The role of protein structure in the mitochondrial import pathway. Unfolding of mitochondrially bound precursors is required for membrane translocation

In order to examine the influence of protein structure on the post-translational import of a protein into mitochondria, the carboxyl-terminal 129 residues of F1-ATPase beta-subunit precursor (511aa) have been replaced with 61 residues of yeast copper metallothionein. Import of the F1 beta-copper metallothionein (beta CuMT) hybrid into mitochondria was as efficient as that of the F1 beta precursor in the absence of copper. Addition of copper to mitochondrial import reactions, which had no significant effect on import of the F1 beta-subunit precursor, blocked import of the beta CuMT protein. This copper-dependent transport block for the beta CuMT precursor occurred after the precursor was bound to mitochondria. Expression of the beta CuMT protein in vivo revealed that beta CuMT would bind copper and allow growth of a copper-sensitive yeast host on an otherwise inhibitory level of the cation as long as it was localized in the cytoplasm. These data indicate that the binding of copper by beta CuMT renders it refractile for partial unfolding which is necessary for its translocation into mitochondria. These observations provide an alternative scheme for the selection of mutants defective in mitochondrial import.     

Protein import into mitochondria in a homologous yeast in vitro system

To study the import of proteins into mitochondria we developed a homologous in vitro system in which mitochondria and cell-free translation extract are both derived from the yeast Saccharomyces cerevisiae. This system allows the synthesis of precursor proteins in the presence of isolated mitochondria and offers a means of analyzing yeast mutants defective in mitochondrial protein import. The in vitro import of an artificial precursor protein into yeast mitochondria in the presence of its substrate analog was analyzed subsequent to synthesis in either a yeast or rabbit reticulocyte cell-free translation reaction. Results suggest that a component(s) present in the yeast cytosolic extract may interact with the precursor protein.     

Coupling of protein synthesis and mitochondrial import in a homologous yeast in vitro system.

We made use of a homologous cell-free mitochondrial protein import system derived from the yeast Saccharomyces cerevisiae to investigate the coupling of protein synthesis and import. Mitochondrial precursor proteins were synthesized in a yeast lysate either in the presence or absence of isolated yeast mitochondria. We were, therefore, able to analyze protein import into mitochondria either in a strictly posttranslational reaction (when isolated mitochondria were added only after protein synthesis has been arrested by the addition of cycloheximide) or in a reaction in which synthesis and import were permitted to occur simultaneously. We found that the import of a precursor protein consisting of the amino-terminal mitochondrial targeting sequence of cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase is very inefficient in a strictly posttranslational reaction, whereas efficient import is observed if precursor synthesis and import are coupled. The same result was obtained when we analyzed the import of bulk endogenous yeast mitochondrial proteins in this system. Finally, we found that the insertion of the yeast outer membrane protein porin is also several times more efficient when synthesis and insertion are coupled.     

Import of an incompletely folded precursor protein into isolated mitochondria requires an energized inner membrane, but no added ATP.

We have studied the post-translational import of incomplete precursor chains into isolated yeast mitochondria. The precursor was a fusion protein containing a mitochondrial presequence attached to mouse dihydrofolate reductase. In vitro-synthesis of the precursor was interrupted by the elongation inhibitor cycloheximide and the arrested nascent chains cosedimenting with ribosomes were released by EDTA. These incomplete chains were efficiently imported by isolated yeast mitochondria; their import resembled that of the complete precursor in requiring an energized inner membrane and a mitochondrial presequence. It differed from that of the completed precursor in its resistance to methotrexate (which only binds to correctly folded dihydrofolate reductase) and its independence of added ATP. The incomplete chains were also more sensitive to proteinase K than the completed precursor. We conclude that the incomplete chains were incompletely folded and suggest that the lack of tight folding caused import into mitochondria to become independent of added ATP. This implies that ATP may participate, directly or indirectly, in the unfolding of the precursor for its transport into mitochondria.     

Duplication of leader sequence for protein targeting to mitochondria leads to increased import efficiency

We describe a novel method for enhancing protein import into mitochondria, by tandemly duplicating the N-terminal cleavable leader peptide using a gene manipulation strategy. The import into isolated yeast mitochondria of passenger proteins (yeast mitochondrial ATP synthase subunits 8 and 9 and some mutagenised derivatives) that show little or no import when endowed with one such leader (that of Neurospora crassa mitochondrial ATP synthase subunit 9) is remarkably improved when the leader is tandemly duplicated. The import of these chimaeric proteins bearing a double leader is so rapid that a series of partially processed precursor intermediates accumulates inside the mitochondria before the final proteolytic release of leader sequences from the passenger proteins. It is considered that the duplicated leader greatly accelerates delivery of the import precursors to outer membrane receptor elements and the associated translocation systems, thereby enhancing precursor uptake into mitochondria.     

Reinvestigation of the requirement of cytosolic ATP for mitochondrial protein import

Protein import into mitochondria requires the energy of ATP hydrolysis inside and/or outside mitochondria. Although the role of ATP in the mitochondrial matrix in mitochondrial protein import has been extensively studied, the role of ATP outside mitochondria (external ATP) remains only poorly characterized. Here we developed a protocol for depletion of external ATP without significantly reducing the import competence of precursor proteins synthesized in vitro with reticulocyte lysate. We tested the effects of external ATP on the import of various precursor proteins into isolated yeast mitochondria. We found that external ATP is required for maintenance of the import competence of mitochondrial precursor proteins but that, once they bind to mitochondria, the subsequent translocation of presequence-containing proteins, but not the ADP/ATP carrier, proceeds independently of external ATP. Because depletion of cytosolic Hsp70 led to a decrease in the import competence of mitochondrial precursor proteins, external ATP is likely utilized by cytosolic Hsp70. In contrast, the ADP/ATP carrier requires external ATP for efficient import into mitochondria even after binding to mitochondria, a situation that is only partly attributed to cytosolic Hsp70.     

Induced tRNA Import into Human Mitochondria: Implication of a Host Aminoacyl-tRNA-Synthetase

In human cell, a subset of small non-coding RNAs is imported into mitochondria from the cytosol. Analysis of the tRNA import pathway allowing targeting of the yeast tRNA^Lys _CUU into human mitochondria demonstrates a similarity between the RNA import mechanisms in yeast and human cells. We show that the cytosolic precursor of human mitochondrial lysyl-tRNA synthetase (preKARS2) interacts with the yeast tRNA^Lys _CUU and small artificial RNAs which contain the structural elements determining the tRNA mitochondrial import, and facilitates their internalization by isolated human mitochondria. The tRNA import efficiency increased upon addition of the glycolytic enzyme enolase, previously found to be an actor of the yeast RNA import machinery. Finally, the role of preKARS2 in the RNA mitochondrial import has been directly demonstrated in vivo, in cultured human cells transfected with the yeast tRNA and artificial importable RNA molecules, in combination with preKARS2 overexpression or downregulation by RNA interference. These findings suggest that the requirement of protein factors for the RNA mitochondrial targeting might be a conserved feature of the RNA import pathway in different organisms.     

Adriamycin, a drug interacting with acidic phospholipids, blocks import of precursor proteins by isolated yeast mitochondria

Acidic phospholipids such as cardiolipin partially unfold an artificial precursor protein which consists of a mitochondrial presequence fused to mouse dihydrofolate reductase (Endo, T., and Schatz, G. (1988) EMBO J. 7, 1153-1158). We now show that import of this precursor protein into isolated yeast mitochondria is blocked by adriamycin, a drug binding to cardiolipin and other acidic phospholipids. This inhibition is lessened if the precursor's dihydrofolate reductase moiety is labilized by point mutations; inhibition is abolished altogether if the "wild-type" precursor is presented to mitochondria in a urea-denatured state. These and other observations suggest that adriamycin interferes with the generation of a translocation-competent, loose structure of the precursor protein. They imply that acidic phospholipids such as cardiolipin participate, directly or indirectly, in the translocation of this fusion protein into isolated mitochondria.     

Nascent polypeptide-associated complex stimulates protein import into yeast mitochondria

To identify yeast cytosolic proteins that mediate targeting of precursor proteins to mitochondria, we developed an in vitro import system consisting of purified yeast mitochondria and a radiolabeled mitochondrial precursor protein whose C terminus was still attached to the ribosome. In this system, the N terminus of the nascent chain was translocated across both mitochondrial membranes, generating a translocation intermediate spanning both membranes. The nascent chain could then be completely chased into the mitochondrial matrix after release from the ribosome. Generation of this import intermediate was dependent on a mitochondrial membrane potential, mitochondrial surface proteins, and was stimulated by proteins that could be released from the ribosomes by high salt. The major salt-released stimulatory factor was yeast nascent polypeptide-associated complex (NAC). Purified NAC fully restored import of salt-washed ribosome-bound nascent chains by enhancing productive binding of the chains to mitochondria. We propose that ribosome-associated NAC facilitates recognition of nascent precursor chains by the mitochondrial import machinery.     

Synthesis of a bovine adrenodoxin precursor in vitro and its import into yeast mitochondria]

The bovine adrenal cortex adrenodoxin gene was inserted into pTZ19 under T7 promoter control. The adrenodoxin mRNA was synthesized with T7 RNA polymerase and then translated in the reticulocyte cell-free translation system. The protein product was identified as the adrenodoxin precursor with molecular weight 22000. The import of the precursor into isolated yeast mitochondria was carried out. The protein was found to be inserted into the trypsin-insensitive compartment of mitochondria via an energy dependent way. This resulted in the processing of the precursor to the 12000-mature form. Thus, the precursor of mammalian adrenodoxin can be normally imported into yeast mitochondria.     

Zinc-dependent intermembrane space proteins stimulate import of carrier proteins into plant mitochondria

Mitochondrial inner membrane carrier proteins are imported into mitochondria from yeast, fungi and mammals by specific machinery, some components of which are distinct from those utilized by other proteins. Import of two different carriers into plant mitochondria showed that one contains a cleavable presequence which was processed during import, while the other imported in a valinomycin-sensitive manner without processing. Mild osmotic shock of mitochondria released intermembrane space (IMS) components and impaired carrier protein import. Adding back the released IMS proteins as a concentrate in the presence of micromolar ZnCl2 stimulated carrier import into IMS-depleted mitochondria, but did not stimulate import of a non-carrier control precursor protein, the alternative oxidase. Anion-exchange separation of IMS components before addition to IMS-depleted mitochondria revealed a correlation between several 9-10 kDa proteins and stimulation of carrier import. MS/MS sequencing of these proteins identified them as plant homologues of the yeast zinc-finger carrier import components Tim9 and Tim10. Stimulation of import was dependent on either Zn2+ or Cd2+ and inhibited by both N-ethylmalamide (NEM) and a divalent cation chelator, consistent with a functional requirement for a zinc finger protein. This represents direct functional evidence for a distinct carrier import pathway in plant mitochondria, and provides a tool for determining the potential function of other IMS proteins associated with protein import.     

Sequencing of the nuclear gene for the yeast cytochrome c1 precursor reveals an unusually complex amino-terminal presequence.

Cytochrome c1 is a component of the mitochondrial respiratory chain in most eukaryotes. The protein is coded by nuclear DNA, synthesized as a larger precursor outside the mitochondria and then cleaved to the mature form in two successive steps during its import into the mitochondria. We have cloned the structural gene for yeast cytochrome c1 by functional complementation of a cytochrome c1-deficient yeast mutant with a yeast genomic library in the yeast-Escherichia coli 'shuttle' vector YEp 13. The complete nucleotide sequence of the gene and of its 5'- and 3'-flanking regions was determined. The deduced amino acid sequence of the yeast cytochrome c1 precursor reveals an unusually long transient amino-terminal presequence of 61 amino acids. This presequence consists of a strongly basic amino-terminal region of 35 amino acids, a central region of 19 uncharged amino acids and an acidic carboxy-terminal region of seven amino acids. This tripartite structure of the presequence resembles that of the precursor of cytochrome c peroxidase and supports a previous suggestion on the import pathways of these two precursors.     

In vitro and in vivo studies on the mitochondrial import of CBS1, a translational activator of cytochrome b in yeast

Summary Translation of mitochondrial cytochrome b mRNA in yeast is activated by the product of the nuclear gene CBS1. CBS1 encodes a 27 kDa precursor protein, which is cleaved to a 24 kDa mature protein during the import into isolated mitochondria. The sequences required for mitochondrial import reside in the amino-terminal end of the CBS1 precursor. Deletion of the 76 amino-terminal amino acids renders the protein incompetent for mitochondrial import in vitro and non-functional in vivo. When present on a high copy number plasmid and under the control of a strong yeast promoter, biological function can be restored by this truncated derivative. This observation indicates that the CBS1 protein devoid of mitochondrial targeting sequences can enter mitochondria in vivo, possibly due to a bypass of the mitochondrial import system.     

A chemically synthesized pre-sequence of an imported mitochondrial protein can form an amphiphilic helix and perturb natural and artificial phospholipid bilayers.

Subunit IV of yeast cytochrome oxidase is made in the cytoplasm with a transient pre-sequence of 25 amino acids which is removed upon import of the protein into mitochondria. To study the function of this cleavable pre-sequence in mitochondrial protein import, three peptides representing 15, 25 or 33 amino-terminal residues of the subunit IV precursor were chemically synthesized. All three peptides were freely soluble in aqueous buffers, yet inserted spontaneously from an aqueous subphase into phospholipid monolayers up to an extrapolated limiting monolayer pressure of 40-50 mN/m. The two longer peptides also caused disruption of unilamellar liposomes. This effect was increased by a diffusion potential, negative inside the liposomes, and decreased by a diffusion potential of opposite polarity. The peptides, particularly the two longer ones, also uncoupled respiratory control of isolated yeast mitochondria. The 25-residue peptide had little secondary structure in aqueous buffer but became partly alpha-helical in the presence of detergent micelles. Based on the amino acid sequence of the peptides, a helical structure would have a highly asymmetric distribution of charged and apolar residues and would be surface active. Amphiphilic helicity appears to be a general feature of mitochondrial pre-sequences. We suggest that this feature plays a crucial role in transporting proteins into mitochondria.     

Yeast mitochondrial ATPase subunit 8, normally a mitochondrial gene product, expressed in vitro and imported back into the organelle.

Subunit 8 of yeast mitochondrial F1F0-ATPase is a proteolipid made on mitochondrial ribosomes and inserted directly into the inner membrane for assembly with the other F0 membrane-sector components. We have investigated the possibility of expressing this extremely hydrophobic, mitochondrially encoded protein outside the organelle and directing its import back into mitochondria using a suitable N-terminal targeting presequence. This report describes the successful import in vitro of ATPase subunit 8 proteolipid into yeast mitochondria when fused to the targeting sequence derived from the precursor of Neurospora crassa ATPase subunit 9. The predicted cleavage site of matrix protease was correctly recognized in the fusion protein. A targeting sequence from the precursor of yeast cytochrome oxidase subunit VI was unable to direct the subunit 8 proteolipid into mitochondria. The proteolipid subunit 8 exhibited a strong tendency to embed itself in mitochondrial membranes, which interfered with its ability to be properly imported when part of a synthetic precursor.     

Protein transport into mitochondria is conserved between plant and yeast species

Protein targeting into plant mitochondria was investigated by in vitro translocation experiments. The precursor of the mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia was synthesized in vitro, translocated to, processed, and assembled in purified Vicia faba mitochondria. Transport (but not binding) required a membrane potential and external nucleotides and was conserved among plant species. beta subunit precursors from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe were imported and correctly processed in plant mitochondria. This translocation used protease-sensitive components of the outer membrane. Conversely, the N. plumbaginifolia beta subunit precursor was efficiently translocated and cleaved in yeast mitochondria. However, a precursor for a chloroplast protein was not targeted to plant or yeast mitochondria. We conclude that the machinery for protein import into mitochondria is specific and conserved in plant and yeast organisms. These results are discussed in the context of a poly- or monophyletic origin of mitochondria.     

Targeting of tRNA into yeast and human mitochondria: the role of anticodon nucleotides

In vivo, yeast mitochondria import a single cytoplasmic tRNA, tRNA(CUU)Lys, while human mitochondria do not import any cytoplasmic tRNA. We have previously demonstrated that both yeast and human isolated mitochondria can specifically internalize tRNA(CUU)Lys, several of its mutant versions and some mutant versions of yeast cytosolic tRNA(UUU)Lys (not imported in vivo). Aminoacylation of tRNA(CUU)Lys by the cytoplasmic lysyl-tRNA synthetase was a prerequisite for its import. Here we are studying the influence of one-base replacements in the anticodon of tRNAs(Lys) on their aminoacylation, on binding to the precursor of the mitochondrial lysyl-tRNA synthetase (carrier protein directing the import), and on the efficiency of import into isolated yeast and human mitochondria. We show that the base U35 is the main identity element for the yeast cytoplasmic lysyl-tRNA synthetase. The single replacement that abolished import was C34G, while all the others only modulated the import efficiency. The need of aminoacylation for import and for interaction with the carrier protein was shown only for a subset of mutant versions, while the others could be recognized and internalized without aminoacylation or in misacylated forms.     

Intramitochondrial sorting of the precursor to yeast cytochrome c oxidase subunit Va

We have continued our studies on the import pathway of the precursor to yeast cytochrome c oxidase subunit Va (pVa), a mitochondrial inner membrane protein. Previous work on this precursor demonstrated that import of pVa is unusually efficient, and that inner membrane localization is directed by a membrane-spanning domain in the COOH- terminal third of the protein. Here we report the results of studies aimed at analyzing the intramitochondrial sorting of pVa, as well as the role played by ancillary factors in import and localization of the precursor. We found that pVa was efficiently imported and correctly sorted in mitochondria prepared from yeast strains defective in the function of either mitochondrial heat shock protein (hsp)60 or hsp70. Under identical conditions the import and sorting of another mitochondrial protein, the precursor to the beta subunit of the F1 ATPase, was completely defective. Consistent with previous results demonstrating that the subunit Va precursor is loosely folded, we found that pVa could be efficiently imported into mitochondria after translation in wheat germ extracts. This results suggests that normal levels of extramitochondrial hsp70 are also not required for import of the protein. The results of this study enhance our understanding of the mechanism by which pVa is routed to the mitochondrial inner membrane. They suggest that while the NH2 terminus of pVa is exposed to the matrix and processed by the matrix metalloprotease, the protein remains anchored to the inner membrane before being assembled into a functional holoenzyme complex.     

Inhibition of the import of mitochondrial proteins by RNase

RNase treatment of a cell-free translation system prevents transport of mitochondrial precursor proteins from that system into isolated yeast mitochondria. This inhibition depends on the presence of ribosomes in the reticulocyte lysate; if they are cleared by centrifugation, RNase treatment does not specifically inhibit protein uptake by mitochondria. Since protein import can occur in the absence of polyribosomes, RNase treatment does not degrade a structure essential for this process. Rather, the inhibition may be an effect of degraded ribosomes.