From the Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan
Received for publication, September 10, 2002, and in revised form, November 11, 2002
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ABSTRACT |
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Mitochondrial processing peptidase (MPP)
recognizes a large variety of basic presequences of mitochondrial
preproteins and cleaves the single site, often including arginine, at
the Nuclear-encoded mitochondrial proteins generally carry
N-terminal transport signal sequences (presequences) to target into mitochondria (1, 2), and inside the organelle, the presequences are
removed by the mitochondrial processing peptidase
(MPP)1 (3-5). The
presequences vary in length and sequence, but each is specifically
recognized during protein transport and the maturation event by
transport receptors and MPP, respectively. Because protease processing
is irreversible, the maturation step should be highly regulated and
specifically catalyzed by MPP for the biosynthesis of mitochondria.
MPP consists of two structurally related subunits, Mammal and fungi MPP subunits are highly homologous to core I and II
proteins in the mitochondrial cytochrome bc1
complex and, in some species of higher plants, the MPP and core
complexes are identical (16, 17). A recent report demonstrated that recombinant bovine core proteins also have processing activity themselves (18). Previous reports of simulation models of rat and
potato MPP structures built using bovine and avian core proteins showed
that the MPP complex forms a crack leading to a large internal cavity
in the dimer (19, 20). A metal binding site of The decrease in the ionic strength dependence of MPP activity suggests
that the enzyme associates with the basic presequence through
electrostatic interaction (22). Indeed, distal and proximal basic amino
acids from cleavage points are required for effective processing of the
presequence (23, 24). The distal basic sites are required as positively
charged amino acids, lysine and arginine, and the positions of the
basic residues in the presequence are not restricted. On the other
hand, the proximal basic site should be an arginine residue located at
the So far, mutation analyses for MPP subunits have been reported (8, 14,
31), and the target residues are usually acidic amino acids because MPP
recognizes basic sites on the presequences. MPP has a large polar
cavity where a variety of presequences can bind; thus, acidic binding
sites should be located inside the cavity. However, the specific acidic
residues involved in binding are largely unknown. In this report, we
focused on acidified sites3
in the cavity expected by the modeling structure of rat MPP and mutated
the residues that are conserved in MPP and the core complex among the
different species. In alanine-scanning mutations, we observed that a
mutant of MPP alternatively cleaves two sites of a preprotein. We
conclude that acidic amino acids in Selection of Acidic Amino Acid Residues Conserved in the MPP
Cavity--
We built a model of rat MPP complex based on structures of
bovine and avian core proteins of cytochrome bc1
complex as reference proteins using the Insight II/Homology Software
Package (Molecular Simulations Inc.) as previously described (19). We
displayed structures of the modeling MPP and bovine core complex using
structure-viewing software, WebLab viewer (Molecular Simulations Inc.),
and picked up acidic amino acids existing in the internal cavities. The
amino acid sequences of the MPP subunits and core proteins were aligned using ClustalW and selected acidic amino acid residues conserved among
MPPs and core proteins from various species. We excluded residues that
had already mutated to neutral ones (8, 14, 31) and finally selected
eight acidic residues lined on the wall of the internal cavity in the
MPP structure.
Mutation, Expression, and Purification of MPP--
To obtain
recombinant MPP, we wanted to purify recombinant rat MPP from
Escherichia coli cells. However, rat Protein Synthesis and Processing of Preproteins--
The genes
of preproteins were cloned into the pGEM-4Z vector (Promega) and
mutated using the QuikChange site-directed mutagenesis system
(Promega). Radiolabeled preproteins were synthesized in rabbit
reticulocyte lysate containing [35S]methionine (TNT
coupled reticulocyte lysate system; Promega). The preprotein and
recombinant MPP were incubated for an appropriate time at 30 °C in a
processing buffer (10 mM HEPES-KOH, pH 7.4, 0.1% Tween 20, and 0.5 mM MnCl2), and the reaction was stopped by adding SDS-PAGE sample buffer (7). The processing product was
separated on SDS-PAGE and visualized using an Imaging Analyzer (Fuji
film; Bas1000). The processing efficiency of MPP was determined by the
quantification of the radioactivity of the cleaved protein in the total
protein using Image Gauge 3.0 (Fuji Photo Film Co., Ltd. and Koshin
Graphic Systems).
Processing of Recombinant p Effect of Substitutions of Negatively Charged Residues Inside the
MPP Cavity on the Processing Activity--
To elucidate characteristic
sites in which MPP interacts with the basic presequence, we focused our
attention on acidic residues that are located inside the central cavity
of MPP. We selected eight acidic residues conserved between MPPs and
core proteins and did alanine-scanning mutations on the MPP subunits.
After each subunit was purified homogeneously with a nickel-chelating column, we then analyzed the processing activity of the in
vitro reconstituted enzyme using various mitochondrial preproteins
with different presequences containing from 9 (in the case of p Mutations at the Glu191 and Asp195 in
At first, we made sure of the location of the rat Competitive Recognition for Two Sites in the Preprotein by the
Mutated MPP--
Because mutations to alanines at Glu191
and Asp195 reduce the interaction of MPP to P2
arginine, the S2-defective mutants of MPP may pass through
P2 arginine, thereby missing an important interaction necessary for correct processing. As a result, the MPPs cause a shift
of the cleavage site to the C-terminal XX
Because the mutated MPP could cleave p Comparison of the Processing Rates between the Cleavages at Normal
and New Sites by the S2-defective Mutants of MPP--
We
have so far analyzed abnormal processing by the
S2-defective mutant of MPP using a preprotein, p Here we described a notable character of the
S2-deficient mutant of MPP that causes not only reduced
recognition at the RX We initially analyzed the effect of alanine mutations for acidic amino
acid residue-conserved inside cavities of MPP and core proteins on the
processing activity toward various preproteins. The relative processing
efficiency of the mutants varied for the preproteins, and therefore, it
is difficult to achieve consensus on the function for each acidic amino
acid in the processing reaction. However, it is notable that the
processing of pAd is sensitive to mutations at multiple acidic sites in
the MPP cavity. Because the pAd has nine basic sites in the long
presequence region containing 50 amino acid residues, such a highly
basic long presequence may require many acidic sites in the cavity to
stabilize the enzyme-substrate complex through ionic interactions.
Indeed, our preliminary experiment shows that long synthetic peptides
based on the presequence of pAd have a much lower value of
Kd than those of the shorter peptide for binding to
MPP. Detailed analysis for each mutated enzyme will be required to
elucidate the functions of the acidic amino acids in the processing mechanism.
The processing of preproteins by S2-deficent MPP indicates
alternate substrate specificity and drastic change of the substrate recognition system of the MPP. It follows that the characterization of
the shift of cleavage described here may give a new insight into the
recognition and cleavage of the presequence. Thereby, we propose a
possible mechanism for the search and determination of processing site
using the substrate binding scaffold and the multiple subsite inside a
MPP cavity. Observations from the crystal structure show two synthetic
prepeptides bound to MPP in an extended conformation. Both peptides
form main-chain hydrogen bonds with the edges of the two 2 position (P2). To elucidate the recognition
and specific processing of the preproteins by MPP, we mutated to
alanines at acidic residues conserved in a large internal cavity formed
by the MPP subunits,
-MPP and
-MPP, and analyzed the processing
efficiencies for various preproteins. We report here that alanine
mutations at a subsite in rat
-MPP interacting with the
P2 arginine cause a shift in the processing site to the
C-terminal side of the preprotein. Because of reduced interactions with
the P2 arginine, the mutated enzymes recognize not only the
N-terminal authentic cleavage site with P2 arginine but
also the potential C-terminal cleavage site without a P2
arginine. In fact, it competitively cleaves the two sites of the
preprotein. Moreover, the acidified site of
-MPP, which binds to the
distal basic site in the long presequence, recognized the authentic
P2 arginine as the distal site in compensation for ionic
interaction at the proximal site in the mutant MPP. Thus, MPP seems to
scan the presequence from
- to
-MPP on the substrate binding
scaffold inside the MPP cavity and finds the distal and P2
arginines on the multiple subsites on both MPP subunits. A possible
mechanism for substrate recognition and cleavage is discussed here
based on the notable character of a subsite-deficient mutant of MPP in
which the substrate specificity is altered.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
- and
-MPP (6, 7). The
-MPPs have a zinc binding motif,
HXXEHX76E, in which histidine and the
final glutamate residues participate in metal binding, and the first
glutamate residue is involved in water activation for the hydrolysis of
the peptide bond (8). Although
-MPP has an active site containing a
zinc ion, the recombinant subunit alone has no apparent peptidase
activity (9-12).
-MPPs are mainly responsible for the binding of
the basic site in the long presequence; a preprotein was cross-linked
to
-MPP (13), and acidic amino acids in the C-terminal domain are
mainly required for binding (14). The cooperative function of the
subunits is involved not only in cleavage but also in the high affinity
interaction to the presequence (15). Thus,
-MPP regulates the
catalytic activity of
-MPP and also participates in substrate binding.
-MPP and a substrate
binding site of
-MPP are located inside the cavity. Moreover, the
wall of the cavity is lined with negatively charged amino acids. More
recently, the crystal structures of recombinant yeast MPP and a
cleavage-deficient mutant of MPP-complexed synthetic presequence
peptides have been determined (21). MPP binds the short peptide in the
extended conformation within a large polar cavity. The substrate
peptide mainly interacts to the
-MPP side, and the N-terminal site
of the peptide almost reaches toward
-MPP. Recognition sites for
residues in the peptide are identified around an active site in the
N-terminal domain of
-MPP.
2 position2
(P2). Hydrophobic and hydrophilic residues at
P1 and P2-P3, respectively, are
also important for effective catalysis (25-27). Thus, a general sequence efficiently cleaved by MPP is represented as the sequence motif (R/K)XnRX
(
and
indicate hydrophobic and hydrophilic residues, respectively).
However, a defect of an element in the motif is allowed without a
complete loss of cleavage by MPP. Besides the motif sequence,
glycine-induced flexible conformation of the presequence is preferred
for processing (28, 29). The requirement of flexibility for processing
was demonstrated as the common structure of the presequence bound on
MPP using fluorescence energy transfer in the analysis (30), and the
extended structure of the presequence was observed in complex crystals
(21). Thus, MPP recognizes the flexible presequence on the
multi-binding site arranged inside a molecular cavity.
-MPP are involved in a
determinant for the specific cleavage of a single site through the
interaction with P2 arginine and discuss the substrate
recognition mechanism.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-MPP was mostly produced as inclusion bodies or insoluble aggregates in the cells, in
contrast to the
-MPP subunit that was mostly found in soluble form,
and the amount of active dimer was insufficient for purification. We
then used yeast
-MPP, most of which was expressed in a soluble monomeric form, as the partner to the rat
-MPP. The yeast
-MPP and rat
-MPP genes were mutated using the QuikChange site-directed mutagenesis system (Promega). Hexahistidine-tagged MPP subunits in the
C termini were individually expressed in bacteria and purified using a
nickel-chelating column, described previously (8). Briefly, each
subunit gene was cloned into the expression vector, pTrc99A (Amersham
Biosciences), and transformed into an E. coli strain BL21
(DE3). The subunits were individually expressed in LB medium for
24 h at 25 °C, and the harvested cells were lysed by
sonication. After centrifugation, the recombinant subunits were
purified from the resultant supernatant using a nickel-chelating Sepharose column (Amersham Biosciences; His trap) until the purity exceeded 95%. We then mixed an equal molar concentration of subunits to reconstitute the active enzyme complex in vitro. The
recombinant MPP exhibited processing activity to various precursor
proteins translated in vitro, and the kinetic parameters for
a synthetic oligopeptide substrate were essentially the same as those
for MPP purified from bovine liver or yeast mitochondria, as previously reported (8, 14).
and the N-terminal Protein
Sequences--
To overproduce the preprotein of yeast
-MPP,
p
, of which mature residues from 144 to 425 were deleted, a
restriction NcoI site and a hexahistidine were introduced at
the N and C termini of the preprotein gene, respectively, using the PCR
method. The gene was subcloned into a protein expression vector,
pET-3d, and the protein was produced in bacteria at 25 °C overnight.
After lysing the cells by sonication, the protein solution was adjusted to 6 M guanidine and loaded on a nickel-chelating Sepharose
column equilibrated with binding buffer (20 mM HEPES-KOH,
pH 7.5, containing 6 M guanidine, 0.5 M NaCl, 5 mM imidazole, and 1 mM 2-mercaptethanol). The
column was washed with binding buffer containing 6 M urea instead of guanidine, and then p
protein was eluted with 20 mM HEPES-KOH, pH 7.5, containing 6 M urea, 500 mM imidazole, and 1 mM 2-mercaptethanol. The
processing reaction was started by a 50× dilution of the purified p
(1 µg) into the processing buffer-premixed recombinant MPP (3 µg).
After incubation for 1 h at 30 °C, the proteins in the reaction
solution were precipitated by trichloroacetic acid and separated by
SDS-PAGE. For the N-terminal sequence analysis, about 30 pmol of
protein on immobilized membrane (Immobilon-PSQ Transfer
Membrane; Millipore) was applied to the Protein Sequencing System 491A
(PerkinElmer Life Sciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) to 50 (in the case of pAd) amino acid residues (Fig.
1A). The relative processing
efficiency of the enzymes varied for the preproteins (Fig.
1B). Generally, mutations in
-MPP caused essentially no or small reductions in the processing efficiency, except for decreases in the processing of pAd by MPP mutated at the Glu383. In
contrast, mutations in
-MPP resulted in a decrease in processing activity for p
, pMDH, and pAd. In particular,
E191A,
D324A,
D413A, and
E420/D421A MPPs showed little processing activity toward pAd; that is, less than 10% of the wild-type MPP. The reduced activity for the preproteins does not result from large defects of MPP
conformation by the mutations because the mutant subunits can associate
with their non-tagged counterpart subunit by pull-down assay using a
nickel-chelating column (data not shown).
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Fig. 1.
Comparison of various mitochondrial
presequences and the processing activity of alanine-mutated MPPs toward
the preproteins. A, comparison of the N-terminal
presequences of four mitochondrial preproteins. The N-terminal amino
acid sequences of the preproteins are shown with the cleavage sites.
The radiolabeled preproteins translated in vitro in
reticulocyte lysate were used in the processing assay as substrate for
MPP. B, effect of mutations on processing activity.
Processing efficiencies toward the preproteins were examined as
described under "Materials and Methods." The amounts of MPP used
for the assay were 12.5, 0.5, 100, and 20 ng for p , pMDH, pSCC, and
pAd, respectively. Around 40-50% of the preproteins were processed by
the amount of wild-type (WT) enzyme. Two-site processing of
p
by
E191A MPP is inserted in the left panel.
p, the precursor form; m, the mature form;
x, the cleavage product newly generated by the mutated
MPP.
-MPP Produced an Abnormal Processing Product of
Preprotein--
When
E191A MPP was reacted with p
, we noticed an
extra processing product (x
) that migrates to a smaller molecular
weight than the mature one (m
) on SDS-PAGE (indicated by an
arrow with x in Fig. 1B). This product
was never observed during incubation with the wild-type MPP (Fig.
1B and Fig. 2A).
The
E191A MPP cleaved another three preproteins at the mature sites
alone (data not shown). The abnormal processing product did not result
from proteolysis by endogenous bacterial protease(s), which may be
contaminated in the
E191A MPP preparation, because no processing
occurs by the subunit only. Thus, the mutated enzyme complex should
cleave p
at the two sites and generate an additional smaller
molecular weight polypeptide with a mature one, indicating that the
substrate specificity seems to be altered by the mutation. It is
notable that a single-site mutation within the protease changes the
cleavage site of substrate. We then analyzed the detailed character of the mutated MPP because it must give us a new insight into the mechanism of recognition and cleavage for presequences by MPP.
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Fig. 2.
Shift of the cleavage site in the preprotein
by the mutated MPPs. A, two-site cleavage of the p by
MPPs mutated at the Glu191 and Asp195. Wild
type (WT) and each mutated MPP (100 ng of protein) were
incubated with radiolabeled p
at 30 °C for 10 min, and the
processing products were subjected to SDS-PAGE and visualized by an
imaging analyzer. The processing efficiency was determined.
p, precursor form (p
); m, mature form (m
);
x, cleavage product newly generated by the mutant MPP
(x
). B, cleavage sites of p
by the mutant MPP.
Recombinant p
protein denatured in 6 M urea (lanes
1, 2, and 4) or no substrate (lanes
3 and 5) was rapidly diluted in the reaction buffer and
incubated with MPP (lanes 2 and 4) or no enzyme
(lane 1) at 30 °C for 1 h. The proteins were
separated on SDS-PAGE gel and stained with Coomassie Brilliant Blue
R-250. The N-terminal amino acid sequence of p
is shown with the
processing sites (arrows with m and x)
that were determined by direct protein sequencing. The P2
and P1'-P2' positions for each cleavage site
are underlined. The underlined sequences denote
two types of processing motifs, RX
and
XX
, by MPP.
Glu191
corresponding to the site in the yeast MPP structure using the recently reported crystal structure of MPP bound with presequence peptides. The
structure denoted that
Glu160 and
Asp164
in the S2 pocket cooperatively bound to the
guanidino group of P2 arginine. These acidic
residues are highly conserved among
-MPPs, corresponding to
Glu191 and Asp195 in rat
-MPP. If the
abnormal processing by
E191A MPP resulted from the partial defect of
the P2-S2 interaction, mutation at the
Asp195 also causes the shift of cleavage. Therefore, we
mutated the Asp195 to an alanine residue (D195A) and to
double alanines with Glu191 (E191A/D195A), and the mutants
of MPP reacted with p
. We observed an extra fragment processed by
D195A MPP in addition to the authentic processed product (Fig.
2A). The two cleaved polypeptides showed similar sizes to
m
and x
fragments processed by E191A MPP, as judged by SDS-PAGE,
suggesting that the cleavage sites are the same, but efficiencies in
the processing at the two sites are different between E191A and D195A
MPPs. Moreover, the double alanine mutant of MPP, E191A/D195A,
converted p
to x
with little m
(Fig. 2A). The
wild-type MPP specifically recognizes and cleaves at the motif in the
presequence, but in the mutants of MPP, the substrate specificity seems
to be lost. To ensure site specificity of the mutated MPP, we
determined the N-terminal amino acid sequences of x
and m
using
recombinant p
purified from bacterial extract. The recombinant
preprotein was incubated with the wild-type or E191A MPP, and the
processing products were separated on SDS-PAGE. The wild-type MPP
produced a single processing product, and the mutated enzyme gave two
products, similar to the cleavage products of the radiolabeled
preprotein substrate (Fig. 2B). Edman sequence analyses for
the two polypeptides revealed that the N-terminal five residues were
YSNIX and LS(S/N)LA for m
and x
, respectively. This
indicates that the cleavage sites were between residues 9 and 10 (RL
YSN) and 20 and 21 (FK
LSS) (Fig. 2B). Thus, the
mutated MPP cleaves at a new site (x
) in addition to the
P2 arginine site (m
). The new processing site is
included in an extended processing signal of no arginine at
P2 with hydrophobic and hydrophilic residues at
P1' and P2'-P3', respectively (the
XX
motif), and this sequence is less efficiently
cleaved than the P2 arginine motif by MPP. Potentially,
p
includes a weak cleavage signal at the C-terminal site from an
authentic RX
cleavage site. The wild type and
any other mutants of MPP described in the previous section never
process the C-terminal site. Why do the S2-defective mutants of MPP cleave at the P2 arginine-deficient site
more dominantly than at the P2 arginine site?
site.
Alternatively, the mutated MPPs may simply cleave m
to x
by
stepwise processing. To ensure whether the S2-defective
mutants of MPP cleave two sites of the preprotein by competitive
processing or stepwise processing, we first analyzed the kinetics of
the processing reaction. The x
polypeptides were produced early in
the processing reaction by E191A or D195A MPPs, and no accumulation of
m
was observed during the initial period of the reaction, even with
the E191A/D195A enzyme (Fig.
3A), suggesting a decreasing
probability of stepwise processing. We next determined if the mutated
MPPs could cleave the mature protein, m
. Then the mutated enzymes
were added to the reaction solution after the accumulation of m
by
processing of the wild-type enzyme alone. Practically no production of
x
was observed (Fig. 3B, lanes 5-7). More
directly, none of the mutants cleaved m
that was constructed with
the deletion of the nine N-terminal residues of p
(Fig.
3B, lanes 10-12). The m
polypeptide was no
longer a substrate for the mutated enzymes. Thus, the E191A and D195A
MPPs seem to cleave the two N-terminal sites of p
competitively
rather than stepwise.
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Fig. 3.
Competitive processing of
p by MPPs mutated at the Glu191
and Asp195. A, kinetic analysis of processing by
the mutated MPPs. The mutated MPPs (25 ng of protein), E191A, D195A,
and E191A/D195A, were incubated with radiolabeled p
at 30 °C for
the indicated times and the processing products on SDS-PAGE. The
processing ratios were quantified using an imaging analyzer.
B, cleavage of m
by the MPPs. Radioactive p
was
incubated with no MPP (lane 1) or 100 ng of wild-type
(WT) MPP (lane 2 and 4-7) at 30 °C
for 30 min to accumulate m
product. Then the reactions were
stopped (lane 1-3) or continued for 10 min (lane
4-7) after the addition of 100 ng of each MPP into the reaction
solutions. The preprotein was reacted with E191A MPP at 30 °C for 30 min, and the incubation was stopped (lane 3). In
vitro translated m
was reacted with no MPP (lane 8)
or 100 ng of MPP proteins (lane 9-10) at 30 °C for 10 min. Each reaction was analyzed on SDS-PAGE and the imaging analyzer.
An asterisk indicates the non-processed product, because it
was translated during protein synthesis in a rabbit reticulocyte lysate
system using the control vector, pGEM-4Z.
to x
but not attack m
,
the nine N-terminal residues of p
are involved in the proteolytic production of x
. We have previously reported that the N-terminal arginines distal to the cleavage site in the long presequence could
interact mainly with the twin acidic site
(Asp390/Glu391) in yeast
-MPP (14). Then we
speculated that arginines in the nine N-terminal residues were
recognized by
-MPP as the distal basic sites. To examine this
hypothesis, the MPP complex was reconstituted with wild-type or mutated
(E390Q/D391N)
-MPP and wild-type or the mutated (E191A)
-MPP, and
then we tested in vitro processing of p
using various
combinations of the wild-type and the mutated subunits (Fig.
4A). The MPP reconstituted
from
E390Q/D391N and the wild-type
-MPP had essentially no effect
on the p
processing activity, the same observation as in our
previous report stating that the mutated MPP complex is insensitive to
processing of such a short presequence. When we used a combination of
E390Q/D391N and
E191A, the reconstituted enzyme cleaved from p
to m
but hardly produced any x
fragments. Individual alanine
substitutions at the two N-terminal arginines (Arg3 and
Arg8) in the p
presequence revealed that
Arg8 is required not only for processing to m
by
wild-type MPP but also for cleavage to x
by
E191A MPP (data not
shown). The data suggest that the mutated MPP recognizes the
Arg8 as the basic distal site on the twin acidic site of
-MPP (Fig. 4B). Thus, the shift of the processing site is
induced by the competitive recognition of the mutated MPP for two sites
in the preprotein. It must be due to both effects, the defective
interaction to P2 arginine on
-MPP and, instead of
P2-S2 interaction, ionic recognition for the
basic distal site on the acidic subsite of
-MPP.
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Fig. 4.
Shift of recognition for the proximal
(P2) arginine of the presequence to an acidified subsite on
the -MPP. A, processing of p
by
MPPs reconstituted from various combinations of the subunits.
Radiolabeled p
was incubated with MPPs (25-200 ng of proteins)
reconstituted from wild-type (WT) or mutated subunits at
30 °C for 30 min. The processing products were separated on SDS-PAGE
and detected using an imaging analyzer. B, a putative model
for interactions between the acidic sites in MPP and the basic sites in
the presequence. The p
protein is illustrated by a thick gray
line. The dashed lines indicate potential interactions
between MPP subunits and arginines in the N-terminal region of p
. In
the middle panel, the arrows show the competitive
processing reaction on the MPP at the two cleavage sites, m
or x.
, as a
substrate. The preprotein has a short N-terminal presequence separated
by a cleavage site with P2 arginine. There is a potential
processing site without P2 arginine in the N-terminal of
the mature protein. The two processing motifs belong to
RX
and XX
, respectively,
but the sequences of P1 to P3' are not
identical to each other (Fig. 2B). In the motifs,
P3' of the former site is asparagine, and the latter site is serine; the latter is the more strict processing signal by MPP. To
compare the rates of the competitive cleavage at the former and latter
sites by the S2-defective mutants of MPP, we constructed cDNA encoding two artificial preproteins, which have identical sequence among the former and latter sites, except for with or without
P2 arginine. One is the pMDH derivative, pMDH2cs, in which a segment of AAS
FSTS was introduced just after the nine residues of
the authentic cleavage site with P2 arginine (Fig.
5A). The other one is pAd2cs,
in which a cleavage signal with P2 arginine is inserted
into the presequence, and the authentic P2 site is mutated
to alanine. As a result, the preprotein has tandem cleavage sites of
RT
LSVS and AT
LSVS (Fig. 5A). Then we analyzed the
processing of the preproteins by the wild-type and
S2-deficient mutant of MPPs. The mutated MPPs, E191A,
D195A, and E191A/D195A, cleaved the preproteins both at the former site
with P2 arginine and at the latter site with no arginine
(Fig. 5B). The E191A and D195A MPPs cleaved at the former
and latter sites in pMDH2cs with about 25 and 10% efficiency,
respectively. The E191A/D195A enzyme converted pMDH2cs to an almost
equal amount of the two polypeptides. Wild-type MPP cleaved pMDH2cs at
almost the former site with slight processing at the latter site. Very
similar processing patterns were observed using pAd2cs, resulting in
cleavage almost at the former site by the wild type, dominant
cleavage at the former site with partial cleavage at the latter site by
E191A and D195A, and even processing at the two sites by E191A/D195A
(Fig. 5B). These results suggest that the
S2-deficient mutant of MPPs frequently passes though former
cleavage signals containing P2 arginine. Thus,
Glu191 and Asp195 of
-MPP function as
determinants for the single site cleavage through the interaction with
P2 arginine, possibly with a scanning of the presequence
from the N-terminal side.
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Fig. 5.
Processing of preproteins that have tandem
cleavage signals,
RX and
XX
.
A, N-terminal amino acid sequences of pMDH2cs and
pAd2cs. Segments introduced into the presequence instead of the
original sequences are shown in italic letters. The
potential cleavage sites are hyphenated and indicated by
arrows with each cleavage motif, P2 arginine
(RX
) and P2 no arginine
(XX
), underlined. B,
two-site cleavage of pMDH2cs and pAd2cs by MPPs mutated at
Glu191 and Asp195. The radiolabeled pMDH or
pMDH2cs were incubated with wild-type (WT) or mutant MPPs
(0.5 ng) at 30 °C for 10 min. In the processing of pAd and pAd2cs,
500 ng of MPP was used and reacted at 30 °C for 30 min.
p, precursor form; m, mature form; x,
cleavage product at C-terminal P2 no-arginine site.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(
and
indicate
hydrophobic and hydrophilic residues, respectively) site but also a
shift of cleavage to the XX
site. In a previous report, alanine mutations at both rat
Glu191 and
Asp195 resulted in no loss of MPP activity toward the
substrate without P2 arginine. Moreover, the processing
activity could be partially compensated for by the
Arg191 enzyme toward P2 acidic preproteins,
suggesting an ionic interaction between the residue Glu191
and P2 (32). The crystal structure of a cleavage-deficient mutant of yeast MPP interacting with the synthetic peptide of the
presequence has recently denoted that the two acidic amino acid
residues corresponding to Glu191 and Asp195 in
rat
-MPP interact with P2 arginine (21). From these
findings, Glu191 and Asp195 at the
S2 of rat MPP are the essential residues required for the
specific determination of the cleavage site.
sheets
(21). One of the sheets is located near the active site, and the other
is in the C domain of
-MPP (strands 1 and
2 of red ribbons in Fig.
6A). Because the N-terminal sides of the peptides of the cytochrome c oxidase subunit IV
presequence almost reach the
-MPP, the edges of the two other
sheets of
-MPP (strands 3 and 4 of
yellow ribbons in Fig. 6A) seem to be available
to bind to longer presequences within the cavity. The proposed
"substrate binding scaffold" formed from the four
sheets inside
the MPP cavity denotes a highly ordered substrate binding state, and it
could lead to the directional insertion of the peptide into the MPP
cavity through a narrow crack between both subunits. The mechanism of
entry of the presequence into the cavity is unknown, but electrostatic
interactions would contribute to capturing the positively charged
presequence into the cavity, because its inside is an
electronegative environment due to the arrangement of many aspartate
and glutamate residues. During the insertion, MPP may scan the extended
substrate from the N to C termini on the substrate binding scaffold and
determine the cleavage site. If a cleavage signal of
RX
is included in a short stretch of the
N-terminal side, as p
and pMDH, the MPP uses the edges of the
strands 1 and 2 to bind the presequence backbone with clipping
P2 arginine to acidic S2 on
-MPP and
immediately cleaving off the peptide (Fig. 6B, upper
panel). Unlike with the shorter peptide, the MPP can scan a longer
presequence to find the cleavage signal with interactions to the edges
of another one or two
strands in
-MPP (Fig. 6B,
lower panel). Moreover, distal basic amino acids bind to
symmetric acidic sites for S2 to clip the N-terminal side
of the longer presequence to strands 3 and 4 on
-MPP, offering
stabilization of the substrate-enzyme complex. Thus, the long
presequence is pasted along the scaffold, clipping the proximal and
distal basic residues to acidic S2 on
-MPP and the
acidic side on
-MPP, respectively. Besides,
S1'-P1', S2'-P2', and
S3'-P3' interactions would also be required for
an overall high affinity Michaelis complex and for the strict
recognition of the processing site. The multiple subsite based on the
scaffold restricts the protein processing and allows a defect of
subsites in the enzyme without a complete loss of processing activity,
although the mutation could cause a shift in the processing site.
View larger version (35K):
[in a new window]
Fig. 6.
Putative model for determination of
the cleavage site using substrate binding scaffold and multiple
subsite inside cavity of MPP. A, structure of yeast MPP
complexed with yeast cytochrome c oxidase subunit IV
presequence. The structure of the MPP subunits (ribbon
models) and cytochrome c oxidase subunit IV (Cox
IV) presequence peptide (stick and space-filling
models) are visualized from x-ray crystallographic data in a
protein data bank (ID code 1HR9) using the WebLab ViewerPro 3.7 software package. The - and
-MPPs are shown in blue
and green ribbons, respectively. Four strands of
yellow ribbons in
-MPP and red ribbons in
-MPP indicate a substrate binding scaffold inside the cavity. The
space-filling models colored in red indicate acidic subsite
residues required for binding to proximal and distal basic residues in
the presequence. B, differential modes for substrate
recognition of MPP toward shorter (upper panel) and longer
(lower panel) presequences. See "Discussion" for
details. The
- and
-MPPs are painted in blue and
green, respectively. The substrate binding scaffold is
indicated by four orange arrows, and the internal acidic
cavity is gradually colored in red. Scissors indicate
the active site of
-MPP. Bold and narrow lines
of preprotein indicate the presequence and mature portion,
respectively. The plus (+) symbols represent basic amino
acid residues in the presequence, and the minus (
) symbols
indicate binding sites of
- and
-MPPs for proximal
(S2) and distal basic sites in the presequence.
It is unknown what regulates the directional insertion of
the presequence into the cavity through a narrow crack and the putative scanning of the sequence. Boteva et al. (33) report ATP and ADT binding on Neurospora MPP subunits. In the report they
indicate putative ATP binding sequences that resemble to some extent
the consensus pattern for "segment A and B" of typical ATP-binding proteins. One of the sequences is part of a Gly-rich loop of about a
20-amino-acid stretch that is highly conserved in -MPPs. A deletion
of the Gly-rich structure in yeast enzyme resulted in a great decrease
in the processing activity with a reduction of substrate binding,
indicating that the loop is essential for presequence cleavage and
binding (19). Most interesting is the crystallographic data showing
weak electron densities for the loop near the cleavage site of the
substrate peptide, suggesting that it is flexible and/or flapping,
interacting with the presequence (21). Additionally, we have observed
an increase in prepeptide cleavage activity of the enzyme in the
presence of ATP and a decrease in the activity after apyrase treatment
of MPP.4 Although there is no
experimental evidence of the linkage between nucleotide binding and the
loop-flapping, nucleotide-induced structure changes in the loop may be
required for the directional insertion of the presequence and for the
movable presequence inside the cavity. Further analyses on protein
structure and the dynamics will contribute to an understanding of a
putative model for the determination of cleavage sites through the
scanning of the presequence on the substrate binding scaffold and
multiple subsite with or without nucleotide binding.
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FOOTNOTES |
---|
* This work was supported in part by Grant-in-aid for Scientific Research 12780456 from the Ministry of Education, Science, Sports, and Culture of Japan (to S. K.) and by a grant-in-aid from Takeda Science Foundation in Japan (to S. K.).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.
To whom correspondence should be addressed. Tel.: 81-92-642-4182;
Fax: 81-92-642-2607; E-mail: s.kitscc@mbox.nc.kyushu-u.ac.jp.
§ Present address: Dept. of Microbiology and Immunology, Shinshu University School of Medicine, Matsumoto 390-8621, Japan.
Published, JBC Papers in Press, November 13, 2002, DOI 10.1074/jbc.M209263200
2 Following Schechter and Berger (34), the enzyme binding sites are denoted S1, S2, ... , Si and S1', S2', ... , Si' away from the scissile peptide bond toward the N and C termini, respectively. Amino acid residues in the substrates are referred to as P1, P2, ... , Pi and P1', P2', ... , Pii', in accordance with the binding site.
3 The residues were numbered according to the full-length MPP precursors.
4 S. Kitada, E. Yamasaki, K. Kojima, and A. Ito, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MPP, mitochondrial
processing peptidase;
-MPP, a
subunit of MPP;
-MPP, a
subunit of MPP;
pAd, a precursor form of Ad;
p
, a precursor form of
-MPP;
pMDH, a precursor form of MDH;
pSCC, a precursor form of
bovine cytochrome P450scc.
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