, the first histidine in this motif is a
tyrosine, it is of interest to see whether replacing this tyrosine
(Tyr57) with histidine will increase enzyme activity. As
shown in Table II, MPP activity
reconstituted from Y57H mutant core I and wild type core II is the same
as that reconstituted from wild type core I and core II. The yield and
purity of all the recombinant mutated core I protein are comparable
with those of wild type core I protein. This suggests that tyrosine and
histidine play similar roles in the active site of MPP. Replacing
Tyr57 with phenylalanine or tryptophan does not affect the
reconstitutive activity of core I, whereas replacing Tyr57
with threonine completely abolishes its reconstitutive activity. This
suggests that the aromatic ring, rather than hydroxy group, at the
position 57 of core I is essential for its reconstitutive activity.
Effect of the amino acid replacement at Tyr57 of core 1-on the
reconstituted activity of MPP
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ACKNOWLEDGEMENT |
---|
We thank Dr. Roger Koeppe for the critical review of this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM 30721, an OARS award for project number HR00-019 from the Oklahoma Center for the Advancement of Science and Technology, and Agriculture Experimental Station Project 1819 at Oklahoma State University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ To whom correspondence is to be addressed.
Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M007128200
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ABBREVIATIONS |
---|
The abbreviations used are:
MPP, mitochondrial
processing peptidase;
HPLC, high performance liquid chromatography;
IPTG, isopropyl -D-thiogalactopyranoside;
ISP, iron-sulfur protein;
pISP, iron-sulfur protein precursor;
PAGE, polyacrylamide gel electrophoresis;
Ni-NTA, nickel-nitrilotriacetic
acid;
PCR, polymerase chain reaction.
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