Factors Governing Nonoverlapping Substrate Specificity by Mitochondrial Inner Membrane Peptidase*

Wentian LuoDagger, Xuemin Chen, Hong Fang, and Neil Green

From the Department of Microbiology and Immunology, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232

Received for publication, October 25, 2002, and in revised form, December 6, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

At least three peptidases are involved in cleaving presequences from imported mitochondrial proteins. One of the peptidase, the inner membrane peptidase, has two catalytic subunits, Imp1p and Imp2p, which are structurally related but functionally distinct in the yeast Saccharomyces cerevisiae. Whereas both subunits are members of the type I signal peptidase family, they exhibit nonoverlapping substrate specificities. A clue to the substrate specificity mechanism has come from our discovery of the importance not only of the -1 and -3 residues in the signal peptides cleaved by Imp1p and Imp2p but also the +1 cargo residues attached to the signal peptides. We specifically find that Imp1p prefers substrates having a negatively charged residue (Asp or Glu) at the +1 position, whereas Imp2p prefers substrates having the Met residue at the +1 position. We further suggest that the conformation of the cargo is important for substrate recognition by Imp2p. A role for the cargo in presequence recognition distinguishes Imp1p and Imp2p from other type I signal peptidases.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mitochondria have an outer and inner membrane enclosing two water-soluble compartments, the intermembrane space and the matrix. This complex membrane structure and the 400 or so proteins residing within mitochondria support a wide range of activities including respiration and oxidative phosphorylation, fatty acid metabolism, and iron homeostasis (1). The mitochondrial genome encodes only 13 translational products in humans, whereas yeast mitochondria make only eight proteins. Therefore, the majority of mitochondrial proteins rely on targeting, translocation, and processing systems to arrive at and be inserted into mitochondria.

Mitochondrial targeting is achieved to a large extent by N-terminal presequences whose cleavage by mitochondrial peptidases is often necessary for the attached proteins (referred to as cargo) to achieve their mature conformations (2). At least three endoproteases are involved in processing presequences. The first is the mitochondrial processing peptidase (MPP)1 that cleaves an N-terminal targeting peptide on a majority of imported mitochondrial proteins (3, 4). The second peptidase is the mitochondrial intermediate peptidase, which cleaves an octapeptide immediately following the MPP-cleaved signal in a subset of mitochondrial proteins (5-7). The third peptidase, the inner membrane peptidase (IMP), cleaves signal peptides from at least four proteins located in the inner membrane and intermembrane space (8-13).

Our understanding of IMP function has come exclusively from studies using the yeast S. cerevisiae. The IMP has three known subunits: Imp1p, Imp2p, and Som1p (11, 12, 14, 15). Imp1p and Imp2p are catalytic subunits and members of the type I signal peptidase (SP) family that includes endoplasmic reticulum (ER) SP and a large number of eubacterial SPs (16). As with most eubacterial SPs and unlike ER SP, Imp1p and Imp2p contain Ser/Lys catalytic dyads (16-18), a finding consistent with the widely accepted endosymbiont hypothesis in which mitochondria evolved from a eubacterial progenitor. Som1p is a small protein (74 amino acids) required for cleavage of signal peptides from a subset of Imp1p substrates (15, 19). The mechanism by which Som1p functions is not known.

The crystal structure of only one type I SP, the leader peptidase from Escherichia coli, has been determined previously (20). Its structure reveals small binding pockets for amino acids located at the -1 and -3 positions upstream from cleavage sites in signal peptides. Similar binding pockets are probably present in the eukaryotic members of the type I SP family, as -1 and -3 amino acids are important for substrate recognition by ER SP (21) and Imp1p (17).

Imp1p and Imp2p exhibit nonoverlapping substrate specificities, which sets them apart from other type I SPs (12). Imp1p cleaves an N-terminal peptide from NADH-cytochrome (cyt) b5 reductase and precursors to cyt b2 (l-lactate dehydrogenase) and subunit II of cyt c oxidase (complex IV in the respiratory chain) (11, 13). Imp2p cleaves the signal peptide from the precursor to cyt c1, a subunit of complex III (12). The cyt b2 and cyt c1 precursors have a bipartite presequence (9, 10). MPP cleaves the mitochondrial-targeting signal from p-cyt b2 and p-cyt c1, generating i-cyt b2 and i-cyt c1 intermediates that are cleaved by Imp1p and Imp2p, respectively.

Almost all of the type I SPs including Imp2p recognize small, uncharged amino acids at the -1 position and, to a lesser extent, -3 positions of signal peptides (17, 21, 22). Imp1p substrates have a Asn-1 (Fig. 1), which is unconventional in substrates of type I SPs. Based on this observation, the prevailing hypothesis for the nonoverlapping substrate specificity mechanism has been that Imp1p cleaves unconventional signal peptides (12). A recent study from our laboratory provides evidence that the -1 residue is not the sole determinant for substrate specificity. Construction of an extensive set of mutations in which the Asn-1 of i-cyt b2 was substituted with 19 amino acids (17) reveals that Imp1p efficiently cleaves signal peptides containing (in addition to Asn) Ala, Ser, Cys, Leu, and Met at the -1 position. This recognition of a Ala-1 means that Imp1p can cleave not only unconventional signal peptides but also conventional signal peptides. Taken together, the data argue that amino acids present within and outside the -1 and -3 positions of the IMP substrates are responsible for nonoverlapping substrate specificity. In agreement with this conclusion, the double mutant i-cyt b2 (Ala-1-Ala -3), which contains -1 and -3 residues like those found in Imp2p substrate i-cyt c1 (Fig. 1), is not cleaved by Imp2p (17).


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Fig. 1.   Amino acid sequences of natural and artificial substrates for Imp1p and Imp2p. The relevant amino acid sequences of Imp1p substrates i-cyt b2, cyt c oxidase subunit II (COXII) and NADH-cyt b5 reductase, and Imp2p substrate i-cyt c1 are depicted. Also depicted are the relevant sequences of artificial substrates constructed in this study. The dots at the N-terminal (NH2) side of the indicated sequences represent the MPP-cleaved presequence, and the dots at the C-terminal (COOH) side represent the cargo. The slash marks the signal peptide cleavage site separating the -1 residue from the +1 residue.

In this study, we have sought to identify presequence and cargo residues important for Imp1p and Imp2p recognition. For simplicity, our efforts are focused on substrates i-cyt b2 and i-cyt c1 that do not require Som1p for their processing (15, 19). Our strategy is to construct a series of mutations and chimeras that allow all or part of these substrates to be switched from one IMP catalytic subunit to the other catalytic subunit.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Strains-- Yeast strain XCY101 (MATalpha imp1::HIS3 ura3-52 leu2-3,112 his3-Delta 200 trp1-Delta 901 suc2-Delta 9 lys2-80), which expresses Imp2p but not Imp1p, was constructed in our laboratory (17). Yeast strain JNY34/pXC3 (imp2 S41A) (MATa imp2Delta -1 ura3-52 trp 1), which expresses functional Imp1p but not Imp2p, has been described previously (17). Yeast strain CVY1 (MATa ura3-52 his3-Delta 200 lys2-80 trp1-Delta 901), which expresses both Imp1p and Imp2p, has been described previously (18).

Plasmid and Site-directed Mutations-- A two-step PCR was used to create mutations as described previously (17). To mutagenize the CYB2 gene encoding p-cyt b2, upstream primer (amino acids1-21, 5'-AAA CTG CAG ATG CTA AAA TAC AAA CCT TTA-3') and downstream primer (amino acids 1753-1773, 5'-CCG GAA TTC TGC ATC CTC AAA TTC TGT TAA-3') were paired with different internal oligonucleotides (Table I). The second-step PCR created mutated full-length CYB2 having a PstI site at the 5' end and a EcoRI site at the 3' end of CYB2 gene. This construct was introduced into pHF454 (2 µm of TRP1), which placed the protein immediately downstream of the ADH1 promoter and upstream of a triple HA epitope. To mutagenize the CYT1-encoding p-cyt c1, procedures were the same as in the mutagenisis of CYB2 with the exception of the use of upstream primer (amino acids 1-23, 5'-AAA CTG CAG ATG TTT TCA AAT CTA TCT AAA CG-3') and downstream primer (amino acids 904-927, 5'-CCG GAA TTC CTT TCT TGG TTT TGG TGG ATT GAA-3'). These primers were paired with internal primers listed in Table I for constructions of mutations. To create the CYT1/CYB2 chimeras, the procedures were the same as in the mutagenesis of CYB2 and CYT1 with the exception of the use of different upstream and downstream primers and the pairing of different internal primers (Table I).

Pulse-Chase Analysis-- Yeast cells were cultured overnight to log phase at 30 °C, and four optical density (600nm) cell equivalents were used for each experiment. Cells were incubated for 60 min at 30 °C in minimal medium lacking methionine and cysteine. Cells expressing i-cyt b2 and chimeric proteins described in the text were labeled with 48 µCi of [35S]methionine/cysteine for 10 min and then subjected to a chase for 30 min in the presence of excess unlabeled methionine/cysteine mixture. Cells expressing i-cyt c1 were labeled with 48 µCi of [35S]methionine/cysteine for 15 min and then subjected to a chase for 30 min in the presence of excess unlabeled methionine/cysteine mixture. Yeast cells were broken using glass beads in 200 µl of 10% trichloroacetic acid. The pellet fraction was boiled in 20 µl of 1× SDS-PAGE sample buffer for 5 min. After centrifugation, the supernatant was mixed with 0.75 ml of phosphate-buffered saline solution containing 1% Triton X-100 and protease inhibitors. After another round of centrifugation, 10 µl of rat anti-HA high affinity monoclonal antibody was added to the supernatant, which was incubated at 4 °C for 3 h. Twenty microliters of agarose-conjugated protein G were added, and incubation continued overnight at 4 °C. After washing of the agarose bead pellet, 20 µl of SDS-PAGE sample buffer was added. After boiling for 5 min, immunoprecipitation was repeated as described above. Ten microliters of the solution was subjected to SDS-PAGE analysis.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Imp2p Recognizes the -1 and -3 Amino Acids in the Signal Peptide of i-cyt c1-- Our published work (17) demonstrates the importance of the -1 and -3 residues in the signal peptide of Imp1p substrate i-cyt b2. To determine whether the -1 and -3 residues are important for cleavage of an Imp2p substrate, we constructed mutations that changed the -1 and/or -3 amino acids of i-cyt c1 to residues found in i-cyt b2 (Table I) (see "Experimental Procedures"). In the pulse-chase experiment depicted in Fig. 2, two results are noteworthy. (i) When the Ala-1 and/or Ala-3 of i-cyt c1 are changed to Asn and/or Ile, respectively, cleavage by Imp2p is blocked (compare lane 6 with lanes 7-9), revealing a critical role for the -1 and -3 amino acids. (ii) When the -1 and -3 residues of i-cyt c1 are changed to Asn and Ile, respectively, Imp1p cannot cleave the protein (lane 5), even though i-cyt b2 has a Asn-1 and a Ile-3 (Fig. 1). Taken together with our prior results (17), substrate specificity is determined not only by the -1 and -3 amino acids but also by amino acids outside the -1 and -3 positions in the IMP substrates.

                              
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Table I
Oligonucleotides used in the construction of mutations and gene fusions
aa, amino acids.


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Fig. 2.   Pulse-chase analysis of p-cyt c1 mutant proteins containing amino acid substitutions at the -1 and -3 positions. Strains JNY34/pXC3 (imp2-(S41A)) (lanes 2-5) and XCY101 (lane 1 and lanes 6-9) were subjected to a 15-min pulse and a 30-min chase using methods described previously (17). Lanes 2 and 6 depict proteins expressed from plasmid pXC1, which encodes wild-type p-cyt c1-(HA) (17). The p-cyt c1 mutations indicated at the top of each lane are encoded by plasmids pXC6 (lanes 3 and 7), pXC7 (lanes 4 and 8), and pXC8 (lanes 5 and 9). The oligonucleotides used in the construction of the mutations are listed in Table I and under "Experimental Procedures." Proteins were precipitated using anti-HA antibodies and resolved using a 10% SDS-PAGE gel. The genotypes of yeast strains are listed under "Experimental Procedures."

Cargo Sequences Are Important for Substrate Recognition by Imp1p-- To address whether cargo sequences are important for substrate specificity, we constructed a gene fusion in which the presequence of p-cyt c1 was fused to the cyt b2 cargo (C1-61B81-591) (Table I). We introduced into this chimera the A-1N substitution, as Imp1p does not efficiently cleave signal peptides containing Ala at both the -1 and -3 positions such as that found in the i-cyt c1 signal peptide (17). The data presented in Fig. 3 show that Imp1p cleaved with good efficiency the C1-61(-1N)B81-591 chimera (compare lane 3 with lane 1, which depicts wild-type cyt b2). In contrast, Imp1p did not efficiently cleave the C1-61B81-591 chimera, which contains the wild-type signal peptide of i-cyt c1 (compare lane 2 with lane 1). In Fig. 2, we showed that Imp1p did not cleave the i-cyt c1 signal peptide (-1N) when it was attached to the i-cyt c1 cargo (lane 3). These results indicate that Imp1p can cleave the i-cyt c1 signal peptide provided it contains Asn-1 and is attached to cyt b2 cargo, thus demonstrating roles for the signal peptide and cargo in the substrate specificity of Imp1p.


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Fig. 3.   Pulse-chase analysis of chimeric proteins containing the presequence of p-cyt c1 and the cargo of p-cyt b2. Strains JNY34/pXC3 (imp2-(S41A)) (lanes 1-3) and XCY101 (lanes 4 and 5) were subjected to a 10-min pulse and a 30-min chase. Lane 1 depicts proteins expressed from plasmid pXC2 that encodes wild-type p-cyt b2-(HA) (17). Lanes 2 and 4 depict proteins expressed from plasmid pXC4 that encodes a chimera containing the presequence of p-cyt c1 (amino acids 1-61) and the cargo of p-cyt b2 (amino acids 81-591). Lanes 3 and 5 depict proteins expressed from plasmid pXC5 that encodes a chimera containing the presequence of p-cyt c1 (containing the Ala-1 to Asn-1 substitution) and the cargo of p-cyt b2. Proteins were precipitated using anti-HA antibodies and resolved using a 7% SDS-PAGE gel. The oligonucleotides used in the construction of the chimeras are listed in Table I and under "Experimental Procedures."

In considering cargo sequences that may be important for substrate recognition by Imp1p, we noted that all of the known Imp1p substrates have a negatively charged amino acid, Glu or Asp, immediately following the cleavage site (the +1 position in the cargo) (Fig. 1). Reasoning that the +1 residue may be important for substrate recognition, we prepared a i-cyt c1 construct in which its Met+1 was changed to Glu (Table I). We also introduced Asn at the -1 position and Ile at the -3 position. Pulse-chase analysis demonstrated that Imp1p cleaved i-cyt c1 (-3I -1N +1E) although slightly inefficiently (Fig. 4A, compare lane 1 with lane 3, which depicts Imp2p cleavage of wild-type i-cyt c1). We confirmed the importance of the +1 residue in Imp1p substrates by constructing a mutation that changed the +1 Glu of i-cyt b2 to Met (Table I) and by showing that Imp1p cleaved this protein inefficiently (Fig. 4B, compare lane 3 with lanes 1 and 2).


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Fig. 4.   The +1 residue is important for substrate recognition by Imp1p. A, strains JNY34/pXC3 (imp2-(S41A)) (lane 1), XCY101 (lane 2), and CVY1 (lane 3) were subjected to a 15-min pulse and a 30-min chase. Lanes 1 and 2 depict proteins expressed from plasmid pWL3 that encodes triple mutant p-cyt c1-(HA) (-3I -1N +1E). Lane 3 depicts proteins expressed from plasmid pWL1 that encodes wild-type p-cyt c1-(HA) (-3A -1A +1M). Proteins were resolved using a 10% SDS-PAGE gel. B, strains CVY1 (lane 1), JNY34/pXC3 (imp2-(S41A)) (lanes 2 and 3), and XCY101 (lanes 4 and 5) were subjected to a 10-min pulse and a 30-min chase. Lanes 1, 2, and 4 depict proteins expressed from plasmid pWL2 that encodes wild-type p-cyt b2-(HA) (+1E). Lanes 3 and 5 depict proteins expressed from plasmid pWL4 encoding p-cyt b2-(HA) containing the +1E to +1M substitution. Proteins were resolved using a 7% SDS-PAGE gel.

Cargo Sequences Are Important for Substrate Recognition by Imp2p-- The pulse-chase depicted in Fig. 3 demonstrated that Imp2p did not cleave chimera C1-61B81-591 (lane 4), suggesting that Imp2p does not recognize the presequence of its natural substrate i-cyt c1 when the presequence is fused to the cyt b2 cargo. We were unable to analyze in vivo a complementary chimera consisting of the p-cyt b2 presequence and cyt c1 cargo, because the fusion protein was not stably expressed in yeast cells. Therefore, we employed the following strategy.

As the +1 residue is important for substrate recognition by Imp1p, we asked whether the +1 residue mediates Imp2p substrate recognition. We constructed a mutation that changed the Met+1 of i-cyt c1 to Glu, a +1 residue found in some Imp1p substrates (Table I) (Fig. 1). As shown in Fig. 5A, this mutation strongly inhibited signal peptide cleavage by Imp2p (compare lanes 3 and 4), revealing an important role for the +1 residue in substrates recognized by Imp2p. We next asked whether the triple mutation (-3A -1A +1M) (Table I) causes Imp1p substrate i-cyt b2 to be switched to Imp2p in a manner similar to the substrate switching produced by a corresponding triple mutation in i-cyt c1 (Fig. 4A). However, Imp2p did not cleave i-cyt b2 containing the triple mutation (Fig. 5B, lane 4). These data demonstrate that although the -1, -3, and +1 residues are important for substrate recognition by Imp2p, residues outside the -3, -1, and +1 positions prevent i-cyt b2 from being switched to Imp2p.


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Fig. 5.   The +1 residue is important for substrate recognition by Imp2p. Strains JNY34/pXC3 (imp2-(S41A)) (Imp1p), and XCY101 (Imp2p) were subjected to a 10-min pulse (A) or a 15-min pulse (B) and a 30-min chase (A and B). A, lanes 1 and 3 depict proteins expressed from plasmid pWL1 that encodes wild-type p-cyt c1-(HA) (+1M). Lanes 2 and 4 depict proteins expressed from plasmid pWL5 that encodes p-cyt c1 containing the +1M to +1E substitution. Proteins were resolved using a 10% SDS-PAGE gel. B, lanes 1 and 3 depict proteins expressed from cells containing plasmid pWL2 that encodes wild-type p-cyt b2-(HA) (-3I -1N +1E). Lanes 2 and 4 depict proteins expressed from plasmid pWL6 that encodes triple mutant p-cyt b2 (-3A -1A +1M). Proteins were resolved using a 7% SDS-PAGE gel.

We decided to utilize our C1-61B81-591 chimera to further define the role of cargo sequences in substrate specificity by Imp2p. Imp2p inefficiently cleaved chimera C1-61B81-591(+1M) in which the Glu1 of the cyt b2 cargo was changed to Met (Fig. 6, lane 5). However, cleavage efficiency dramatically increased by an insertion mutation [+1M] that placed a Met residue between the Ala-1 of the i-cyt c1 signal peptide and Glu1 of the i-cyt b2 cargo in chimera C1-61[+1M]B81-591 (lane 6). The amino acid sequence of the fusion joint and amino acid changes are shown in Fig. 1. This result indicated that a spacing of the cyt b2 cargo from the i-cyt c1 signal peptide by a single Met residue led to efficient cleavage of the chimera by Imp2p.


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Fig. 6.   Amino acid spacing of the cargo is important for substrate recognition by Imp2p. Strains JNY34/pXC3 (imp2-(S41A)) (lanes 1-3), and XCY101 (lanes 4-6) were subjected to a 10-min pulse and a 30-min chase. Lanes 1 and 4 depict proteins expressed from plasmid pWL2 that encodes wild-type p-cyt b2-(HA). Lanes 2 and 5 depict proteins expressed from plasmid pWL7 that encodes a chimera containing the presequence of p-cyt c1-(1-61) and the cargo of p-cyt b2-(81-591) with the exception that the cargo has a +1E to +1M substitution. Lanes 3 and 6 depict proteins expressed from plasmid pWL8 that encodes a chimera containing the presequence of p-cyt c1-(1-61), which is separated from the cargo of p-cyt b2-(81-591) by a +1M insertion. Proteins were resolved using a 7% SDS-PAGE gel.

Although the data presented in Fig. 6 demonstrate the importance of cargo sequences in switching a substrate from Imp1p to Imp2p, we still do not know whether residues outside the -1 and -3 positions in the p-cyt b2 presequence contribute to substrate specificity. To address this issue, we constructed a mutation that introduced a single Met residue between the p-cyt b2 presequence and the cyt b2 cargo (Table I). We also constructed mutations that introduced Ala residues at the -1 and -3 positions of the p-cyt b2 presequence. Despite having the appropriate -1 and -3 residues and the required Met1 spacing residue, Imp2p did not cleave i-cyt b2 (-3A -1A [+1M]) (Fig. 7, lane 3), suggesting that the presequence of p-cyt b2 has residues outside the -1 and -3 positions that prevent cleavage by Imp2p.


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Fig. 7.   Pulse-chase analysis of p-cyt b2 in which its cargo is separated from the presequence by a Met residue. Strains JNY34/pXC3 (imp2-(S41A)) (lanes 1 and 2) and XCY101 (lane 3) were subjected to a 10-min pulse and a 30-min chase. Lane 1 depicts proteins expressed from plasmid pWL2 that encodes wild-type p-cyt b2-(HA). Lanes 2 and 3 depict proteins expressed from plasmid pWL9 that encodes p-cyt b2-(HA) containing an insertion of a Met residue between the presequence and cargo [+1M] and amino acid substitutions that replace the natural Ile-3 and Asn-1 with Ala residues. Proteins were resolved using a 7% SDS-PAGE gel.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Multiple factors contribute to nonoverlapping substrate specificity. We will discuss separately the roles played by the signal peptide and the attached cargo.

Role of Signal Peptides-- Results from a previous study (17) and this study (Fig. 2) demonstrate the importance of -1 and -3 residues for signal peptide recognition by Imp1p and Imp2p. As with most type I SPs, Imp2p recognizes small, uncharged amino acids at the -1 and -3 positions (Figs. 1 and 2). Imp1p differs from Imp2p in that Imp1p substrates have a Asn-1 (Fig. 1). This unusual -1 residue contributes to nonoverlapping substrate specificity because Imp2p cannot cleave i-cyt c1 when the Ala-1 is substituted with Asn (Fig. 2, lane 7). The -3 residue also contributes to substrate specificity, because Imp2p cannot cleave i-cyt c1 when its Ala-3 is substituted with Ile (Fig. 2, lane 8), the residue found at the -3 position in Imp1p substrate i-cyt b2 (Fig. 1). Similarly, Imp1p cannot efficiently cleave i-cyt b2 when Ala residues are placed at the -1 and -3 positions, although Imp1p can efficiently cleave i-cyt b2 when an Ala residue is present at either the -1 position or -3 position (17). Thus, the -1 and -3 residues play an important role in the nonoverlapping substrate specificity exhibited by Imp1p and Imp2p.

The presequence of p-cyt b2 plays a further undefined role in substrate specificity. As shown in Fig. 7, we are unable to switch Imp1p substrate i-cyt b2 to Imp2p even when Ala residues are present at the -1 and -3 positions and the appropriate spacing mutation is present in the sequence of the cyt b2 cargo (described below). The p-cyt b2 and p-cyt c1 have bipartite presequences, and both presequences exhibit hydrophobic amino acid stretches that are typical of signal peptides cleaved by type I SPs (23). The p-cyt b2 presequence is longer (80 residues) than the p-cyt c1 presequence (61 residues), although it is presently unclear whether this difference contributes to nonoverlapping substrate specificity.

Role of Cargo-- Cargo sequences are generally not important for substrate recognition by most type I SPs. Indeed, ER SP can cleave signal peptides cotranslationally (i.e. before a significant portion of the cargo has been synthesized) (24). However, our results show that cargo sequences play an essential role in nonoverlapping substrate specificity. The clearest example of this role comes from our results showing the importance of the +1 residue in the cargo. Imp1p substrates have a negatively charged residue at the +1 position, whereas the Imp2p substrates have a Met residue at the +1 position (Fig. 1). When these +1 residues are interchanged, cleavage efficiency of i-cyt b2 (by Imp1p) and i-cyt c1 (by Imp2p) is significantly reduced (Figs. 4B and 5A).

The +1 residue is generally thought to play no role in substrate recognition by type I SPs, because most of the enzymes in this family cleave signal peptides irrespective of the +1 residue (21, 22, 25-27). An important exception is that Pro is not tolerated at the +1 position in substrates of type I SPs. The crystal structure of leader peptidase from E. coli reveals binding pockets for the -1 and -3 residues but no such site for the +1 residue (20). Therefore, Pro probably alters the conformation of the cleavage site rather than interfere with a specific interaction between enzyme and +1 residue. One interpretation for the importance of +1 residues in IMP substrates is that, unlike other type I SPs, Imp1p and Imp2p have specific binding sites for the +1 residues. However, at this time, we cannot rule out the possibility that the +1 residues confer specific conformations necessary for substrate cleavage by Imp1p and Imp2p.

The cyt b2 cargo presents additional features that direct it to Imp1p and away from Imp2p. We find that when the cyt b2 cargo is fused directly to the i-cyt c1 signal peptide, cleavage by Imp2p is blocked even when the Glu1 is changed to Met (Fig. 6). However, Imp2p can cleave this chimera if a Met residue is present between the i-cyt c1 signal peptide and cyt b2 cargo (Fig. 6). This means that the spacing of the cyt b2 cargo by one amino acid from the i-cyt c1 signal peptide is sufficient for cleavage. From these data, we conclude that the conformation of the cyt b2 cargo inhibits presequence recognition by Imp2p. This conclusion is supported by prior data indicating that the attachment of heme c to the i-cyt c1 apoprotein is necessary for cleavage of the signal peptide by Imp2p (28), suggesting that the conformation of the i-cyt c1 apoprotein interferes with Imp2p recognition. These data lead us to suggest that signal peptides of Imp2p substrates and perhaps Imp1p substrates are cleaved after at least a portion of the cargo has been translocated across the outer mitochondrial membrane.

The unusual substrate preferences of Imp1p and Imp2p offer some hope that anti-microbial drugs specifically inhibiting eubacterial SPs can be found. Most eubacterial type I SPs have a Ser/Lys catalytic dyad, whereas ER SP catalytic site contains His (18). It may be possible to exploit this difference in the development of compounds specifically inhibiting eubacterial SPs (29, 30). One problem with this approach is that Imp1p and Imp2p also have Ser/Lys dyads (17). However, our observed role for cargo sequences, particularly the +1 residues, in the recognition of substrates by the IMP is clearly unique in the type I SP family. Therefore, it is plausible that drugs specifically targeting eubacterial SPs can be developed.

    ACKNOWLEDGEMENTS

We thank Reeha Arunkumar for technical assistance and Haobo Liang for critical reading of this paper.

    FOOTNOTES

* This work was supported by grants from the NSF (to N. G. and H. F.), NSF Career Award 9985079 (to H. F.), and American Heart Association Established Investigator Award (to N. G.).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.

Dagger To whom correspondence should be addressed: Dept. of Microbiology and Immunology, School of Medicine, Vanderbilt University, Nashville, TN 37232-2363. Tel.: 615-343-2233; Fax: 615-343-7392; E-mail: wentian.luo@vanderbilt.edu.

Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M210916200

    ABBREVIATIONS

The abbreviations used are: MPP, mitochondrial processing peptidase; ER, endoplasmic reticulum; IMP, inner membrane protease; cyt, cytochrome; SP, signal peptidase; HA, hemagglutinin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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