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Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-2063, USA
(RECEIVED July 26, 2006; FINAL REVISION August 31, 2006; ACCEPTED August 31, 2006)
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Abstract |
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-ATPase that were important for Tom20 binding were found at the C-terminus of the leader. In contrast, it was the leucines in the N-terminus of the leader of ornithine transcarbamylase that were essential for binding. Modeling the peptides to the structure of Tom20 showed that the hydrophobic residues from the three proteins could all fit into the hydrophobic binding pocket. The mutants of pALDH that did not bind to Tom20 were still imported in vivo in transformed HeLa cells or in vitro into isolated mitochondria. In contrast, the mutant from pOTC was imported less well (
50%) while the mutant from F1-ATPase was not imported to any measurable extent. Binding to Tom20 might not be a prerequisite for import; however, it also is possible that import can occur even if binding to a receptor component is poor, so long as the leader binds tightly to another component of the translocator. Keywords: Tom20; bacteria two-hybrid; leader sequence; mitochondria protein import
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Although hundreds of leader peptides recognize the TOM and TIM complexes, no amino acid sequence similarity exists among the leaders. However, it has been noted that the leader peptides are often rich in positive charges and have the ability to form an amphiphilic helix (von Heijne 1986; Endo et al. 1989; Karslake et al. 1990; Thornton et al. 1993; Hammen 1994; Wang and Weiner 1994; Jarvis et al. 1995; Neupert 1997). For example, the leader from rat aldehyde dehydrogenase (pALDH) is a 19–amino-acid peptide that can form a helix–linker–helix conformation in trifluoro ethanol (Karslake et al. 1990; Thornton et al. 1993). This leader peptide contains two arginine residues in each half of the sequence (Fig. 1). When the N-terminal arginine residues were substituted by neutral glutamine residues (R3Q,R10Q double mutant), import was severely affected (Hammen et al. 1996). This was not found when the C-terminal arginine residues were mutated. Removing the linker causes the remaining residues to form a continuous helix (Thornton et al. 1993) that compensated for the loss of positive charges so that the R3Q,R10Q double mutant without linker was imported 60% compared to the native leader (Hammen et al. 1996).
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In the present study, we employed a bacteria two-hybrid assay to determine if modified leader peptides can bind to Tom20. Both leucine to alanine and arginine to glutamine mutants of pALDH leader peptide were studied. It will be shown that the bacteria two-hybrid system is a convenient way to screen for the ability of a modified leader to bind to a Tom component, but the ability to bind is not predictive as to whether or not the modified leader will function in import. pOTC and pF1-ATPase leader peptides and their mutants were also included in this study to determine if the proposed Tom20 binding region is critical for import. Lastly, using the coordinates for Tom20 it was possible to determine whether the leaders from other peptides bind in a similar manner as does pALDH.
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Results |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Positive charges
To assess the ability of the R3Q,R10Q double mutant of the pALDH leader that was imported poorly (Hammen et al. 1996), to bind to Tom20, a bacterial two-hybrid assay was employed. Positive controls were performed by using the peptide corresponding to native pALDH that has been shown to bind to Tom20 (Schleiff et al. 1999; Abe et al. 2000; Mukhopadhyay et al. 2002). A positive interaction was based upon observing a red colony on MacConkey plates (Fig. 2A-1). A negative control was a peptide that corresponded to the mature part of pALDH, residues 20–39, which was shown previously to not interact with Tom20 (Mukhopadhyay et al. 2002) or the peptide just corresponding to T18 of adenylate cyclase (Fig. 2A-1). These peptides did not show red colonies on the plates in the two-hybrid assay. It was not possible to quantify binding, but the intensity of the color was an indication of the strength of binding. Five R/Q mutant peptides were used. All four single-point mutants (R3Q, R10Q, R14Q, and R17Q) interacted as well as the native leader. The double mutant R14Q,17Q that was a functional import leader also bound. Of greater importance was finding that the double mutant R3Q,R10Q, which barely could be imported, also bound like the other peptides (data not shown). Thus, the inability of the latter construct to support import was not related to a lack of binding to Tom20.
Hydrophobic residues
The NMR model for the binding of the pALDH leader sequence to Tom20 leads one to conclude that the leader requires hydrophobic interactions with leucine residue located at the C-terminal domain of the leader (Fig. 2B-1; Abe et al. 2000). Consistent with this model, both the L2A and L6A mutant peptides were found to produce red colonies on MacConkey plates, showing that the N-terminal mutations did not affect the binding of the leader peptide to Tom20. The side chains of leucine 15, 18, and 19 of bound pALDH are located in the hydrophobic pocket of Tom20 (Fig. 2B-1). Mutating L19 to alanine resulted in a less intense red colony than did L2A or L6A, suggesting that L19A mutation disrupted hydrophobic interactions with Tom20, which can be seen in Figure 2B-2. Leucine 15 of the pALDH leader sequence is located within 4 Å from residues I74, L110, and T113 of Tom20. It can be anticipated that mutating L15 to alanine will destroy much of the hydrophobic interactions (Fig. 2B-2). Indeed, L15A mutant peptide did not produce any red colonies on MacConkey plates, showing that this residue is essential for Tom20 binding through hydrophobic interactions (Fig. 2A-2). The double mutant of L15A, L19A, as could be expected from Figure 2B-2, did not produce red colonies.
Tom20 binding to modified pOTC leader peptides as determined by bacteria two-hybrid assay
The pOTC leader peptide is 32 amino acids long, and a secondary structure prediction model shows that the N-terminal region can form an 
-helical conformation (Fig. 1). The structure of Tom20 bound with pOTC leader was not solved, but chemical shift data indicated that the N-terminal leucine residues might be involved in Tom20 binding ( Muto et al. 2001). To examine this, leucine residues at positions 5, 8, and 9 were all mutated to alanine to produce the triple mutant (L5A,L8A,L9A) and was employed in the bacteria two-hybrid assay. The native pOTC leader peptide produced red colonies indicating that it bound to Tom20. In contrast, the triple mutant leader produced a less intense red color, indicating that it bound poorly with Tom20. The result showed that the N-terminal leucine residues are important for Tom20 binding, just as were the C-terminal residues of pALDH, and binding can be detected by the bacteria two-hybrid assay.
In vitro import of mutant pALDH leaders into HeLa cell mitochondria
It could be rationalized that binding to Tom20 is a prerequisite for import, though a leader that binds to it still might not be imported. The ability of the modified leaders possessing L to A mutations to be imported into isolated mitochondria was determined. The L2A mutant was previously found to be imported into isolated mitochondria but with a much lower efficiency compared to the native leader (Hammen et al. 1999). In the present study, the L6A mutant was found to be imported less well than the native, in spite of it being able to bind to Tom 20 as shown in Figure 3A. Substitutions of leucines in the C-terminal domain of the leader led to some unexpected import results. The L19A mutant that bound weakly was imported, and the L15A mutant that did not bind well to Tom20 was also imported. These data show that the ability of a leader peptide to bind to Tom20 is not a predictor of import. L18A and L15A,L18A double mutant that were not used in bacteria two-hybrid assay were also imported efficiently (Fig. 3A). The import result is summarized in Figure 3B.
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Finding import of proteins in HeLa cell does not allow one to determine that the rates of import were same for each leader. It is possible that the mutant leaders were imported at a much lower rate than the native leader. To examine this, an ER signal peptide was fused to the C-terminus of native and mutant precursor proteins as we have previously done to establish a cotranslational import model (Mukhopadhyay et al. 2004), the idea being that if the pre-protein were free in cytosol it could ultimately be found in both mitochondria and ER. If the protein followed a cotranslational import it would be found only in mitochondria. Alternatively, it is possible that if a precursor protein coming off the ribosome bound poorly to Tom20, it could then go into the cytosol and be imported more slowly using a post-translational pathway. Slow import could allow the ER signal at the C-terminal end of the protein to bring the precursor to the ER. When L15A-EGFP-ER, L18A-EGFP-ER, and L15A,L18A-EGFP-ER were expressed in HeLa cells, only mitochondrial localization was observed just as was found with the native leader (Fig. 4A-1). To reconfirm mitochondrial localization, cells expressing L15A-EGFP-ER were stained with Mitotracker Red. Images from Mitotracker Red–stained mitochondria (Fig. 4A-2) were merged with images from mitochondria expressing the chimeric proteins, and the results verified that the chimeric protein was located in HeLa cell mitochondria. This finding verified that although the mutated leader peptides did not interact with Tom20 in the binding assay, they were imported as efficiently into mitochondria, as was the native leader.
In vivo import of the mutant pOTC and pF1 -ATPase leader into HeLa cell mitochondria
To determine if the reduced interaction of the mutated leader with Tom20 from the bacterial two-hybrid assay could affect import, the pOTC leader peptide, attached to EGFP, was employed in in vivo import in HeLa cells. The native leader was imported efficiently to mitochondria, as no fluorescence was found in cytosol. The cells expressing the mutant leader showed that the green fluorescence was not exclusively localized to the organelles, as some fluorescence was also found in the cytosol (Fig. 4B-1), suggesting that the mutant was not totally imported. The construct with dual leaders also produced some nonmitochondrial fluorescence.
It was shown by NMR study that the C-terminal region of yeast F1 -ATPase presequence might be involved in Tom20 binding. The potential hydrophobic residues of this leader peptide are tryptophan, cysteine, and methionine at positions 29, 32, and 33, respectively (Fig. 1). These residues were changed to alanine to disrupt Tom20 binding, and in vivo import efficiency was determined. The mutant leader fused with EGFP was not imported, while the native leader fused with EGFP was efficiently imported into HeLa cell mitochondria (Fig. 4B-2). An ER signal was attached to the C-terminus of mutant pF1-EGFP to make mutant pF1-EGFP-ER. This construct was transformed to HeLa cells and the expressed proteins were now only found in ER, suggesting that by preventing Tom20 interactions, the ability of the mutant leader for mitochondrial import was totally lost. The results also showed that unlike pALDH leader, the import of F1 -ATPase is strictly dependent on Tom20 binding.
Confocal microscopy of in vivo import of pALDH and pOTC leaders fused to EGFP
In addition to fluorescent microscopy, confocal microscopy was used to confirm the localization of the green fluorescence. L15A-EGFP-ER, L18A-EGFP-ER, and L15A,L18A-GFP-ER were used in the confocal study. Each protein, after expression, was found primarily in the mitochondria (Fig. 4C). EGFP-ER was used as a control and after expression was found only in ER. These results confirmed the fluorescent microscopy data that showed the mutated pALDH leaders that did not bind Tom20 well were imported efficiently into mitochondria. In contrast, although the mutant pOTC-EGFP was localized in mitochondria, substantial amounts of the fluorescence were also observed in cytosol (Fig. 4C). Native pOTC-EGFP, however, was found only in mitochondria (Fig. 4C). Again, the confocal microscopy image was consistent with what was found with fluorescent microscopy.
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Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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What is unique about mitochondrial leader sequences is that there is essentially no sequence identity or similarity between the hundreds of known leaders. Virtually all contain positive charges and, for a limited number whose structures have been determined, form short stable amphiphilic helices (Endo et al. 1989a,b; Karslake et al. 1990). The TOM proteins were found to possess patches of negative charges, so an "acid chain" hypothesis was presented (Schatz 1997; Komiya et al. 1998). It basically proposed that the driving force to bring the leader sequence from one TOM protein to the next was the increased binding due to electrostatic interactions. Data, though, have been presented to suggest that this might not be the major driving force (Nargang et al. 1998; Mukhopadhyay et al. 2003).
Investigators have used different approaches to investigate the role of the various amino acid residues in the leader. Often mutant leaders were produced and their ability to bind to the soluble portion of Tom20 was assessed (Brix et al. 1999). We too have used this approach (Schleiff et al. 1999). It was not known, though, if there is a correlation between the ability of a modified leader to bind Tom20 and its ability to serve as a leader sequence to bring a protein into mitochondria. We found a 12–amino acid peptide using phage display that bound tightly to Tom20 but did not function as a mitochondrial leader (Mukhopadhyay et al. 2005). It could be argued that a leader might bind poorly to one component of the TOM complex but could be passed to the next member so long as there was a net negative 
G for the entire process. If this were the case, then it is possible that the various TOM components might bind either to different portions of the leaders or with different affinities so measuring binding to Tom20 may not be indicative to the ability of a leader to be imported.
The binding of leader peptides to Tom20 have been studied using a NMR technique (Abe et al. 2000). First, they solved the structure of the binary complex with the leader from rat liver pALDH. Second, they used the same soluble portion of Tom20 but looked at chemical shifts of various residues of different peptides to predict which residues were important (Muto et al. 2001). We took advantage of these studies to investigate binding of mutated leaders to Tom20. Both of our studies pointed to the importance of hydrophobic interactions between the leaders and Tom20. Neither study found that salt bonds existed as would be necessary for the acid chain hypothesis.
Though both NMR studies showed that hydrophobic residues in the leader interacted with Tom20, the residues were not located in the same portion of different leader sequences. For pALDH, the important leucine residues were in the C-terminal domain, while those for pOTC were in the N-terminal domain. Similarly, hydrophobic residues in other leaders they investigated were differentially distributed as shown in Figure 1. To be discussed is the fact that it is possible to model the binding of each peptide to Tom20 to show that indeed there is a critical hydrophobic interaction between each leader and Tom20.
Here, we took advantage of a known bacterial two-hybrid system (Karimova et al. 1998) to assess leader binding to Tom20. In this system, the target proteins are placed at the N-terminal of T18 (one part of adenylate cyclase). The more classical yeast two-hybrid system has the target peptide cloned to the C-terminal of the DNA binding protein; hence, it would not mimic a precursor protein. Hybrid assays do not produce quantitative data but do allow for the rapid screening of potential binding. The intensity and time for color formation is a qualitative measure of binding.
To verify that the leucine residues identified from the NMR studies were indeed important for leader binding to Tom20, both mutant leaders from pALDH and pOTC were employed. The alanine mutants of either one, corresponding to leucine residues identified by NMR, did not produce color in the binding assay, indicative of no or weak binding. Mutations of other leucines did not affect the binding of those peptides to Tom20 in the two-hybrid assay system.
The EGFP constructs that possessed leader sequences from pALDH that did not appear to bind to Tom20 were all imported in vivo into HeLa cell mitochondria. Even by using a dual leader it appeared that the mutant leaders were imported as well as the native leader. Microscopy analysis of EGFP with the modified pOTC leader revealed that a portion of the fluorescence was not localized to mitochondria. Though not quantified, it can be estimated by visualization that as much as 90% of the pALDH construct was imported but perhaps just 50% of pOTC was imported. In contrast, mutation to the Tom20 binding region of F1 -ATPase leader produced a protein that was not imported.
The hydrophobic pocket in Tom20 is shown in Figure 2B-1. It appears that leucine 15 of pALDH leader is located within close proximity from threonine 113, leucine 110, and isoleucine 74 of Tom20. By replacing leucine 15 with alanine in the pALDH leader, much of the hydrophobic interaction would be destroyed (Fig. 2B-2). We cannot tell from our data whether the first 14 residues of the pALDH leader associate weakly with Tom20 prior to the leucine residue binding to the hydrophobic pocket.
Using the published structure of Tom20 bound with pALDH leader we modeled the binding of the other peptides that originally were employed in the NMR study. Since only chemical shift data were presented for non-pALDH leaders, it was not possible to know the actual structure of those peptides. We assumed that the other peptides would form the same helical structure as did the leader from ALDH. For each peptide, the interaction between the hydrophobic residues in the leader and the residues from Tom20 as shown in Figure 2B was maintained. Thus, it appears that there is critical hydrophobic residue in the leader that must fit into the hydrophobic domain on Tom20.
Previous studies from our laboratory have shown that for import to occur it was necessary to have a helix at the N-termini of the pALDH leader (Wang and Weiner 1993). The L15A mutant that still maintained its helical structure (unpublished circular dichroism studies) was imported even though it did not bind to Tom20. To explain why import did not correlate well with Tom20 binding we propose two propositions. The first is that the mutant simply bypasses Tom20 and uses an alternative import pathway. It is known that the mitochondrial carrier proteins, such as ATP/ADP carrier proteins, are imported in a Tom20-independent pathway (Rehling et al. 2003). These proteins, though, do not possess the typical N-terminal leader sequences; instead they possess internal targeting sequence. Surprisingly, Lithgow et al. (1994) showed that deletion of Tom20 was not lethal to yeast and suggested that other TOM components could substitute for Tom20. There are, however, no data in the literature to show that a mutation to a precursor protein that uses Tom20 can be made to bypass the receptor component.
An alternative suggestion is that the leaders actually bind to Tom20, some weakly, but then are bound more tightly to next component of the translocation apparatus using a different portion of the leader sequence. This implies that a mutant leader may interact weakly with Tom20, but, so long as it can be rapidly transferred to the next component of the translocator, it could be imported.
We show that the mutated pOTC still could bind weakly to Tom20. For pOTC, the Tom20 binding domain is the hydrophobic residues located in the N-terminal helical domain. This again shows the importance of helicity and hydrophobicity in Tom20 binding. Although the structure of pOTC leader bound with Tom20 is not known, it can be rationalized that removing these hydrophobic residues may cause the leader to dissociate rapidly from Tom20. However, since a significant amount of the mutant was imported, the bulk of the protein that bound weakly to Tom20 could have been transferred to the next TOM component, assuming that a different portion of the leader is involved in the next binding and that Tom20 was not bypassed.
Although the wild-type F1-ATPase was imported into HeLa cell mitochondria, the mutant was not imported to any measurable extent. We took advantage of the dual signal approach (Mukhopadhyay et al. 2004) to see if pF1-ATPase mutant that did not bind to Tom20 nor was imported into mitochondria could translocated to another organelle. The mutant dual leader construct, pF1-EGFP-ER, was expressed in HeLa cells and found in the ER. Finding that the protein was located primarily in the ER shows that this protein, unlike pALDH, could not be imported into mitochondria once it went into the cytosol. We cannot explain why mutant F1-ATPase behaved differently than did mutant pALDH or pOTC. One possible reason is that the same domain on the former was also needed to bind to other receptor components.
It will be necessary to determine the binding of mutants to the various TOM and TIM components to determine if indeed different segments of the leaders interact with the various translocator components. Using the two-hybrid assay it is possible now to use different leaders and examine if the Tom20 binding motif is necessary for import. An advantage of using the bacterial two-hybrid assay to assess binding is that pure peptides or proteins are not required, thus making it easier to use many recombinantly expressed mutants of the various leaders.
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Materials and methods |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Construction of plasmids for bacteria two-hybrid
Gene encoding Tom20(2–28) was fused to the C-terminus of the T25 in the vector pKT25 to make the fusion protein Tom20-T25. To detect the expression level of fusion protein by Western blot, a c-myc tag was inserted between them. cDNA encoding pALDH(1–19), pOTC(1–36), and mutant peptides from pALDH and pOTC were cloned to the N-terminus of the T18 of vector pUT18. For control experiments, peptides corresponding to the amino acids 20–39 of pALDH leader were fused to T18. T18 alone was also used as a control. To detect the expression level by Western blot, a flexible peptide linker (SGGGGSGG) followed by HA-tag was inserted between them. The mutations (R to Q and L to A) were performed by PCR.
Vector construction for in vitro and in vivo import
pALDH was originally cloned into pGEM3Z. Each mutation (L to A) in the plasmid was performed by PCR. These constructs were used for in vitro import studies. For in vivo import studies, the cDNA corresponding to the leader sequence and some mature portions of pALDH and pOTC were fused to the cDNA corresponding to EGFP to make pALDH-EGFP and pOTC-EGFP, respectively. These plasmids and the plasmids with ER constructs were described in a previous study (Mukhopadhyay et al. 2004). Each construct was confirmed by the DNA Sequencing Center at Purdue University.
In vitro import
In vitro import was performed as described previously (Mukhopadhyay et al. 2005). Briefly, TNT (Promega) synthesized precursor proteins were incubated with mitochondria isolated from HeLa cells for 30 min at 30°C. After incubation, proteinase K was added to digest nonimported proteins. Mitochondria were centrifuged and were dissolved in SDS-PAGE buffer. 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 Molecular Dynamics Inc. The amount of import was estimated by comparing the radioactivity of imported proteins to total radioactivity in translated products.
Cell line and culture conditions
HeLa cells were provided by Dr. Steven Broyles (Department of Biochemistry, Purdue University). The HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum, 10 µg/mL gentamicin at 37°C, and 5% CO2. Transfection was performed as described previously (Mukhopadhyay et al. 2004).
Direct observation of EGFP fluorescence in cultured cells
HeLa cells were plated on coverslips in six-well plates in 3 mL of growth medium (DMEM plus 10% calf serum) 1 d before transfection. The cells were 40%–80% confluent on the day of transfection. The cells were transfected with 1 µg of plasmid DNA at 37°C using the FuGene 6 Transfection Reagent from Roche. Between 16 h and 24 h after transfection, the cells were washed twice with phosphate-buffered saline (PBS), fixed on the coverslips with 4% paraformaldehyde in PBS at room temperature for 30 min, and washed twice with PBS. Cells were observed with an Olympus BX60 fluorescence microscope. When mitochondria were to be stained, 20 nM MitoTracker Red (Molecular Probes) was added to the medium and incubated for 20 min before fixation.
Confocal microscopy
When cells were visualized under confocal microscopy, the cells were grown in four-well chambers (Lab-Tek II chambered cover glass) and were directly observed through the confocal microscope.
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Footnotes |
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Abbreviations: pALDH, precursor rat aldehyde dehydrogenase; pOTC, precursor human ornithine trans carbamylase; TOM, translocase outer membrane; TIM, translocase inner membrane; EGFP, enhanced green fluorescent protein; pALDH-EGFP, the leader sequence of pALDH plus 20 mature amino acids fused to EGFP; pOTC-EGFP, the leader sequence of pOTC plus five mature amino acids fused to EGFP; F1 ATPase, the leader sequence of F1 plus two mature amino acids were fused to EGFP; F1 ATPase-EGFP mutant, F1 -EGFP W29A, C32A, M33A; EGFP-ER, the ER signal is fused to the C-terminus of EGFP.
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062462006.
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Acknowledgments |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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References |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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Brix, J., Rudiger, S., Bukau, B., Schneider-Mergener, J., and Pfanner, N. 1999. Distribution of binding sequences for the mitochondrial import receptors Tom20, Tom22, and Tom70 in a presequence-carrying preprotein and a non-cleavable preprotein. J. Biol. Chem. 274: 16522–16530.
Endo, T., Eilers, M., and Schatz, G. 1989a. Binding of a tightly folded artificial mitochondrial precursor protein to the mitochondrial outer membrane involves a lipid-mediated conformational change. J. Biol. Chem. 264: 2951–2956.
Endo, T., Shimada, I., Roise, D., and Inagaki, F. 1989b. N-terminal half of a mitochondrial presequence peptide takes a helical conformation when bound to dodecylphosphocholine micelles: A proton nuclear magnetic resonance study. J. Biochem. 106: 396–400.
Goping, I.S., Millar, D.G., and Shore, G.C. 1995. Identification of the human mitochondrial protein import receptor, huMas20p. Complementation of mas20 in yeast. FEBS Lett. 373: 45–50.[CrossRef][Medline]
Hammen, P.K., Gorenstein, D.G., and Weiner, H. 1994. Structure of the signal sequences for two mitochondrial matrix proteins that are not proteolytically processed upon import. Biochemistry 33: 8610–8617.[CrossRef][Medline]
Hammen, P.K., Waltner, M., Hahnemann, B., Heard, T.S., and Weiner, H. 1996. The role of positive charges and structural segments in the presequence of rat liver aldehyde dehydrogenase in import into mitochondria. J. Biol. Chem. 271: 21041–21048.
Hammen, P.K., Heard, T.S., Waltner, M., and Weiner, H. 1999. The loss in hydrophobic surface area resulting from a Leu to Val mutation at the N-terminus of the aldehyde dehydrogenase presequence prevents import of the protein into mitochondria. Protein Sci. 8: 890–896.[Abstract]
Hoogenraad, N.J., Ward, L.A., and Ryan, M.T. 2002. Import and assembly of proteins into mitochondria of mammalian cells. Biochim. Biophys. Acta 1592: 97–105.[Medline]
Igura, M., Ose, T., Obita, T., Sato, C., Maenaka, K., Endo, T., and Kohda, D. 2005. Crystallization and preliminary X-ray analysis of mitochondrial presequence receptor Tom20 in complexes with a presequence from aldehyde dehydrogenase. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 61: 514–517.[CrossRef]
Jarvis, J.A., Ryan, M.T., Hoogenraad, N.J., Craik, D.J., and Høj, P.B. 1995. Solution structure of the acetylated and noncleavable mitochondrial targeting signal of rat chaperonin 10. J. Biol. Chem. 270: 1323–1331.
Jensen, R.E. and Dunn, C.D. 2002. Protein import into and across the mitochondrial inner membrane: Role of the TIM23 and TIM22 translocons. Biochim. Biophys. Acta 1592: 25–34.[Medline]
Johnston, A.J., Hoogenraad, J., Dougan, D.A., Truscott, K.N., Yano, M., Mori, M., Hoogenraad, N.J., and Ryan, M.T. 2002. Insertion and assembly of human tom7 into the preprotein translocase complex of the outer mitochondrial membrane. J. Biol. Chem. 277: 42197–42204.
Karimova, G., Pidoux, J., Ullmann, A., and Ladant, D. 1998. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc. Natl. Acad Sci. 98: 5752–5756.
Karslake, C., Piotto, M., Pak, Y.K., Weiner, H., and Gorenstein, D.G. 1990. 2D NMR and structural model for a mitochondrial signal peptide bound to a micelle. Biochemistry 29: 9872–9878.[CrossRef][Medline]
Koehler, C.M. 2004. New developments in mitochondrial assembly. Annu. Rev. Cell Dev. Biol. 20: 309–335.[CrossRef][Medline]
Komiya, T., Rospert, S., Koehler, C., Looser, R., Schatz, G., and Mihara, K. 1998. Interaction of mitochondrial targeting signals with acidic receptor domains along the protein import pathway: Evidence for the "acid chain" hypothesis. EMBO J. 17: 3886–3898.[CrossRef][Medline]
Lithgow, T., Junne, T., Wachter, C., and Schatz, G. 1994. Yeast mitochondria lacking the two import receptors Mas20p and Mas70p can efficiently and specifically import precursor proteins. J. Biol. Chem. 269: 15325–15330.
Mukhopadhyay, A., Avramova, L.V., and Weiner, H. 2002. Tom34 unlike Tom20 does not interact with the leader sequences of mitochondrial precursor proteins. Arch. Biochem. Biophys. 400: 97–104.[CrossRef][Medline]
Mukhopadhyay, A., Heard, T.S., Wen, X., Hammen, P.K., and Weiner, H. 2003. Location of the actual signal in the negatively charged leader sequence involved in the import into the mitochondrial matrix space. J. Biol. Chem. 278: 13712–13718.
Mukhopadhyay, A., Ni, L., and Weiner, H. 2004. A co-translational model to explain the in vivo import of proteins into HeLa cell mitochondria. Biochem. J. 382: 385–392.[CrossRef][Medline]
Mukhopadhyay, A., Ni, L., Yang, C.S., and Weiner, H. 2005. Bacterial signal peptide recognizes HeLa cell mitochondrial import receptors and functions as a mitochondrial leader sequence. Cell. Mol. Life Sci. 62: 1890–1899.[CrossRef][Medline]
Muto, T., Obita, T., Abe, Y., Shodai, T., Endo, T., and Kohda, D. 2001. NMR identification of the Tom20 binding segment in mitochondrial presequences. J. Mol. Biol. 306: 137–143.[CrossRef][Medline]
Nargang, F.E., Rapaport, D., Ritzel, R.G., Neupert, W., and Lill, R. 1998. Role of the negative charges in the cytosolic domain of TOM22 in the import of precursor proteins into mitochondria. Mol. Cell. Biol. 18: 3173–3181.
Neupert, W. 1997. Protein import into mitochondria. Annu. Rev. Biochem. 66: 863–917.[CrossRef][Medline]
Pfanner, N. and Geissler, A. 2001. Versatility of the mitochondrial protein import machinery. Nat. Rev. Mol. Cell Biol. 2: 339–349.[CrossRef][Medline]
Rehling, P., Pfanner, N., and Meisinger, C. 2003. Insertion of hydrophobic membrane proteins into the inner mitochondrial membrane–a guided tour. J. Mol. Biol. 326: 639–657.[CrossRef][Medline]
Rehling, P., Brandner, K., and Pfanner, N. 2004. Mitochondrial import and the twin-pore translocase. Nat. Rev. Mol. Cell Biol. 5: 519–530.[CrossRef][Medline]
Schatz, G. 1996. The protein import system of mitochondria. J. Biol. Chem. 271: 31763–31766.
Schatz, G. 1997. Just follow the acid chain. Nature 388: 121–122.[CrossRef][Medline]
Schleiff, E., Heard, T.S., and Weiner, H. 1999. Positively charged residues, the helical conformation and the structural flexibility of the leader sequence of pALDH are important for recognition by hTom20. FEBS Lett. 461: 9–12.[CrossRef][Medline]
Suzuki, H., Okazawa, Y., Komiya, T., Saeki, K., Mekada, E., Kitada, S., Ito, A., and Mihara, K. 2000. Characterization of rat TOM40, a central component of the preprotein translocase of the mitochondrial outer membrane. J. Biol. Chem. 275: 37930–37936.
Suzuki, H., Maeda, M., and Mihara, K. 2002. Characterization of rat TOM70 as a receptor of the preprotein translocase of the mitochondrial outer membrane. J. Cell Sci. 115: 1895–1905.
Taylor, R.D. and Pfanner, N. 2004. The protein import and assembly machinery of the mitochondrial outer membrane. Biochim. Biophys. Acta 1658: 37–43.[Medline]
Thornton, K., Wang, Y., Weiner, H., and Gorenstein, D.G. 1993. Import, processing, and two-dimensional NMR structure of a linker-deleted signal peptide of rat liver mitochondrial aldehyde dehydrogenase. J. Biol. Chem. 268: 19906–19914.
Truscott, K.N., Brandner, K., and Pfanner, N. 2003. Mechanisms of protein import into mitochondria. Curr. Biol. 13: 326–337.
von Heijne, G. 1986. Mitochondrial targeting sequences may form amphiphilic helices. EMBO J. 5: 1335–1342.[Medline]
Wang, Y. and Weiner, H. 1993. The presequence of rat liver aldehyde dehydrogenase requires the presence of an -helix at its N-terminal region which is stabilized by the helix at its C termini. J. Biol. Chem. 268: 4759–4765.
Wang, Y. and Weiner, H. 1994. Evaluation of electrostatic and hydrophobic effects on the interaction of mitochondrial signal sequences with phospholipid bilayers. Biochemistry 33: 12860–12867.[CrossRef][Medline]
Yano, M., Kanazawa, M., Terada, K., Takeya, M., Hoogenraad, N., and Mori, M. 1998. Functional analysis of human mitochondrial receptor Tom20 for protein import into mitochondria. J. Biol. Chem. 273: 26844–26851.
Yano, M., Hoogenraad, N., Terada, K., and Mori, M. 2000. Identification and functional analysis of human Tom22 for protein import into mitochondria. Mol. Cell. Biol. 20: 7205–7213.
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