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Originally published In Press as doi:10.1074/jbc.M406915200 on July 14, 2004

J. Biol. Chem., Vol. 279, Issue 38, 39396-39400, September 17, 2004
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Cargo Sequences Are Important for Som1p-dependent Signal Peptide Cleavage in Yeast Mitochondria*

Haobo Liang, Wentian Luo, Neil Green, and Hong Fang{ddagger}

From the Department of Microbiology and Immunology, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232-2363

Received for publication, June 21, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The inner membrane protease (IMP) has two catalytic subunits, Imp1p and Imp2p, that exhibit nonoverlapping substrate specificity in mitochondria of the yeast Saccharomyces cerevisiae. The IMP also has at least one noncatalytic subunit, Som1p, which is required to cleave signal peptides from a subset of Imp1p substrates. To understand how Som1p mediates Imp1p substrate specificity, we addressed the possibility that Som1p functions as a molecular chaperone, which binds to specific substrates and directs them to the catalytic site. Our results show that cargo sequences attached to the signal peptide are important for Som1p-dependent presequence cleavage; however, no specific cargo sequence is required. Indeed, we show that a substrate normally destined for Imp2p is cleaved in a Som1p-dependent manner when the substrate is directed to Imp1p. These results argue against the notion that Som1p is a molecular chaperone. Instead, we propose that the cargo of some Imp1p substrates can assume a conformation incompatible with presequence cleavage. Som1p could thus act through Imp1p to improve cleavage efficiency early during substrate maturation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitochondria consist of two hydrophobic compartments, the outer and inner membranes, and two hydrophilic compartments, the matrix and intermembrane space, which hold proteins that perform important cellular functions including oxidative phosphorylation, tri-carboxylic acid cycle, and fatty acid oxidation (1). Among the hundreds of proteins that reside within the mitochondrial compartments, only 13 (in human) or 8 (in yeast) are encoded by the mitochondrial genome. All other proteins are encoded within the nucleus and translated in the cytoplasm. To achieve mitochondrial targeting, many proteins have N-terminal signal peptides that must be cleaved following membrane translocation. The mitochondrial processing peptidase (MPP)1 cleaves the signal peptides from most of these proteins (2). A smaller set of mitochondrial proteins has a signal peptide required for sorting to the intermembrane space and inner membrane following mitochondrial targeting and translocation. This signal peptide is cleaved by the inner membrane peptidase (IMP).

Study of the IMP in the yeast Saccharomyces cerevisiae has revealed three distinct subunits, Imp1p, Imp2p, and Som1p (3–5). Imp1p and Imp2p are catalytic subunits and members of the type I signal peptidase family, which includes endoplasmic reticulum (ER) signal peptidase, chloroplast thylakoid processing peptidase, and many eubacterial signal peptidases (6). Imp1p and Imp2p exhibit nonoverlapping substrate specificity (4). Imp1p cleaves signal peptides from nuclearly encoded cytochrome b2 precursor (p-cyt b2), the precursor to NADH-cytochrome-b5 reductase (pre-cyt b5-red), the precursor to glycerol-3-phosphate dehydrogenase (pre-Gut2p) and mitochondrially encoded pre-cytochrome c oxidase subunit 2 (pre-Cox2p) (3, 7–9). Imp2p cleaves the signal peptide from nuclearly encoded cytochrome c1 precursor (p-cyt c1) (4). Nonoverlapping substrate specificity derives in large part from the fact that Imp1p does not follow the "–3, –1 rule" in which small uncharged amino acids are required at the P3 and P1 positions in signal peptide cleavage sites (10). Instead, Imp1p tolerates a variety of amino acid residues at the P1 position and requires a negatively charged residue at the P'1 position in its substrates (11, 12).

The third IMP subunit, Som1p, binds to Imp1p and is required for signal peptide cleavage from two Imp1p substrates, pre-cyt b5-red and pre-Cox2p (5, 13). Other known Imp1p substrates, p-cyt b2 and pre-Gut2p, can be cleaved in the absence of Som1p (5, 9). This behavior indicates that noncatalytic subunit Som1p exerts a novel level of IMP substrate specificity, one that differentiates between Imp1p substrates. Here, we consider whether Som1p is a molecular chaperone that binds to specific substrates and delivers them to Imp1p.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains, Plasmids, Media, and Antibodies—Yeast strains used in this study were BY4741 (MATa his3{Delta}1 leu2{Delta}0 met15{Delta}0 ura3{Delta}0) (Invitrogen), HFY501 (MATa som1::LEU2 his3{Delta}1 leu2{Delta}0 met15{Delta}0 ura3{Delta}0) (prepared for this study), XCY101 (MAT{alpha} imp1::HIS3 ura3–52 leu2–3,112 his3-{Delta}200 trp1-{Delta}901 suc2-{Delta}9 lys2–80) (11), and JNY34 (MATa imp2{Delta}-1 ura3–52 trp1) (11). Plasmid pXC3 carries a mutated IMP2 gene (11). This mutation results in a S41A substitution that inhibits Imp2p catalytic activity but allows Imp2p to be expressed and bind to Imp1p. Binding of Imp2p to Imp1p is needed to stabilize Imp1p in yeast cells (4). Media used to support yeast cell growth have been described previously (14). Anti-HA high affinity rat monoclonal antibody and protein G-conjugated agarose beads (Roche Applied Science) were used for immunoprecipitation.

Disruption of SOM1 Gene—Oligonucleotides containing sequences flanking the SOM1 and the LEU2 genes were used to amplify the LEU2 gene using PCR. PCR product was introduced into strain BY4741 with selection for Leu-positive colonies. Recombination of the LEU2 gene into the SOM1 chromosomal locus was then confirmed by diagnostic PCR. The disruption removed the entire SOM1 open reading frame from the genome of strain BY4741.

Construction of HA-tagged Proteins—A DNA segment encoding three consecutive HA epitopes (15) followed by a stop codon (TAA) was inserted between the EcoRI and KpnI restriction sites of plasmids pHF454 (2µ, TRP1, ADH1 promotor) and pHF455 (2µ, URA3, ADH1 promotor) (16). Genes encoding mutant and fusion proteins without translational stop codons were synthesized using two-step PCR (16). The genes were then inserted into the above mentioned plasmids between the ADH1 promotor (17) and the DNA segment encoding the triple HA epitopes. Sequences of oligonucleotides used in this study are available on request.

Pulse Labeling and Immunoprecipitation—Methods similar to those described previously were used (14). Briefly, cells were grown at 30 °C to log phase (A600 = 1.5) in appropriate media. Cells were then shifted to media lacking methionine and cysteine for 1 h and pulse-labeled for 10 min with 60 µCi/ml [35S]Met (PerkinElmer Life Sciences). Cells were harvested and broken by glass beads. Total proteins were precipitated by 10% trichloroacetic acid, resuspended in 20 µl of SDS-PAGE sample buffer, and placed in a boiling water bath for 5 min. This mixture was diluted with 1 ml of phosphate-buffered saline/Triton X-100 (0.1%), and cell debris was removed by centrifugation. The protein solution was incubated overnight with 1.6 µg/ml anti-HA antibodies at 4 °C. This incubation was followed by incubation (2 h) with 20 µl of protein G-agarose beads (Roche Applied Science). Protein G beads were washed twice with cold phosphate-buffered saline/Triton X-100 (0.1%) and then twice with distilled water. 20 µl of SDS-PAGE sample buffer was added to the protein-bead slurry. The mixture was placed in a boiling water bath (5 min) and loaded onto an SDS-gel.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cargo Sequences Influence Som1p-dependent Presequence Cleavage—Som1p is needed for Imp1p cleavage of pre-cyt b5-red but not p-cyt b2 (5). Based on this difference, we reasoned that pre-cyt b5-red has a sequence, which is not present in p-cyt b2, that is responsible for Som1p-dependent cleavage. To determine whether the putative sequence is located within the presequence or cargo of pre-cyt b5-red, we constructed a chimera that contained the cargo of pre-cyt b5-red fused to the presequence of p-cyt b2. The p-cyt b2 presequence is actually composed of two signal peptides fused together in tandem (18). MPP cleaves a mitochondrial targeting signal peptide to generate i-cyt b2, and then Imp1p cleaves the second signal peptide to generate mature cyt b2. The p-cyt b2/cyt b5-red chimera, tagged with three HA epitopes at its C terminus, was expressed in strains BY4741 (wild type) and HFY501 ({Delta}som1). Yeast strain genotypes can be found under "Experimental Procedures." Cells were then subjected to a 10-min pulse with radio-labeled methionine, and proteins were precipitated from cell extracts with anti-HA antibodies (see under "Experimental Procedures"). As shown in Fig. 1, cleavage of i-cyt b2 signal peptide from i-cyt b2/cyt b5-red was Som1p-dependent. Because this signal peptide can be cleaved from the cyt b2 cargo independently of Som1p (5), the data support the notion that amino acid sequences located within the cargo of pre-cyt b5-red are important for Som1p-dependent cleavage.



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  		FIG. 1.
Cargo sequence determines whether the signal peptide cleavage is dependent on Som1p. HA-tagged p-cyt b2/cyt b5-red was expressed in strain BY4741 (WT, wild type) (lane 1 HFY501 ({Delta}som1) (lane 2). Cells were subjected to a 10-min pulse, and proteins were precipitated by anti-HA antibodies and resolved by a SDS-10% gel.

 
Som1p Facilitates Imp1p Substrate Recognition without Requirement for a Specific Substrate Sequence—We next sought to identify specific cargo sequences involved in presequence cleavage from the p-cyt b2/cyt b5-red chimera. To this end, we introduced distinct internal and C-terminal deletions into the cyt b5-red cargo (Fig. 2A) and expressed the constructs in strains BY4741 (wild type) and HFY501 ({Delta}som1). Cells were then examined by pulse labeling as described above. To our surprise, the mutated forms of i-cyt b2/cyt b5-red were cleaved in a Som1p-dependent manner (Fig. 2B). This result suggests that no specific cargo sequence is required for Som1p-dependent cleavage. Cleavage efficiency, however, was reduced when a region immediately flanking the Imp1p cleavage site was removed from the chimera (Fig. 2B, lanes 3 and 4). To examine this region in more detail, we designed a new set of constructs containing smaller internal deletions (10 or 11 amino acids) (Fig. 3A). As shown by pulse labeling, Som1p clearly was needed to cleave signal peptides from this new set of mutant chimeras (Fig. 3B). These results thus suggest that no specific cargo sequence is required for Som1p-dependent substrate cleavage, although it is plausible that deletion of cargo residues located near the cleavage site affect the conformation of the cleavage site and thus partially inhibit presequence cleavage in some protein contexts.



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  		FIG. 2.
Partial deletion of cyt b5 -red cargo does not block the Som1p-dependent signal peptide cleavage of p-cyt b2/cyt b5-red. A, design of internal and C-terminal deletions of cyt b5-red cargo are shown. Dashed lines represent the deleted regions. B, HA-tagged p-cyt b2/cyt b5-red constructs were expressed in strains BY4741 (WT, wild type) (odd-numbered lanes) and HFY501 ({Delta}som1) (even-numbered lanes), and strains were subjected to pulse labeling as described in Fig. 1. Proteins were resolved on a SDS-10% gel (lanes 1–10) or a SDS-12% gel (lanes 11–12).

 



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  		FIG. 3.
Deletion of cargo residues next to the Imp1p cleavage site in the p-cyt b2/cyt b5-red chimera does not prevent Som1p-dependent cleavage. A, design of short internal deletions of cyt b5-red cargo is shown. B, HA-tagged p-cyt b2/cyt b5-red constructs were expressed in strains BY4741 (WT, wild type) (odd-numbered lanes) and HFY501 ({Delta}som1) (even-numbered lanes), and strains were subjected to pulse labeling as described in Fig. 1. Proteins were resolved by a SDS-10% gel.

 
Next, we examined the role of cargo sequences using p-cyt b5-red, a natural Imp1p substrate. This protein is identical to the above-mentioned chimera with respect to the cargo sequence but differs, of course, in presequence composition. A series of internal and C-terminal deletions was constructed in the p-cyt b5-red cargo (Fig. 4A), and Imp1p cleavage was examined in cells that have or lack Som1p. Although most of the mutations did not prevent Som1p-dependent cleavage (Fig. 4B), residues located close to the cleavage site were important (Fig. 4B, lanes 3 and 4). These residues were nonessential for presequence cleavage in the chimeric protein context (Fig. 2B), suggesting that cargo residues located near the cleavage site can affect presequence cleavage in some protein contexts. Taken together, our data suggest that no specific cargo sequence is required for Som1p-dependent presequence cleavage.



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  		FIG. 4.
Cargo sequences are not important for Som1p-dependent processing of p-cyt b5-red except those flanking the cleavage site. A, design of cargo deletions in p-cyt b5-red is shown. B, HA-tagged p-cyt b5-red constructs were expressed in strains BY4741 (WT, wild type) (odd-numbered lanes) and HFY501 ({Delta}som1) (even-numbered lanes), and strains were subjected to pulse labeling as described in Fig. 1. Proteins were resolved on a SDS-10% gel (lanes 1–10) or SDS-12% gel (lanes 11–12). *, p-cyt b5-red molecules that were transported to the ER and glycosylated.

 
p-Cyt b5-red Can Be Directed to Both the Mitochondria and ER—Fig. 4B depicts protein bands (*) with molecular weights that are greater than that of p-cyt b5-red. These bands disappear when yeast cells are treated with tunicamycin prior to the pulse (data not shown). Tunicamycin inhibits Asn-linked glycosylation in the ER (19), indicating that the bands with greater molecular mass represent p-cyt b5-red that has been directed to the ER and glycosylated. This conclusion is supported by our inspection of the p-cyt b5-red sequence, which identified four consensus Asn-linked glycosylation sites. We also observed differently sized glycosylated protein bands with different cargo deletions (Fig. 4B, compare lane 5 with lane 7). This result is consistent with deletion of one or more of the consensus glycosylation sites. The data presented in Fig. 4B thus demonstrate that p-cyt b5-red can be targeted both to mitochondria and the ER. Our data further suggest that the p-cyt b5-red presequence but not the p-cyt b2 presequence exhibits this novel dual targeting, because glycosylated cyt b5-red cargo was not seen when the presequence of p-cyt b2 was used to target the p-cyt b2/cyt b5-red chimera to mitochondria (Fig. 2B). The p-cyt b5-red presequence has a rather long hydrophobic stretch consisting of ~20 amino acid residues (8). This unusually long hydrophobic stretch is probably responsible for ER targeting, whereas distinct uncharacterized sequences in the p-cyt b5-red presequence are likely to be responsible for mitochondrial targeting. We do not know whether ER targeting occurs naturally in yeast cells that express wild type p-cyt b5-red, and it has not been reported previously. However, our overexpression of p-cyt b5-red using the ADH1 promotor to facilitate protein detection (see under "Experimental Procedures") may contribute to the ER targeting seen in this study.

A Mutant Form of p-Cyt c1 Can Be Cleaved in a Som1p-dependent Manner—Previous work from our laboratory showed that Imp2p substrate p-cyt c1 could be switched to Imp1p when the P3, P1, and P'1 residues of p-cyt c1 were changed to Ile, Asn, and Glu (INE), respectively (12). The presequence of p-cyt c1 consists of a bipartite signal peptide (20). MPP cleavage of p-cyt c1 generates i-cyt c1, and IMP cleavage of i-cyt c1 generates cyt c1. To determine whether Som1p is needed for cleavage of i-cyt c1 when it is switched to Imp1p, we prepared a DNA construct encoding a truncated form of p-cyt c1 (p-cyt c1(INE)), lacking its C-terminal 87 residues, a region that includes the transmembrane segment of i-cyt c1 (20). We employed this truncation to facilitate detection of the protein, because the truncated protein was expressed at higher levels than full-length p-cyt c1 in yeast mitochondria (data not shown).

The p-cyt c1(INE) was expressed in strains BY4741 (wild type), XCY101 ({Delta}imp1), HFY501 ({Delta}som1), and JN34 ({Delta}imp2)/pXC3. Plasmid pXC3 carries a mutation that results in a S41A substitution in the Imp2p protein. This mutation inhibits Imp2p catalytic activity but allows Imp2p to bind to and stabilize Imp1p (11). As such, strain JN34/pXC3 has Imp1p activity but no Imp2p activity. As shown by pulse labeling (Fig. 5), i-cyt c1(INE) was processed by Imp1p but not by Imp2p (Fig. 5, lanes 2 and 3). This result is consistent with our previous study, which showed that full-length i-cyt c1 could be switched from Imp2p to Imp1p by incorporating INE residues at the cleavage site (12). Importantly, when Imp1p cleaved i-cyt c1(INE), it did so in a Som1p-dependent manner (Fig. 5, compare lane 2 with lane 4). As Imp2p does not need Som1p to cleave wild type i-cyt c1 (5), it is highly unlikely that i-cyt c1(INE) would have a sequence that interacts with Som1p only when Imp1p cleaves the precursor artificially as a result of mutation at the presequence cleavage site. Thus, taken together with data presented above, our results strongly argue against a model in which Som1p directly interacts with an Imp1p substrate to facilitate its cleavage.



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  		FIG. 5.
Signal peptide of p-cyt c1(INE) is cleaved by Imp1p in a Som1p-dependent manner. Truncated p-cyt c1(INE) (residues 1–222) tagged with three HA epitopes was expressed in strains BY4741 (WT, wild type), JNY34 ({Delta}imp2)/pXC3 (Imp1p), XCY101 ({Delta}imp1) (Imp2p), and HFY501 ({Delta}som1) (lanes 1–4, respectively). Cells were subjected to a 10-min pulse, and proteins were precipitated using anti-HA antibodies. Proteins were resolved using a SDS-12% gel.

 
To further support our conclusion that cargo sequences are important for Imp1p substrate cleavage, we designed a fusion protein that contained the bipartite presequence of p-cyt c1(INE) fused to the cyt b2 cargo. The signal peptide of i-cyt c1(INE) is cleaved in a Som1p-dependent manner (Fig. 5), whereas the signal peptide of i-cyt b2 is cleaved in a Som1p-independent manner (5). We reasoned that, if cargo sequences dictate Som1p-dependence, the i-cyt c1(INE)/cyt b2 chimera would be cleaved independently of Som1p. To test this idea, the chimera was expressed in strains BY4741 (wild type), JN34 ({Delta}imp2)/pXC3, XCY101 ({Delta}imp1), and HFY501 ({Delta}som1), and cells were subjected to pulse labeling. As shown in Fig. 6, the signal peptide of i-cyt c1(INE)/cyt b2 was indeed cleaved in a Som1p-independent manner (lanes 2 and 4). These results support our argument that cargo sequences play a critical role in determining whether an Imp1p substrate needs Som1p to be cleaved.



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  		FIG. 6.
Signal sequence of p-cyt c1(INE)/cyt b2 is cleaved by Imp1p in a Som1p-independent manner. HA-tagged p-cyt c1 (INE)/cyt b2 was expressed in strains BY4741, JNY34/pXC3, XCY101, and HFY501 (lanes 1–4, respectively). Cells were subjected to a 10-min pulse. Proteins were precipitated using anti-HA antibodies and resolved by a SDS-7% gel. The band labeled i-cyt c1 (INE)/cyt b2 actually consists of two closely spaced bands. The upper band corresponds to p-cyt c1(INE)/cyt b2 with a bipartite presequence.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Som1p is one of three known subunits, the others being Imp1p and Imp2p, of the yeast IMP. Imp1p and Imp2p are catalytic subunits with nonoverlapping substrate specificity (4), and both subunits are members of the type I signal peptidase family (6). Although the function of Som1p remains largely unknown, previous studies (5, 9, 13) have shown that Som1p is required to cleave two of the four known Imp1p substrates. Here, we have tested a model in which Som1p acts as a molecular chaperone that binds to the signal peptide or attached cargo of an Imp1p substrate and directs the substrate to Imp1p. We have tested this model by constructing and examining two chimeras. One chimera has the signal peptide of a Som1p-independent substrate fused to the cargo of a Som1p-dependent substrate (Fig. 1). The second chimera has a configuration similar to the first except its signal peptide and cargo come from substrates that display the opposite requirements for Som1p (Fig. 6). When these chimeras are tested for Som1p-dependent cleavage, results are consistent with the idea that cargo sequences play the critical role.

To identify cargo sequences that putatively bind to Som1p, we have examined a series of internal and C-terminal deletions in chimeric and natural Imp1p substrates (Figs. 2, 3, 4). To our surprise, the mutated proteins are cleaved in a Som1p-dependent manner, with one exception (Fig. 4, lanes 3 and 4). The exception occurs when a deletion removes cargo residues located next to the Imp1p cleavage site. This mutation inhibits Imp1p cleavage in the presence or absence of Som1p. However, deletion of cargo residues from a related protein does not prevent Som1p-dependent cleavage (Fig. 2, lanes 3 and 4). We therefore suggest that this deletion alters the conformation of the cleavage site, rendering it inaccessible to Imp1p. The possibility remains, however, that Som1p binds to sequences from disparate regions of the folded or partially folded protein cargo, and Som1p binding can still occur when the binding site is partially removed from the cargo. We do not favor this idea based on our analysis of a novel Imp1p substrate generated for this study. This novel substrate has been produced by changing the cleavage site residues of an Imp2p substrate so that the substrate can be cleaved by Imp1p. The substrate p-cyt c1 is normally cleaved by Imp2p in a Som1p-independent manner, but when the substrate is switched to Imp1p, the substrate shows Som1p dependence (Fig. 5). We consider it unlikely that p-cyt c1 would have a Som1p-binding site used only when it is artificially switched from Imp2p to Imp1p. For these reasons, we suggest that Som1p does not bind to Imp1p substrates and thus is not a molecular chaperone.

In a previous study, it has been shown that Som1p binds to Imp1p (5). This binding is required for stabilization of Som1p but not Imp1p in yeast cells. Because of this close association between Som1p and Imp1p, we suggest that Som1p binding could be needed to maintain Imp1p in a functionally efficient state. This model predicts that some Imp1p substrates (i.e. those substrates that require Som1p for signal peptide cleavage) can be processed only when Imp1p operates with high efficiency. Because our study clearly demonstrates that cargo sequences play the major role in dictating Som1p dependence, cargo sequences may interfere with presequence cleavage. Interference could occur when the cargo folds into a conformation that renders the cleavage site inaccessible following membrane translocation of the precursor polypeptide. In the presence of Som1p, Imp1p would, according to our interpretation, be able to recognize the precursor and cleave its presequence early during the folding process. It then follows that the conformations of different precursors would influence the Som1p requirement differently. This model thus suggests that, rather than binding substrates directly, Som1p acts through Imp1p to ensure cleavage of a structurally diverse set of protein substrates.


   FOOTNOTES
 
* This work was supported by National Science Foundation CAREER Award 9985079 (to H. F.) and American Heart Association Grant-in-aid Award 0355321B (to N. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 615-343-2233; Fax: 615-343-7392; E-mail: hong.fang{at}vanderbilt.edu.

1 The abbreviations used are: MPP, mitochondrial processing peptidase; IMP, inner membrane protease; ER, endoplasmic reticulum; cyt, cytochrome; red, reductase; pre-Gut2p, glycerol-3-phosphate dehydrogenase precursor; pre-Cox2p, pre-cytochrome c oxidase subunit 2; HA, hemagglutinin. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Morgan-Hughes J. A. (1994) in Myology (Engel, A. G., and Franzini-Armstrong, C., eds) 2nd Ed., pp. 1610–1660, McGraw-Hill, New York
  2. Verner, K., and Schatz., G. (1988) Science 241, 1307–1313[Abstract/Free Full Text]
  3. Pratje, E., and Guiard, B. (1986) EMBO J. 5, 1313–1317[Medline] [Order article via Infotrieve]
  4. Nunnari, J., Fox, T.D., and Walter, P. (1993) Science 262, 1997–2004[Abstract/Free Full Text]
  5. Jan, P.-S., Esser, K., Pratje, E., and Michaelis, G. (2000) Mol. Gen. Genet. 263, 483–491[CrossRef][Medline] [Order article via Infotrieve]
  6. Paetzel, M., Karla, A., Strynadka, N. C., and Dalbey, R. E. (2002) Chem. Rev. 102, 4549–4580[CrossRef][Medline] [Order article via Infotrieve]
  7. Pratje, E., Mannhaupt, G., Michaelis, G., and Beyreuther, K. (1983) EMBO J. 2, 1049–1054[Medline] [Order article via Infotrieve]
  8. Hahne, K., Haucke, V., Ramage, L., and Schatz, G. (1994) Cell 79, 829–839[CrossRef][Medline] [Order article via Infotrieve]
  9. Esser, K., Jan, P. S., Pratje, E., and Michaelis, G. (2004) Mol. Genet. Genomics 271, 616–626[CrossRef][Medline] [Order article via Infotrieve]
  10. von Heijne, G. (1986) Nucleic Acids Res. 14, 4683–4690[Abstract/Free Full Text]
  11. Chen, X., VanValkenburgh, C., Fang, H., and Green, N. (1999) J. Biol. Chem. 274, 37750–37754[Abstract/Free Full Text]
  12. Luo, W., Chen, X., Fang, H., and Green, N. (2003) J. Biol. Chem. 278, 4943–4948[Abstract/Free Full Text]
  13. Esser, K., Pratje, E., and Michaelis, G. (1996) Mol. Gen. Genet. 252, 437–445[Medline] [Order article via Infotrieve]
  14. Mullins, C., Lu, Y., Campbell, A., Fang, H., and Green, N. (1995) J. Biol. Chem. 270, 17139–17147[Abstract/Free Full Text]
  15. Wilson, I. A., Niman, H. L., Houghten, R. A., Cherenson, A. R., Connolly, M. L., and Lerner, R. A. (1984) Cell 37, 767–778[CrossRef][Medline] [Order article via Infotrieve]
  16. Liang, H., VanValkenburgh, C., Chen, X., Mullins, C., Van Kaer, L., Green, N., and Fang, H. (2003) J. Biol. Chem. 278, 50932–50939[Abstract/Free Full Text]
  17. Bennetzen, J. L., and Hall, B. D. (1982) J. Biol. Chem. 257, 3018–3025[Abstract/Free Full Text]
  18. Guiard, B. (1985) EMBO J. 4, 3265–3272[Medline] [Order article via Infotrieve]
  19. Mullins, C., Meyer, H.-A., Hartmann, E., Green, N., and Fang, H. (1996) J. Biol. Chem. 271, 29094–29099[Abstract/Free Full Text]
  20. Sadler, I., Suda, K., Schatz, G., Kaudewitz, F., and Haid, A. (1984) EMBO J. 3, 2137–2143[Medline] [Order article via Infotrieve]

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