Investigations on the in vitro import ability of mitochondrial precursor proteins synthesized in wheat germ transcription-translation extract

Mitochondrial precursor proteins synthesized in rabbit reticulocyte lysate (RRL) are readily imported into mitochondria, whereas the same precursors synthesized in wheat germ extract (WGE) fail to be imported. We have investigated factors that render import incompetence from WGE. A precursor that does not require addition of extramitochondrial ATP for import, the F(A)d ATP synthase subunit, is imported from WGE. Import of chimeric constructs between precursors of the F(A)d protein and alternative oxidase (AOX) with switched presequences revealed that the mature domain of the F(A)d precursor defines the import competence in WGE as only the construct containing the presequence of AOX and mature portion of F(A)d (pAOX-mF(A)d) could be imported. Import competence of F(A)d and pAOX-mF(A)d correlated with solubility of these precursors in WGE, however, solubilization of import-incompetent precursors with urea did not restore import competence. Addition of RRL to WGE-synthesized precursors did not stimulate import but addition of WGE to the RRL-synthesized precursors or to the over-expressed mitochondrial precursor derived from the F1beta ATP synthase precursor inhibited import into mitochondria. The dual-targeted glutathione reductase precursor synthesized in WGE was imported into chloroplasts, but not into mitochondria. Antibodies against the 14-3-3 guidance complex characterized for chloroplast targeting were able to immunoprecipitate all of the precursors tested except the F(A)d ATP synthase precursor. Our results point to the conclusion that the import incompetence of WGE-synthesized mitochondrial precursors is not presequence dependent and is a result of interaction of WGE inhibitory factors with the mature portion of precursor proteins.     

Identification of Signals Required for Import of the Soybean FAd Subunit of ATP Synthase into Mitochondria

The requirements for protein import into mitochondria was investigated by using the targeting signal of the F(A)d subunit of soybean mitochondrial ATP synthase attached to two different passenger proteins, its native passenger and soybean alternative oxidase. Both passenger proteins are soybean mitochondrial proteins. Changing hydrophobic residues at positions -24:25 (Phe:Leu), -18:19 (Ile:Leu) and -12:13 (Leu:Ile) of the 31 amino acid cleavable presequence gave more than 50% inhibition of import with both passenger proteins. Some other residues in the targeting signal played a more significant role in targeting of one passenger protein compared to another. Notably changing positive residues (Arg, Lys) had a greater inhibitory affect on import with the native passenger protein, i.e. greater inhibition of import with F(A)d mature protein was observed compared to when alternative oxidase was the mature protein. When using chimeric passenger proteins it was shown that the nature of the mature protein can greatly affect the targeting properties of the presequence. In vivo investigations of the targeting presequence indicated that the presequence of 31 amino acids could not support import of GFP as a passenger protein. However, fusion of the full-length F(A)d coding sequence to GFP did result in mitochondrial localisation of GFP. Using the latter fusion we confirmed the critical role of hydrophobic residues at positions -24:25 and -18:19. These results support the proposal that core mitochondrial targeting features exist in all presequences, but that additional features exist. These features may not be evident with all passenger proteins.     

NMR solution structure of the mitochondrial F1beta presequence from Nicotiana plumbaginifolia

We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F1beta subunit from Nicotiana plumbaginifolia. A general method for purification of presequences is presented. The method is based on overexpression of a mutant precursor containing a methionine residue introduced at the processing site, followed by CNBr-cleavage and purification of the presequence on a cation-exchange column. The F1beta presequence, 53 amino acid residues long, retained its native properties as evidenced by inhibition of in vitro mitochondrial import and processing at micromolar concentrations. CD spectroscopy revealed that the F1beta presequence formed an alpha-helical structure in membrane mimetic environments such as SDS and DPC micelles (approximately 50% alpha-helix), and in acidic phospholipid bicelles (approximately 60% alpha-helix). The NMR solution structure of the F1beta presequence in SDS micelles was determined on the basis of 518 distance and 21 torsion angle constraints. The structure was found to contain two helices, an N-terminal amphipathic alpha-helix (residues 4-15) and a C-terminal alpha-helix (residues 43-53), separated by a largely unstructured 27 residue long internal domain. The N-terminal amphipathic alpha-helix forms the putative Tom20 receptor binding site, whereas the C-terminal alpha-helix is located upstream of the mitochondrial processing peptidase cleavage site.     

Peptide library approach with a disulfide tether to refine the Tom20 recognition motif in mitochondrial presequences

Many mitochondrial matrix and inner-membrane proteins are synthesized in the cytosol as precursor proteins with an N-terminal presequence, and are imported into the mitochondria. Although no distinct sequence homology has been found among mitochondrial presequences, Tom20, a general import receptor in the outer mitohcondrial membrane, binds to presequences, and distinguishes mitochondrial proteins from non-mitochonrial proteins. The recently determined structure of the cytosolic domain of Tom20 (DeltaTom20) in a complex with the presequence of rat aldehyde dehydrogenase (ALDH) showed that a short stretch of the presequence forms an amphiphilic helix, and its hydrophobic surface interacts with the hydrophobic-binding groove of Tom20. The following NMR analyses revealed a common five-residue pattern for Tom20 binding in five different presequences. To refine the common amino acid motif for the recognition by Tom20, we introduced a new peptide library approach in this study: we prepared a mixture of ALDH presequence variants, tethered these peptides to DeltaTom20 in a competitive manner by an intermolecular disulfide bond, and determined the relative affinities by MALDI-TOF mass spectrometry. We successfully deduced a refined, common motif for the recognition by Tom20, and found that the segment consisting of residues 14-20 of the ALDH presequence was locally optimized in the sequence space, with respect to Tom20 binding.     

Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with a cleavable N-terminal presequence and are imported into mitochondria. We report here the NMR structure of a general import receptor, rat Tom20, in a complex with a presequence peptide derived from rat aldehyde dehydrogenase. The cytosolic domain of Tom20 forms an all alpha-helical structure with a groove to accommodate the presequence peptide. The bound presequence forms an amphiphilic helical structure with hydrophobic leucines aligned on one side to interact with a hydrophobic patch in the Tom20 groove. Although the positive charges of the presequence are essential for import ability, presequence binding to Tom20 is mediated mainly by hydrophobic rather than ionic interactions.     

Functions of outer membrane receptors in mitochondrial protein import

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins and are imported into mitochondria. The targeting signals for mitochondria are encoded in the presequences or in the mature parts of the precursor proteins, and are decoded by the receptor sites in the translocator complex in the mitochondrial outer membrane. The recently determined NMR structure of the general import receptor Tom20 in a complex with a presequence peptide reveals that, although the amphiphilicity and positive charges of the presequence is essential for the import ability of the presequence, Tom20 recognizes only the amphiphilicity, but not the positive charges. This leads to a new model that different features associated with the mitochondrial targeting sequence of the precursor protein can be recognized by the mitochondrial protein import system in different steps during the import.     

Active unfolding of precursor proteins during mitochondrial protein import.

Precursor proteins made in the cytoplasm must be in an unfolded conformation during import into mitochondria. Some precursor proteins have tightly folded domains but are imported faster than they unfold spontaneously, implying that mitochondria can unfold proteins. We measured the import rates of artificial precursors containing presequences of varying length fused to either mouse dihydrofolate reductase or bacterial barnase, and found that unfolding of a precursor at the mitochondrial surface is dramatically accelerated when its presequence is long enough to span both membranes and to interact with mhsp70 in the mitochondrial matrix. If the presequence is too short, import is slow but can be strongly accelerated by urea-induced unfolding, suggesting that import of these 'short' precursors is limited by spontaneous unfolding at the mitochondrial surface. With precursors that have sufficiently long presequences, unfolding by the inner membrane import machinery can be orders of magnitude faster than spontaneous unfolding, suggesting that mhsp70 can act as an ATP-driven force-generating motor during protein import.     

Functional Analysis of the N-Terminal Prepeptides of Watermelon Mitochondrial and Glyoxysomal Malate Dehydrogenases*

Mitochondrial and glyoxysomal malate dehydrogenase (mMDH; gMDH; L-malate: NAD⁺ oxidoreductase; EC 1.1.1.37) of watermelon (Citrullus vulgaris) cotyledons are synthesized with N-terminal cleavable presequences which are shown to specify sorting of the two proteins. The two presequences differ in length (27 or 37 amino acids) and primary structure. Precursor proteins of the two isoenzymes with site-directed mutations in their presequences and hybrid precursor proteins with reciprocally exchanged presequences were analyzed for proper import using two approaches, namely in vitro using isolated watermelon organelles or in vivo after synthesis in the heterologous host, Hansenula polymorpha. The mitochondrial presequence is essential and sufficient to target the mature glyoxysomal isoenzyme into mitochondria (Gietl et al., 1994). As to the function of the mitochondrial presequence a substitution of ^?3R (considered important for one step precursor cleavage in yeast and mammals) with ^?3L permitted import into mitochondria but cleavage of the transit peptide and conversion into active mature enzyme was impeded. Substitution of ^?13R^?12S (in a sequence reminiscent of the octapeptide motif serving as a substrate for the mammalian and yeast intermediate peptidase) into ^?13L¹²F permitted mitochondrial import and processing like the wild type transit peptide. Purified rat mitochondrial processing protease, which can effect single step cleavage of mitochondrial protein precursors, cleaves in vitro translated watermelon mMDH precursor into its mature form. The glyoxysomal presequence is essential and sufficient to target the mature mitochondrial isoenzyme into peroxisomes of Hansenula polymorpha, but these peroxisomes lack a processing enzyme to cleave the presequence (Gietl et al., 1994). We here show that isolated watermelon organelles also import the hybrid proteins in vitro and process the glyoxysomal presequence. Site directed mutations within the conserved RI-X₅-HL-motif impede efficiency of import and cleavage by watermelon organelles.     

NMR identification of the Tom20 binding segment in mitochondrial presequences

Many mitochondrial proteins are synthesized in the cytosol as precursors with N-terminal presequences, and are imported into mitochondria with the aid of translocator protein complexes containing presequence-binding proteins. Tom20, a receptor protein which functions in an early step of the mitochondrial protein import, recognizes presequences with divergent amino acid sequences. Here, we report the identification of the segments involved in binding to Tom20 in mitochondrial presequences. We monitored the chemical shift perturbation of the NMR signals of five different 15N-labeled presequence peptides by the addition of the cytosolic receptor domain of rat or yeast Tom20. The perturbed segments occupy different positions, either near the N terminus or at the C terminus, in the presequences. Spin label experiments revealed that this is not due to different orientations of the presequence peptides bound to Tom20. The results presented here will offer a starting point to perform detailed analyses of Tom20-binding elements by systematic amino acid replacements.     

Discrete mutations in the presequence of potato formate dehydrogenase inhibit the in vivo targeting of GFP fusions into mitochondria

Most mitochondrial proteins are encoded by the nucleus, translated in the cytosol, and imported. Mitochondrial precursors generally contain their targeting information in a cleavable N-terminal presequence, which is rich in hydroxylated and positively charged residues and can form amphiphilic alpha-helices. We report the in vivo targeting of green fluorescent protein (GFP) by the FDH presequence, as well as several truncated or mutated variants. Some of these mutations modify the amphiphilicity of the predicted alpha-helix. The removal of the first two residues abolishes import and some single amino acid mutations strongly inhibit import. Such strong effects on import had not been observed in similar studies on other plant mitochondrial presequences, suggesting that the FDH presequence is a particularly good model for functional studies.     

Transport of proteins into yeast mitochondria

The amino-terminal sequences of several imported mitochondrial precursor proteins have been shown to contain all the information required for transport to and sorting within mitochondria. Proteins transported into the matrix contain a matrix-targeting sequence. Proteins destined for other submitochondrial compartments contain, in addition, an intramitochondrial sorting sequence. The sorting sequence in the cytochrome c1 presequence is a stop-transport sequence for the inner mitochondrial membrane. Proteins containing cleavable presequences can reach the intermembrane space by either of two pathways: (1) Part of the presequence is transported into the matrix; the attached protein, however, is transported across the outer but not the inner membrane (eg, the cytochrome c1 presequence). (2) The precursor is first transported into the matrix; part of the presequence is then removed, and the protein is reexported across the inner membrane (eg, the precursor of the iron-sulphur protein of the cytochrome bc1 complex). Matrix-targeting sequences lack primary amino acid sequence homology, but they share structural characteristics. Many DNA sequences in a genome can potentially encode a matrix-targeting sequence. These sequences become active if positioned upstream of a protein coding sequence. Artificial matrix-targeting sequences include synthetic presequences consisting of only a few different amino acids, a known amphiphilic helix found inside a cytosolic protein, and the presequence of an imported chloroplast protein. Transport of proteins across mitochrondrial membranes requires a membrane potential, ATP, and a 45-kd protein of the mitochondrial outer membrane. The ATP requirement for import is correlated with a stable structure in the imported precursor molecule. We suggest that transmembrane transport of a stably folded precursor requires an ATP-dependent unfolding of the precursor protein.     

Import into mitochondria of precursors containing hydrophobic passenger proteins: pretreatment of precursors with urea inhibits import

We have studied the import into isolated yeast mitochondria of three hydrophobic passenger proteins attached to the N-terminal cleavable presequence of mitochondrial ATPase subunit 9 from Neurospora crassa. One natural precursor (pN9) contained N. crassa subunit 9; two chimaeric precursors, N9L/Y8-1 and N9L/Y9-2, respectively contained yeast mitochondrial ATPase subunits 8 and 9. In the absence of urea, pN9 and N9L/Y8-1 are imported efficiently but N9L/Y9-2 is not imported. After pretreatment of precursors in 4 M urea, binding of pN9 to mitochondria is marginally affected while its import is substantially inhibited; the binding to mitochondria of chimaeric proteins, N9L/Y8-1 and N9L/Y9-2, is greatly enhanced but no import is observed. This behaviour of import precursors containing hydrophobic passenger proteins is contrasted with that of a hydrophilic chimaeric precursor pCOXIV-DHFR, whose binding and import are enhanced by pretreatment with a high concentration of urea (8 M). The import of N9L/Y8-1 is very sensitive to the presence of low concentrations of urea in the import reaction mixture, and is abolished above 0.5 M urea although precursor binding to mitochondria is increased. By contrast, neither the import nor binding of pCOXIV-DHFR is affected directly by urea up to 0.8 M. These deleterious effects of urea on import of the chimaeric precursors N9L/Y8-1 and N9L/Y9-2 are interpreted in terms of a non-productive binding of these precursors to mitochondria, brought about by exposure of their hydrophobic domains resulting from urea unfolding. The generalization that membrane translocation of mitochondrial import precursors is enhanced by their prior unfolding in urea thus does not apply in the case of these precursors containing hydrophobic passenger proteins.     

Targeting and translocation of proteins into the hydrogenosome of the protist Trichomonas: similarities with mitochondrial protein import.

Trichomonads are early-diverging eukaryotes that lack both mitochondria and peroxisomes. They do contain a double membrane-bound organelle, called the hydrogenosome, that metabolizes pyruvate and produces ATP. To address the origin and biological nature of hydrogenosomes, we have established an in vitro protein import assay. Using purified hydrogenosomes and radiolabeled hydrogenosomal precursor ferredoxin (pFd), we demonstrate that protein import requires intact organelles, ATP and N-ethylmaleimide-sensitive cytosolic factors. Protein import is also affected by high concentrations of the protonophore, m-chlorophenylhydrazone (CCCP). Binding and translocation of pFd into hydrogenosomes requires the presence of an eight amino acid N-terminal presequence that is similar to presequences found on all examined hydrogenosomal proteins. Upon import, pFd is processed to a size consistent with cleavage of the presequence. Mutation of a conserved leucine at position 2 in the presequence to a glycine disrupts import of pFd into the organelle. Interestingly, a comparison of hydrogenosomal and mitochondrial protein presequences reveals striking similarities. These data indicate that mechanisms underlying protein targeting and biogenesis of hydrogenosomes and mitochondria are similar, consistent with the notion that these two organelles arose from a common endosymbiont.     

Isolated plant mitochondria import chloroplast precursor proteins in vitro with the same efficiency as chloroplasts.

Most chloroplast and mitochondrial proteins are synthesized with N-terminal presequences that direct their import into the appropriate organelle. In this report we have analyzed the specificity of standard in vitro assays for import into isolated pea chloroplasts and mitochondria. We find that chloroplast protein import is highly specific because mitochondrial proteins are not imported to any detectable levels. Surprisingly, however, pea mitochondria import a range of chloroplast protein precursors with the same efficiency as chloroplasts, including those of plastocyanin, the 33-kDa photosystem II protein, Hcf136, and coproporphyrinogen III oxidase. These import reactions are dependent on the Deltaphi across the inner mitochondrial membrane, and furthermore, marker enzyme assays and Western blotting studies exclude any import by contaminating chloroplasts in the preparation. The pea mitochondria specifically recognize information in the chloroplast-targeting presequences, because they also import a fusion comprising the presequence of coproporphyrinogen III oxidase linked to green fluorescent protein. However, the same construct is targeted exclusively into chloroplasts in vivo indicating that the in vitro mitochondrial import reactions are unphysiological, possibly because essential specificity factors are absent in these assays. Finally, we show that disruption of potential amphipathic helices in one presequence does not block import into pea mitochondria, indicating that other features are recognized.     

Euglena gracilis and Trypanosomatids Possess Common Patterns in Predicted Mitochondrial Targeting Presequences

Euglena gracilis possessing chloroplasts of secondary green algal origin and parasitic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi and Leishmania major belong to the protist phylum Euglenozoa. Euglenozoa might be among the earliest eukaryotic branches bearing ancestral traits reminiscent of the last eukaryotic common ancestor (LECA) or missing features present in other eukaryotes. LECA most likely possessed mitochondria of endosymbiotic ??-proteobacterial origin. In this study, we searched for the presence of homologs of mitochondria-targeted proteins from other organisms in the currently available EST dataset of E. gracilis. The common motifs in predicted N-terminal presequences and corresponding homologs from T. brucei, T. cruzi and L. major (if found) were analyzed. Other trypanosomatid mitochondrial protein precursor (e.g., those involved in RNA editing) were also included in the analysis. Mitochondrial presequences of E. gracilis and these trypanosomatids seem to be highly variable in sequence length (5?C118 aa), but apparently share statistically significant similarities. In most cases, the common (M/L)RR motif is present at the N-terminus and it is probably responsible for recognition via import apparatus of mitochondrial outer membrane. Interestingly, this motif is present inside the predicted presequence region in some cases. In most presequences, this motif is followed by a hydrophobic region rich in alanine, leucine, and valine. In conclusion, either RR motif or arginine-rich region within hydrophobic aa-s present at the N-terminus of a preprotein can be sufficient signals for mitochondrial import irrespective of presequence length in Euglenozoa.     

Role of the Negative Charges in the Cytosolic Domain of TOM22 in the Import of Precursor Proteins into Mitochondria

TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing a tom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutant tom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.     

Hydrophobic residues within the predicted N-terminal amphiphilic alpha-helix of a plant mitochondrial targeting presequence play a major role in in vivo import

A deletion and mutagenesis study was performed on the mitochondrial presequence of the beta-subunit of the F(1)-ATP synthase from Nicotiana plumbaginifolia linked to the green fluorescent protein (GFP). The various constructs were tested in vivo by transient expression in tobacco protoplasts. GFP distribution in transformed cells was analysed in situ by confocal microscopy, and in vitro in subcellular fractions by Western blotting. Despite its being highly conserved in different species, deletion of the C-terminal region (residues 48-54) of the presequence did not affect mitochondrial import. Deletion of the conserved residues 40-47 and the less conserved intermediate region (residues 18-39) resulted in 60% reduction in GFP import, whereas mutation of conserved residues within these regions had little effect. Further shortening of the presequence progressively reduced import, with the construct retaining the predicted N-terminal amphiphilic alpha-helix (residues 1-12) being unable to mediate mitochondrial import. However, point mutation showed that this last region plays an important role through its basic residues and amphiphilicity, but also through its hydrophobic residues. Replacing Arg4 and Arg5 by alanine residues and shifting the Arg5 and Leu6 (in order to disturb amphiphilicity) resulted in reduction of the presequence import efficiency. The most dramatic effects were seen with single or double mutations of the four Leu residues (positions 5, 6, 10 and 11), which resulted in marked reduction or abolition of GFP import, respectively. We conclude that the N-terminal helical structure of the presequence is necessary but not sufficient for efficient mitochondrial import, and that its hydrophobic residues play an essential role in in vivo mitochondrial targeting.     

A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23.

Proteins imported into the mitochondrial matrix are synthesized in the cytosol with an N-terminal presequence and are translocated through hetero-oligomeric translocase complexes of the outer and inner mitochondrial membranes. The channel across the inner membrane is formed by the presequence translocase, which consists of roughly six distinct subunits; however, it is not known which subunits actually form the channel. Here we report that purified Tim23 forms a hydrophilic, approximately 13-24 A wide channel characteristic of the mitochondrial presequence translocase. The Tim23 channel is cation selective and activated by a membrane potential and presequences. The channel is formed by the C-terminal domain of Tim23 alone, whereas the N-terminal domain is required for selectivity and a high-affinity presequence interaction. Thus, Tim23 forms a voltage-sensitive high-conductance channel with specificity for mitochondrial presequences.     

Targeting of a chemically pure preprotein to mitochondria does not require the addition of a cytosolic signal recognition factor.

To analyze the role of cytosolic cofactors in mitochondrial protein targeting, we prepared a chemically pure mitochondrial preprotein. When diluted out of 7 M urea, this precursor protein was efficiently imported into mitochondria without the addition of cytosolic cofactors. Extensive prewashing of mitochondria (up to 2 M KCl) did not reduce its import. Import of the purified precursor showed the characteristics of authentic mitochondrial import including use of the receptor MOM19, requirement for a membrane potential, and proteolytic processing. When the precursor was preincubated at a low concentration of urea, cytosolic cofactors were needed to preserve its import competence. We conclude that targeting of this preprotein via the mitochondrial master receptor MOM19 does not require a cytosolic signal recognition factor; cytosolic cofactors apparently have chaperone-like functions in mitochondrial protein uptake. Moreover, we found that a cleavable presequence was sufficient to direct protein import via MOM19. Together with the cofactor-independent function of MOM19, it is thus conceivable that MOM19 functions as mitochondrial presequence receptor.