Location of the Actual Signal in the Negatively Charged Leader Sequence Involved in the Import into the Mitochondrial Matrix Space*,

Abhijit MukhopadhyayDagger, Thomas S. HeardDagger, Xiaohui WenDagger, Philip K. Hammen, and Henry Weiner§

From the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-2063

Received for publication, December 13, 2002, and in revised form, January 16, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Proteins destined for the mitochondrial matrix space have leader sequences that are typically present at the most N-terminal end of the nuclear-encoded precursor protein. The leaders are rich in positive charges and usually deficient of negative charges. This observation led to the acid-chain hypothesis to explain how the leader sequences interact with negatively charged receptor proteins. Here we show using both chimeric leaders and one from isopropyl malate synthase that possesses a negative charge that the leader need not be at the very N terminus of the precursor. Experiments were performed with modified non-functioning leader sequences fused to either the native or a non-functioning leader of aldehyde dehydrogenase so that an internal leader sequence could exist. The internal leader is sufficient for the import of the modified precursor protein. It appears that this leader still needs to form an amphipathic helix just like the normal N-terminal leaders do. This internal leader could function even if the most N-terminal portion contained negative charges in the first 7-11 residues. If the first 11 residues were deleted from isopropyl malate synthase, the resulting protein was imported more successfully than the native protein. It appears that precursors that carry negatively charged leaders use an internal signal sequence to compensate for the non-functional segment at the most N-terminal portion of the protein.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Most proteins found in the mitochondrial matrix space are encoded by nuclear genes and are synthesized on cytosolic polysomes. The preproteins typically carry N-terminal targeting sequences (leader sequence) that direct the proteins to the translocase of the outer mitochondrial membrane (TOM),1 which consists of receptors and a general import pore (1-5). A common feature of leader sequences is the frequent occurrence of basic residues, particularly arginine, a residue that is found three times more often in the leaders than proteins (6). Because TOM proteins are negatively charged, an acid-chain hypothesis was developed to suggest that positive charges in the leader sequences are necessary for TOM to interact with negatively charged receptor proteins (7).

The leader sequence for pALDH has been studied extensively in our laboratory. As shown in Fig. 1, the leader is composed of 19 amino acids that can be induced into a helix-linker-helix amphiphilic structure in the presence of trifluoroethanol or detergent micelles (8). Although each helical segment contains an equal number of positive charges, the N-terminal helical segment has been shown to be necessary to efficiently target the leader to the matrix space (9). If both of the arginines of the N-helical segment are substituted with glutamine, the ability to be imported is essentially eliminated, showing that the net positive charge in the N-helical segment is more important than the total positive charge throughout the leader (6). Deletion of the linker results in a non-processable leader that is longer in helical content (10) and is more capable of importing ALDH than is the native sequence in a cell-free import assay (6). The enhanced stability of the linker-deleted leader has been used successfully to study structural aspects as a compensating factor for the loss of positive charges resulting from arginine to glutamine mutational substitutions. The linker-deleted structure can import a "passenger" protein even if both of the N-terminal arginine residues (Arg-3 and Arg-10) are mutated to glutamines (6). On the basis of the positive charge versus structural compensation model developed from our previous studies, glutamic and aspartic acid residues were systematically introduced into the pALDH leader to ascertain how negative charges may be tolerated. It was observed that when serine was replaced with glutamic acid, the S7E mutant pALDH was imported to an extent similar to pALDH; however, the R3Q,S7E double mutant was poorly imported (11). This result implies that only a net positive charge was required for the proper import of a precursor into the matrix. Although negatively charged amino acids typically are not found in leader sequences, chaperonin 10 (12) and rhodanese (13) each contain one negative charge. Different forms of yeast IPMS (coded by Leu4 and Leu9) contain one and two negative charges, respectively, in their leader sequences (14-16). Mitochondrial IPMS provided us with a good model to study the mechanism of import of a natural protein with negative charges within the mitochondrial leader sequence.

In this study, we used several chimeric and mutant forms of pALDH and IPMS containing neutral or negative charges in their leader sequences. We will show that some precursors could be imported to the mitochondrial matrix with a leader sequence that is not present at the most N-terminal portion.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasmid Constructions-- The native and mutant leader sequences of ALDH in pGEM-3Z were amplified with a flanking SphI site using native or mutant pALDH as a template. PCR products and the vector containing pALDH or mutant pALDH were digested with SphI, and the vector was subsequently treated with alkaline phosphatase and ligated to the PCR products. The orientation was confirmed by sequencing. LEU4 and LEU9 were amplified with Vent polymerase from genomic DNA isolated from Saccharomyces cerevisiae (ATCC 24657). These two genes were ligated into the pGEM-3Z plasmid vector, respectively, between the KpnI and HindIII sites for in vitro transcription/translation and further mutational studies. The IPMS/ALDH and Leu4/Leu9 chimeras were constructed by three separate polymerase chain reactions that sequentially added additional coding fragments of the LEU4 and LEU9 genes to DNA, encoding either the mature portion of pALDH or the short form Leu4 or Leu9 protein (14). The final PCR products were ligated to the pGEM-3Z plasmid between either the KpnI and HindIII sites or the NcoI and HindIII sites. All constructs were sequenced at the DNA sequencing facility at either Purdue University or Iowa University.

In Vitro Mitochondrial Import-- Mitochondria were isolated from S. cerevisiae (ATCC 24657) and stored at -70 °C until used (17). Radiolabeled protein synthesis and in vitro import were performed as described previously (18). Visualization and analysis of import results from the SDS-PAGE were performed by using the PhosphorImager System. Import data were analyzed with the ImageQuant software from Amersham Biosciences.

Proteolysis Analysis of Leu4 and Leu9-- Radiolabeled protein synthesis was performed as described in a reaction system with a total volume of 12.5 µl. The protein synthesis was terminated by placement of the reaction mixture on ice and the addition of ice-cold water to a final volume of 50 µl. Before the addition of proteinase K, an 8-µl aliquot was removed. Next, 6 µl of 20 mg/ml proteinase K was added to the remaining 42 µl. At different time intervals, 10 µl of the protease digestion sample was removed, and 3 µl of 100 mM phenylmethyl-sulfonyl-fluoride was immediately added to terminate proteolysis. An equal volume of 2× SDS treatment buffer was added. The samples were heated to 100 °C for 5 min and then loaded on a 10% SDS-PAGE.

NMR Spectrometry-- Two-dimensional NMR spectra were obtained using a Varian Unity+ 600-MHz instrument. A peptide corresponding to the N-terminal 21 residues of Leu4 was prepared at the Purdue University Biochemistry Department and was purified by reverse-phase high pressure liquid chromatography. The peptide was dissolved in equal volumes of phosphate buffer (50 mM phosphate, 50 µM EDTA, 50 µM azide, pH 5.2) and d3-TFE to a final concentration of ~1.8 mM. Chemical shifts were referenced to 2,2-dimethyl-2-silapentanesulfonic acid. Spectra were obtained at temperatures ranging from 10 to 35 °C. Clean TOCSY spectra (19) used 30 and 70 ms mixing times, whereas mixing times (tau m) for NOESY spectra were 200 ms. Amide exchange experiments were performed using a Varian VXR 500-MHz instrument. The NMR sample described above was lyophilized and was redissolved in a (1:1) mixture of 2H2O and d3-TFE. The sample was immediately placed in the magnet, and five NOESY spectra (tau m = 150 ms) were acquired at 2-h intervals. Amide cross-peaks were observed in only the first two NOESY data sets. The residues for which NH protons were observed are identified in Fig. 3B. All two-dimensional transformations were performed using Varian software from Varian Associates, Inc.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preproteins Can Be Imported to the Mitochondrial Matrix Space with a Leader Sequence Not Present at the Very N Terminus-- A modified pALDH leader with the R3Q,R10Q double mutation (B in Fig. 1) was shown to be barely capable of allowing a preprotein to be imported to the mitochondria (6). This leader was fused to the N terminus of pALDH to make chimeric protein C (Fig. 1). The double leader strategy was used previously to study the processing of the leader sequence by MPP (18). Here the purpose of using it was to test whether or not a leader could be functional if it were not present at the most N-terminal portion of a protein. We did not expect that chimeric protein C would be imported into the mitochondrial matrix, because we and others have shown that the positive charges are needed at the N terminus of the presequence, presumably to interact with the TOM complex (6, 11, 20, 21). However, chimeric protein C was found to be imported to the same extent as pALDH (Fig. 2I, lane 4). Two different mechanisms can be evoked to explain the import of chimeric protein C. For the first mechanism it is suggested that when an R3Q,R10Q-containing leader is fused with another pALDH leader, the second helical segment of the first leader and the first helix of the second leader form a long continuous helix (helix 2 in Fig. 1C) that might be responsible for import. For the second mechanism it is suggested that although the native leader was not present at the N terminus of C, it served as an internal leader sequence.


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Fig. 1.   Leader sequence of rat liver pALDH and schematic diagram of chimeric proteins. The sequences of native pALDH and the linker deleted (-RGP) leaders are shown. Below the leaders are the chimeric proteins used in this study with numbered boxes to indicate helical domains. The percentage import is relative to pALDH, defined as 100%. A, pALDH with the helix-linker-helix motif. B, pALDH containing an R3Q,R10Q double mutation. C, leader sequence of pALDH with an R3Q,R10Q double mutation fused to pALDH. D, leader sequence of pALDH with an R3Q,S7E double mutation fused to pALDH. E, leader sequence of pALDH with an R3Q,R10Q double mutation fused to another R3Q,R10Q double mutant pALDH. G, the R3Q,R10Q-containing leader of pALDH fused to pALDH with its C-terminal helix deleted. H, an R3Q,R10Q-containing leader was fused with pALDH with a proline-glycine linker between them. I, an R3Q,R10Q-containing leader was fused with another R3Q,R10Q-containing pALDH with a proline-glycine linker between them. J, an R3Q,R10Q double mutant leader fused to pALDH (through a proline-glycine linker) with its C-terminal helix deleted. K, an R3Q,R10Q double mutant leader fused to another R3Q,R10Q double mutant leader (through a proline-glycine linker) that had the C-terminal helix deleted. L, an R3Q,R10Q,R14Q,R17Q-containing leader fused to another R3Q,R10Q leader (through a proline-glycine linker) that had the C-terminal helix deleted.


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Fig. 2.   In vitro import of chimeric pALDH proteins. Import was performed as described under "Materials and Methods." I, lanes 1 and 2 represent pALDH (A), lanes 3 and 4 represent chimera C, lanes 5 and 6 represent chimera D, and lanes 7 and 8 represent chimera E. Lanes 1, 3, 5, and 7 are for newly translated proteins, and lanes 2, 4, 6, and 8 are for those found after import into mitochondria representing imported protein. II, lanes 1 and 2 contain pALDH (A) and lanes 3 and 4 contain chimera G. Lanes 1 and 3 are translated proteins and lanes 2 and 4 contain protein bands found after import. III, lanes 1, 3, and 5 are translated proteins of chimeras A, H, and I, respectively, and lanes 2, 4, and 6 are the bands found after import of A, H, and I, respectively. IV, lanes 1 and 3 contain A and J, respectively, and lanes 2 and 4 represent the imported bands of A and J, respectively. V, lanes 1 and 3 represent translated proteins of A and K, respectively, and lanes 2 and 4 represent protein found after import of A and K. VI, lanes 1 and 3 represent translated proteins of A and L, respectively, and lane 2 represents protein found after import of A. No imported protein was observed with L (lane 4).

To determine which mechanism for the import of chimeric protein C was valid, construct E (Fig. 1) was used. It contained two R3Q,R10Q leader sequences fused back to back, so that it possessed two non-functioning leaders, and was shown to be imported as well as pALDH (Fig. 2I, lane 8) as we reported previously (18). Thus, for chimeric protein E the second long helix (helix 2 in Fig. 1E) and the third helix might have formed an efficient leader. To determine the individual contribution of each helix and the specific region that acted as a leader, we made chimeric protein G (Fig. 1). Chimeric G had one neutral helix at the N terminus followed by a long helical segment. When chimeric protein G was added to isolated mitochondria, it was imported to the matrix space and was processed (Fig. 2II, lane 4). This result shows that the third helix in chimera E was not necessary; the helix, which is a longer version of helix 2 in chimera B, acted as a leader in the presence of a non-charged helical domain at the most N-terminal portion of the preprotein.

From the data in Fig. 2II, we concluded that an internal long helix was necessary for the import of G. Proline and glycine were introduced to disrupt helix 2 in both constructs C and E to produce H and I, respectively (Fig. 1). Pure MPP cleaved at the two processing sites that were present in each construct (data not shown), confirming that the long helix was disrupted because MPP cannot cleave when the residues at the processing site are in a helical environment (18, 22). Constructs H and I were imported and processed in the mitochondria (Fig. 2III, lanes 4 and 6). Although the long helix was disrupted, the two smaller helices in H and I (helix 2 and 3, Fig. 1) could act as a leader because of their similarity to the helix-linker-helix motif found in pALDH. Two more constructs were made to determine the importance of the fourth helix as well as the internal helix-linker-helix motif (helix 2 and 3). We removed the fourth helix from H and I to make J and K, respectively (Fig. 1). Both J and K were imported into mitochondria (Fig. 2IV, lane 4 and 2V, lane 4, respectively). The second and third small helices could form the helix-linker-helix motif and act as a leader, because both precursors had an unfavorable helix at the most N-terminal portion. Import again appeared to be a result of an internal functioning leader.

In chimeric protein K, the second helix had two positive charges. To study their importance, these residues were mutated to glutamines to produce construct L (Fig. 1). Construct L contained three small neutral helices and it was found, as expected, not to be imported into mitochondria (Fig. 2VI, lane 4). No import of L was found, which implies that the internal helix-linker-helix motif still needed some positive charges.

Our previous study showed that the S7E mutant of the ALDH leader was imported along with the native pALDH. However, when the double mutation R3Q,S7E was made, it imported just 16% compared with pALDH, which shows that a net positive charge was necessary in the first helix of pALDH for it to be imported well. Removal of the RGP linker to make the leader a continuous helix (R3Q,S7E (-RGP)) allowed import of as much as 50% compared with pALDH (11). Thus, the positive charge deficiency could be overcome by introducing a long helix; in those studies, the long helix was present at the very N terminus. To increase negative charges at the very N terminus of a precursor protein we made a double mutation, R3Q,S7E, at the N terminus of the first leader of a two-leader containing ALDH to produce D (Fig. 1). When chimeric protein D was incubated with mitochondria, it was imported (Fig. 2I, lane 6), which indicates that not only a neutral helix (chimera C) but also a negatively charged one (chimera D) could be tolerated at the most N-terminal portion as long as an internal leader was present.

All of the complex leaders used in this study were artificial, but their presence allowed us to conclude that a neutral or even a negatively charged helix could be tolerated at the most N-terminal portion if a leader-like structure was present distal to the N terminus. This finding is consistent with the proposal for the first mechanism that an internal leader-like structure can be either a long helix or a helix-linker-helix motif. To test the conclusions obtained using the artificial leaders, a natural preprotein with a negative charge leader sequence IPMS was employed.

Two-dimensional NMR of the IPMS4 Leader Residues 1-21-- The N-terminal residues of the LEU4 and LEU9 gene are illustrated in Fig. 3A. The sequences before the second boldface methionine residue are considered the mitochondrial leaders. The Leu4 short form starts at Met-31, and it is assumed that for Leu9 the short form would start at Met-30. Both leaders possess glutamates within the first 11 residues of the leader sequence, with the Leu4 leader having two and glutamates Leu9 having just one. The first 11 side-chain residues of Leu4 do not possess a net positive charge. The leader was predicted to be a long helix by secondary structure prediction methods (14). The helix would be disrupted by the pair of proline residues. To investigate the conformation of the N terminus of Leu4, a peptide corresponding to the first 21 residues was studied by two-dimensional NMR. As with peptides that were studied previously by NMR in our laboratory (6, 8, 10), a tyrosine-alanine-amide sequence was added to the C-terminal portion of the peptide.


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Fig. 3.   A, comparison of the N-terminal residues of Leu4 and Leu9 long forms. The leader sequences of the LEU4 and LEU9 gene long form products are illustrated. The sequences before the second boldface methionine are considered to be mitochondrial leaders. LEU4 and LEU9 gene short form products have the second boldface methionine as their first amino acid residue, respectively. Negatively charged glutamate residues are also presented in bold. B, interresidue NOEs observed for IPMS4 (1-23) in 50% TFE. Two-dimensional NOESY spectra with 200 ms mixing times were obtained using a Varian Unity+ 600-MHz instrument. Unfilled bars indicate interactions that could not be resolved. The heights of the bars in the row labeled NHi-NHi+1 indicate relative intensity. The row labeled NHExch indicates amide resonances that were observed 1 h after the peptide was dissolved in 50% TFE. C, in vitro mitochondrial import of pALDH, Leu4, and Leu9. Lanes 1-3 contain pALDH, lanes 4-6 contain Leu4, and lanes 7-9 contain Leu9. The protein bands in lanes 1, 4, and 7 represent the TNT-synthesized products; lanes 2, 5, and 8 represent the proteins after import; and lanes 3, 6, and 9 represent the supernatant after the precipitation of mitochondria. The absence of protein bands in lanes 3, 6, and 9 indicates that unimported proteins were completely digested by the added trypsin.

High-resolution homonuclear two-dimensional NMR spectra were obtained at 600 MHz in the presence of 50% TFE, which induced a secondary structure that was not observed in aqueous buffer. Proton resonances were assigned from TOCSY experiments (30 and 70 ms) at 20 °C, and NOESY experiments were used to establish sequential resonance assignments. Complete chemical shift assignments are available as Supplemental Material. Secondary structure was assessed from inter-residue interactions observed in the NOESY spectra illustrated in Fig. 3B. These data were indicative of a helical structure extending from Val-2 through Tyr-22, essentially the entire length of the peptide. Compared with standard random coil values (23), chemical shifts of Halpha were consistent with the general impression formed from analysis of the NOESY data. The segment from K-3 to H-12 exhibited chemical shifts more closely associated with helical character. Amide proton exchange experiments revealed that the region from Ile-7 to Glu-11 was most resistant to exchange with 2H in the solvent. These data provide an image of IPMS4 (1-21) that can be induced to form a continuous helical conformation in TFE that is most stable in the first 10-12 residues.

In Vitro Import of Leu4 and Leu9 into Yeast Mitochondria-- To characterize the leaders of Leu4 and Leu9, in vitro import assays were carried out under the conditions described under "Materials and Methods." Rat pALDH was included as a positive control. The number of potential radiolabeling sites in pALDH and in Leu4 and Leu9 long forms was nearly the same (pALDH contains 10 methionines, and the two Leu sequences contain 9 and 12 methionines, respectively).

Compared with pALDH (Fig 3C, lane 1), the in vitro translation of Leu4 or Leu9 produced two protein bands (Fig. 3C, lanes 4 and 7). The lower protein band was postulated to be a product of translation starting from the second methionine (Met-31 and Met-30, respectively). The imported protein band of Leu9 (lane 8) had a molecular weight almost identical to the long form precursor protein, a result that is consistent with the leader being non-processable (14). pALDH was imported and processed as expected (lane 2). To our surprise, import was not observed with Leu4 (lane 5). This result was inconsistent with previously published data (14, 24). To verify that import of Leu4 long form did not occur, radiolabeled protein was added such that if 1% of the total counts provided in the assay were taken up by mitochondria, it would be observable after a 12-18- h phosphorimage exposure. No import was observed under these conditions.

Cell-free Synthesis and Import of Leu4 and Leu9 Chimeric Proteins-- To investigate the behavior of the Leu4 leader, a leader exchange experiment was performed with the Leu4 purported leader (first 30 amino acids) fused to the Leu9 short form (Fig. 4A). The chimeric Leu4-Leu9 protein was found to be imported to the mitochondrial matrix space (Fig. 4B, lane 5). On the basis of this result, it was concluded that the first 30 amino acid residues of Leu4 could target a passenger protein into mitochondria under in vitro conditions. This observation is in contrast to what was found for the native protein where the same 30 residues were ineffective in targeting its own short form protein into isolated mitochondria under the same experimental system. To address this discrepancy, two more chimeric protein constructs were made. Here, either the Leu9 leader or the pALDH leader was fused to the Leu4 short form as illustrated in Fig. 4A. The import results of these two constructs were quite different in that the Leu9 leader could not import the short form of Leu4, whereas the pALDH leader could (Fig. 4B, lanes 2 and 8). To assess the import ability of Leu4 and Leu9 leaders, the N-terminal 40 residues of Leu4 long form and a corresponding N-terminal 39 residues of Leu9 long form were fused to the mature portion of ALDH, creating a Leu4(1-40)ALDH chimera and a Leu9(1-39)ALDH chimera (Fig. 5). The reason we included the additional 10 residues was that the most N-terminal portion of a mature mitochondrial protein appears to affect structure and import capability (25, 26). Both of these constructs could be imported into mitochondria (Fig. 6A, lanes 2 and 11). These results showed that the N-terminal 40 and 39 residues of Leu4 and Leu9 can function as leader sequences.


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Fig. 4.   A, schematic illustrations of the native and the IPMS chimeras. The long form Leu4 and Leu9 are represented by two connected boxes. The first rectangle represents the mitochondrial leader sequence, whereas the second rectangle indicates the short form protein. The chimeric IPMS proteins were constructed by exchanging the leaders of Leu4 and Leu9 and reconnecting the Leu4 leader before the Leu9 short form and the Leu4 leader before the Leu9 short form. The pALDH-Leu4 was constructed by placing the first 19 residues of pALDH, designated as pALDH leader, before the Leu4 short form. Import of proteins is based on a comparison with Leu9, which is defined as 100%. B, in vitro import of IPMS chimeras. Chimeric proteins synthesized in the TNT system are illustrated. Lanes 1-3 represent Leu9-Leu4 (L9-L4), lanes 4-6 represent Leu4-Leu9 (L4-L9) and lane 7-9 represent pALDH-Leu4. Lanes 1, 4 and 7 represent the TNT-synthesized products. The corresponding proteins after import are shown in lanes 2, 5, and 8, respectively. All of the chimeric proteins were completely digested by trypsin, because no bands were found from the corresponding supernatant after precipitation of the mitochondria in lanes 3, 6, and 9, respectively.


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Fig. 5.   IPMS-ALDH chimeras. Both the first 40 residues of Leu4 long form and the first 39 residues of Leu9 long form were truncated into several fragments. Each of these fragments was placed before the mature portion of pALDH, forming five different Leu4-ALDH chimeras and five different Leu9-ALDH chimeras. Import of protein is based on Leu4 or Leu9, defined as 100%.


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Fig. 6.   Mitochondrial import of different Leu-ALDH chimeras. The first 40 amino acid residues of Leu4 long form or the first 39 amino acid residues of Leu9 long form were truncated into several fragments and fused to mature ALDH separately (Fig. 5). The expressed proteins are shown as follows. A, lanes 1-3 are Leu4(1-40)ALDH, lanes 4-6 are Leu4(1-21)ALDH, lanes 7-9 represent Leu4(Delta 1-11)ALDH, lanes 10-12 represent Leu9(1-39)ALDH, lanes 13-15 represent Leu9(1-20)ALDH, and lanes 16-18 represent Leu9(Delta 1-11)ALDH. Lanes 1, 4, 7, 10, 13, and 16 represent the TNT-synthesized products. Lanes 2, 5, 8, 11, 14, and 17 represent the protein bands after import. Lanes 3, 6, 9, 12, 15, and 18 represent the samples containing the supernatants after mitochondrial precipitation. B, lanes 1-3 represent Leu4(Delta 1-21)ALDH, lanes 4-6 represent Leu9(Delta 1-20)ALDH; lanes 7-9 represent Leu4(12-21)ALDH, and lanes 10-12 represent Leu9(12-20)ALDH. Lanes 1, 4, 7, and 10 represent the TNT-synthesized products, lanes 2, 5, 8, and 11 represent the protein bands after mitochondrial import, and lanes 3, 6, 9, and 12 represent the samples containing supernatant after mitochondrial precipitation.

Characterization of the Leu4 and Leu9 Leaders-- Although the first 11 residues of Leu4 do not possess a net positive charge, the inducible long helical structure may compensate for the charge deficiency similar to that shown with the ALDH constructs (11). Because the primary sequences of the Leu9 and Leu4 leaders are nearly identical, the first 20 residues of Leu9 were presumed to also be capable of forming an alpha -helical structure. To localize the leader fragment most responsible for the import signal, two other chimeras, Leu4(1-21)ALDH and the corresponding Leu9(1-20)ALDH (Fig. 5), were constructed and subjected to the import assay. To our surprise, these two chimeras were barely imported into mitochondria (Fig. 6A, lanes 5 and 14).

To determine the locations of the mitochondrial targeting information within the IPMS leaders, the 40 and 39 N-terminal residues of Leu4 and Leu9 were manipulated (Fig. 5). When the first 11 residues were deleted, the remaining leader segments of both Leu4 and Leu9 could import ALDH as efficiently as, or even better than, the full-length IMPS leader (Fig. 6A, lanes 8 and 17). When the first 21 residues of Leu4 or the first 20 residues of Leu9 were removed, import again became poor (Fig. 6B, lanes 2 and 5). Neither the short fragment of residues 12-21 of the Leu4 leader nor residues 12-20 of the Leu9 leader could act as a successful mitochondrial targeting signal on their own (Fig. 6B, lanes 8 and 11). On the basis of the manipulation and subsequent import results, it is concluded that the most N-terminal 11 residues of both Leu4 and Leu9 leaders are not required to target mature ALDH into mitochondria, whereas the remaining leader portion, residues 11-30, is necessary for passenger protein import.

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

During the past decade much has been learned about the leader sequences of precursor proteins that are destined for the mitochondrial matrix space (3, 27). No reports exist, however, to explain how a leader that does not possess a net positive charge at its N terminus is imported. During our studies with the positive charge in the ALDH leader, we often found that if the leader was made more helical it would compensate for the removal of positive charges (6). No actual structure for a precursor protein that possesses a cleavable leader sequence has been determined. NMR has been used to show that a number of peptides corresponding to leader sequences can indeed form alpha  helices when bound to micelles or when in a hydrophobic environment (6, 8, 10, 28). Recently the structure of the aldehyde dehydrogenase leader bound to TOM20 was shown to be helical, supporting the notion that helicity is indispensable for its function (29).

A few precursor proteins do not possess positive charges in what could be considered to be the classical N-terminal leader sequence. For example, the non-processed precursors rhodanese (30) and chaperonin10 (31, 32) have no positive charges in the first eight residues and actually have a negative charge in the leader. Leu4 leader has one lysine residue but two negative charges in its first 11 residues. These leader peptides were all found to be helical, consistent with our notion that structure can somehow overcome positive charge deficiency. Even if structure could overcome this situation, it is not apparent how the TOM complex, which contains patches of negative charges (7), can recognize the atypical leader.

We took advantage of the two-leader strategy that we used previously to investigate the processing of leader peptides by MPP (18). Here a modified non-functional leader was fused to the N termini of a native or modified precursor protein. In this way, it was possible to ascertain what part of the leader was actually needed by the import apparatus.

Previously, it was shown that the R3Q,R10Q double mutant pALDH was not imported (6). Here we report that if this modified leader was fused to the R3Q,R10Q pALDH, import was restored. This shows that two non-functional leaders could actually be used to import a protein. The reason this double leader was functional in import could be that the internal leader-like structures shown in Fig. 1 actually were the portion of the precursor that was involved in the translocation process. No such structural motif existed in the original R3Q,R10Q double mutant pALDH we previously investigated. From the various chimeric precursors used, it is concluded that an internal helix that is either a long continuous amphipathic helix or part of the helix-linker-helix motif could act as the actual leader. No attempt was made to determine how distal from the N terminus such a structure would function.

Using the strategy described above, it was possible to show that the most N-terminal, non-functional segment could even possess a negative charge such as found in the R3Q,S7E double mutant of pALDH. This finding can then be used to explain how a natural precursor protein could possess a net negative charge at the start of the N-terminal sequence. It can be argued that the import apparatus simply ignores the unfavorable region and interacts with the positively charged stable helical domain that follows.

To test for the above mentioned model we used Leu4 and Leu9, natural yeast proteins that possess negatively charged amino acids in the most N-terminal segment of their leader sequence. NMR spectroscopy was used to show that, as expected, the peptide corresponding to the leader of Leu4 formed a stable alpha  helix. Before the yeast genome was obtained, only one form of the enzyme was known. The second gene product, Leu9 (which has 80% identity with Leu4), was identified later, but the protein was not characterized (15). First, in vitro import was used to show that Leu9, like Leu4, is a mitochondrial protein. Unexpectedly, the Leu4 precursor was not imported under conditions that allowed the Leu9 to be imported. A chimeric protein was made by fusing the purported leader segment of Leu4 or Leu9 to mature ALDH. When the first 21 residues of Leu4 (those found to be helical) were used, no import was found. But if the first 31 residues were used, import could occur. In a similar manner, the first 30 residues of Leu9 could import of mature ALDH, whereas the first 20 could not.

We were concerned with the fact that we could not obtain import with Leu4 as others had reported (14, 24). The previously published experimental procedure used total RNA for translation (14, 24) and antibodies (33) to precipitate imported proteins. In contrast, the current experiments all used pure cDNAs. Given the fact that the Leu9 gene was not identified at the time the initial experiments were performed and that total RNA was used, it is possible that the Leu9 gene product was detected rather than the product for Leu4. We conclude that under the in vitro experimental procedures used in this study, Leu4 was not imported to the mitochondrial matrix space.

The N-terminal portion of Leu4 and Leu9 provided a 21- or 20-residue helix thought to provide the essential structure for a region that does not possess a net positive charge. Our data revealed that the N-terminal region containing the negative charges formed the most stable part of the helix. However, the region that seemed most responsible for import of Leu4 or Leu9 was not the most helical portion of the leader, because the targeting signal seemed to appear after residue 11. It is generally accepted that before import to the mitochondria preproteins remain unfolded. A proteolysis assay was performed with the TNT-synthesized Leu4. Proteinase K proteolysis results indicated that the Leu4 was more resistant to digestion than was the Leu4(Delta 1-11) protein (data not shown). A possible explanation for this phenomenon is that the most N-terminal 11 residues of Leu4 contribute to the overall structure of the protein. Without these residues the native folding of these proteins might be changed, as reflected by the instability of the protein and the proteinase K sensitivity. Therefore, the proteolysis results showed that Leu4 was folded and hence not imported to the mitochondria in an in vitro experiment. Some proteins that fold rapidly have been found to be imported to the mitochondria by a co-translocation pathway. The pALDH leader fused to green fluorescent protein (a protein that folds rapidly) was shown to be imported to the mitochondrial matrix space by a co-translational pathway (34). Similarly, in vivo Leu4 could be translocated to the mitochondria through a co-translational pathway.

The acid-chain hypothesis was based on the fact that the leader possessed positive charges at its N terminus, which could be important for binding with the TOM complex. However, it has been shown that the leader sequence of pALDH binds TOM20 through hydrophobic interactions (29). TOM20 is the first receptor to be recognized by leader sequence and does not appear to depend on the positive charges of the leader sequence. It is possible that the other TOM protein may interact with positive charges present in leader sequence. Here we show that if the N-terminal segment is not positive, the translocator appears to recognize not the N termini but the region of the leader sequences that are positively charged. It has been shown that inner and intermembrane space proteins use an internal signal after the most N-terminal matrix space targeting signal (35, 36). How the first 11 residues in the case of Leu4 enter the translocation pore cannot be explained at this time. If the model of import were co-translational and not post-translational as we suggest, then it is possible that an unknown cytosolic factor is necessary to insert the proteins into the TOM apparatus. Although the data presented in this study cannot explain how the precursor with negative charges interacts with the TOM proteins, they do show that the information for import need not lie at the very N terminus of the precursor protein.

    ACKNOWLEDGEMENT

We thank Professor Guntar Kohlhaw, Purdue University, for his help and advice when working with IPMS.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants AA10795 and GM 53269. This is journal paper 17006 from the Purdue University Agriculture Experiment Station.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.

The on-line version of this article (available at http://www.jbc.org) contains Table 1.

Dagger These authors contributed equally to this work.

§ To whom correspondence should be addressed: Dept. of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, IN 47907-2063. Tel.: 765-494-1650, Fax: 765-494-7897; E-mail: Hweiner@purdue.edu.

Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M212743200

    ABBREVIATIONS

The abbreviations used are: TOM, translocase of the mitochondrial outer membrane; pALDH, precursor aldehyde dehydrogenase; IPMS, isopropylmalate synthase; MPP, mitochondrial processing peptidase; TFE, trifluoroethanol; long form, preprotein containing the leader plus the mature part of IPMS; short form, IPMS containing only the mature portion of IPMS; IPMS4(1-21), a synthetic peptide corresponding to the N-terminal 21 residues of Leu4; NOESY, Nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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