A synthetic signal peptide blocks import of precursor proteins destined for the mitochondrial inner membrane or matrix

A peptide corresponding to amino acids 1-27 of preornithine carbamyltransferase (pOCT) has been chemically synthesized. When added to energized mitochondria in vitro, 20 microM of the peptide, designated pO(1-27), resulted in a collapse of the electrochemical potential across the mitochondrial inner membrane. This effect on transmembrane potential was not observed, however, when pO(1-27) was added to energized mitochondria under conditions that support in vitro import of precursor proteins (i.e. in the presence of reticulocyte lysate). The latter finding, therefore, made possible an examination of the ability of pO(1-27) to block import of homologous and heterologous proteins into the organelle. At 5-10 microM, pO(1-27) prevented import of pOCT in vitro; inhibition was overcome by increasing the concentration of pOCT. In contrast, pO(16-27), a peptide corresponding to amino acids 16-27 of pOCT and exhibiting a charge:mass ratio similar to pO(1-27) had no such inhibitory effect. pO(1-27) blocked import of other unrelated precursor proteins destined either for the mitochondrial matrix (pre-malate dehydrogenase and a hybrid protein containing the signal sequence of pre-carbamyl phosphate synthetase) or for the mitochondrial inner membrane (pre-thermogenin).     

Identification of hydrophobic residues in the signal sequence of mitochondrial preornithine carbamyltransferase that enhance the rate of precursor import

Previous studies employing circular dichroism and resonance energy transfer techniques have demonstrated that the signal peptide of mitochondrial preornithine carbamyltransferase (pOCT) has the potential to interact with the surface of an anionic phospholipid membrane via a short amphiphilic helical domain. Here we have used predictive secondary structure computations as a guide to localize the putative membrane binding region in the pOCT signal sequence and demonstrate that replacement of leucine residues at positions 5, 8, and 9 with the less hydrophobic residue, alanine, significantly reduces the rate of precursor import (4-5-fold compared to wild type); the amino acid substitutions had little effect, however, on the ability of a mitochondrial matrix extract to process the mutant precursor polypeptide. The mutant precursor bound to anionic liposomes with a lower affinity compared to wild-type pOCT and was inhibited to a lesser extent than pOCT during import into mitochondria in the presence of varying concentrations of liposomes. Taken together, the results suggest that this small region of the pOCT signal sequence, containing a limited number of critical hydrophobic residues, contributes to the optimal rate of precursor import, perhaps by functioning as a membrane surface-seeking entity.     

The interaction of a synthetic mitochondrial signal peptide with lipid membranes is independent of transbilayer potential.

We have used fluorescence measurements and assays of vesicle disruption (contents leakage) to monitor the interaction between lipid vesicles and a synthetic peptide corresponding to the N-terminal 27 amino acids of rat mitochondrial pre-ornithine carbamyltransferase (pOCT). This peptide and two fluorescent derivatives bind reversibly to vesicles composed of neutral and anionic phospholipids with increasing affinity as the proportion of anionic lipids in the vesicles increases. The affinity of the peptide for lipid vesicles is unaffected by the presence of a transbilayer potential (inside negative) of at least -80 mV across the vesicle membranes. Our results support the proposal that the signal sequence of pOCT may promote an initial association of the precursor protein with mitochondrial membranes prior to binding to a specific receptor. However, we find no evidence that the pOCT signal sequence can subsequently undergo transfer into or across the lipid bilayer, even in the presence of a transmembrane potential of the magnitude previously found to support the import of precursor proteins into mitochondria.     

A signal sequence domain essential for processing, but not import, of mitochondrial pre-ornithine carbamyl transferase

Studies using deletion mutagenesis indicate that a processing recognition site lies proximal to the normal cleavage position between gln32 and ser33 of pre-ornithine carbamyl transferase (pOCT). pOCT cDNA was manipulated to delete codons specifying amino acids 22-30 of the signal sequence. The mutant precursor, designated pOCT delta 22-30, was imported to the matrix compartment by purified mitochondria, but remained largely unprocessed; the low level of processing that was observed did not involve the normal cleavage site. Several manipulations performed downstream of the normal pOCT processing site (deletion, substitution, and hybrid protein constructions) affected neither import nor correct processing. Our data suggest that domains specifying import and processing site recognition may be functionally segregated within the signal peptide; that processing is not requisite for import of pOCT; and that a proximal region, not involving the normal signal peptide cleavage site, is required for processing site recognition.     

Role for the mitochondrial inner membrane in the maturation of the precursor to ornithine carbamyl transferase

The precursor to ornithine carbamyl transferase (pOCT) is cleaved at two N-terminal sites when imported into intact mitochondria but only at the N-proximal site when incubated with a membrane-free mitochondrial lysate or matrix fraction. Disruption of the mitochondrial membrane system by sonication, freeze-thaw, or lysis with non-ionic detergents blocks the processing of pOCT to its mature form. Mitoplasts prepared from protease-inactivated, import-incompetent mitochondria recover full processing activity; disruption of the inner membrane impairs the maturation process i.e. causes the loss of the mitoplasts' ability to transform pOCT into OCT. The data reveal a dependency of a maturation event on a "specific" interaction between a precursor protein and the mitochondrial inner membrane probably to position and/or to expose the correct N-distal cleavage site of the presequence.     

Expression of nuclear genes encoding the urea cycle enzymes, carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase, in rat liver and intestinal mucosa

RNA dot-blot, quantitative electron microscope immunocytochemistry, and electrophoretic immunoblotting techniques were employed to investigate the expression of carbamoyl-phosphate synthetase I (CPS) and ornithine carbamoyl transferase (OCT) genes in rat liver and intestinal mucosa. Comparing only those cell types in the two tissues which express these enzymes, we show that the concentration of CPS and OCT in hepatocyte mitochondria is 2.3-times and 1.2-times greater, respectively, than in intestinal epithelial cell mitochondria. As a percentage of total tissue protein, however, liver homogenates contain 10-20 times more CPS and 5-10 times more OCT than is found in intestinal mucosa. These relatively large differences in enzyme protein levels between the two tissues are not reflected by differences in their mRNA levels. As a percentage of total translational activity in vitro (based on incorporation of [35S]methionine), total liver mRNA directed synthesis of about twice as much precursor CPS (pCPS) and precursor OCT (pOCT) than did equivalent amounts of mRNA from intestinal mucosa. The ratio of pCPS and pOCT mRNA levels between the two tissues (2:1, liver:intestinal mucosa) was confirmed by dot-blot and Northern hybridizations employing specific cDNA probes. The sizes of the respective mRNAs were the same for the two tissues: about 6000 residues for pCPS mRNA and about 1700 residues for pOCT mRNA.     

Effect of deletions within the leader peptide of pre-ornithine transcarbamylase on mitochondrial import

The uptake of the cytoplasmically synthesized mammalian enzyme, ornithine transcarbamylase, into mitochondria is directed by an N-terminal peptide of 32 amino acids. We have investigated some of the structural requirements for the import of the enzyme from rat liver into isolated mitochondria and into mitochondria of COS cells transfected with cDNA encoding the precursor form of ornithine transcarbamylase. Deletion of 21 amino acids from the N terminus of the leader peptide blocked the import of the precursor; deletion of 5 amino acids at positions 15-19 from the N terminus of the leader peptide had no deleterious effect on the import of the enzyme, nor on the processing and assembly of subunits in mitochondria. The region deleted contained three of eight basic residues in the leader peptide suggesting that specific structural elements containing basic residues, rather than the general basic nature of the leader, may be involved in mitochondrial import.     

Liver mitochondrial aldehyde dehydrogenase: in vitro expression, in vitro import, and effect of alcohols on import

An in vitro expression plasmid (pGRAP) that contained the cDNA coding for the rat mitochondrial aldehyde dehydrogenase precursor was constructed, mRNA was synthesized then translated, and the in vitro synthesized precursor of aldehyde dehydrogenase was used in an in vitro import assay. As expected the 19 amino acid signal peptide of the precursor allowed import of the precursor into rat liver mitochondria. This in vitro system was used to examine the effect of alcohols on import. It was found that the alcohols (ethyl, butyl, hexyl, and octyl) tested inhibited the import of the aldehyde dehydrogenase precursor. Pretreatment of the mitochondria with alcohol was responsible for the inhibition. The inhibition appeared to be relatively specific for pre-aldehyde dehydrogenase as the precursor of ornithine transcarbamylase was still imported in the presence of alcohols. Of potential physiological significance was finding that ethanol inhibited import in a dose-response fashion; 50% inhibition occurred at 75 mM, a concentration achievable during the ingestion of alcohol. In addition, the concentrations of alcohols required to produce an inhibitory effect on import decreased as the hydrocarbon chain length of alcohols increased. The inhibitory effect of alcohols appeared to be specific as other solvents examined did not inhibit import. We postulate that alcohols may perturb the mitochondrial membrane and affect the receptor-translocator necessary for the import of the aldehyde dehydrogenase precursor.     

Mitochondrial import of the human chaperonin (HSP60) protein

The mitochondrial import of a member of the "chaperonin" group of proteins which play an essential role in the import of protein into organelles and their subsequent proper folding has been examined. The cDNA for human hsp60 (synonyms: GroEL homolog, P1) was transcribed and translated in vitro and its import into isolated rat heart mitochondria examined. The protein was converted into a mature form of lower molecular mass (= 58 kDa) which was resistant to trypsin treatment. The import of human hsp60 into mitochondria was inhibited in the presence of an uncoupler and also no import occurred when the N-terminal presequence was lacking. These results indicate that the chaperonin protein(s) are transported into mitochondria by a process similar to other imported mitochondrial proteins. Our results also indicate that although the P1 protein precursor was efficiently imported into mitochondria, in comparison to precursors of other mitochondrial proteins (viz. ornithine carbamoyltransferase and uncoupling protein) much less binding of pre P1 to mitochondria was observed. The significance of this latter observation at present is unclear.     

Presence of the cytosolic factor stimulating the import of precursor of mitochondrial proteins in rabbit reticulocytes and rat liver cells

Previously we purified a cytosolic factor that stimulates the import of the extrapeptide (the synthetic peptide of the presequence of ornithine aminotransferase) into the mitochondrial matrix (Ono, H., and Tuboi, S., 1988, J. Biol. Chem. 263, 3188-3193). In this work this cytosolic factor was shown also to stimulate the import of the precursors of ornithine aminotransferase, a large subunit of succinate dehydrogenase, and sulfite oxidase. The amounts of these precursors bound to the outer mitochondrial membrane were increased by this cytosolic factor, suggesting that the cytosolic factor participates in the recognition step in the import process of the precursor protein. When the cytosolic factor was applied to an ATP-agarose column, the import-stimulating activity was recovered entirely in the unadsorbed fraction. Immunochemical studies showed that in these conditions the 70-kDa heat shock-related protein (Hsp 70) was present exclusively in the fraction adsorbed to the ATP-agarose column. The cytosolic factor is thus different from the 70-kDa heat shock-related protein, which was identified as a factor required for the import of mitochondrial proteins in yeast. The cytosolic factor was also detected in the cytosol of rat liver cells, and a considerable amount of this factor was recovered from rat liver mitochondria by washing them with high salt buffer, suggesting that the cytosolic factor has affinity to the outer mitochondrial membrane and binds to its receptor on the membrane. From these results, we conclude that the cytosolic factor forms a complex with the precursor of mitochondrial protein and then this complex binds to the outer mitochondrial membrane, probably via the receptor of the cytosolic factor.     

The requirement of heat shock cognate 70 protein for mitochondrial import varies among precursor proteins and depends on precursor length.

The cytosolic heat shock cognate 70-kDa protein (hsc70) is required for efficient import of ornithine transcarbamylase precursor (pOTC) into rat liver mitochondria (K. Terada, K. Ohtsuka, N. Imamoto, Y. Yoneda, and M. Mori, Mol. Cell. Biol. 15:3708-3713, 1995). The requirement of hsc70 for mitochondrial import of various precursor proteins and truncated pOTCs was studied by using an in vitro translation import system in which hsc70 was completely depleted. hsc70-dependent import of pOTC was about 60% of the total import, while import of the aspartate aminotransferase precursor, the serine:pyruvate aminotransferase precursor, and 3-oxoacyl coenzyme A thiolase was about 50, 30, and 0%, respectively. The subunit sizes of these four precursor proteins were 40 to 47 kDa. When pOTC was serially truncated from the COOH terminal, the hsc70 requirement decreased gradually and was not evident for the shortest truncated pOTCs of 90 and 72 residues. These truncated pOTCs were imported and proteolytically processed rapidly in 0.5 to 2 min at 25 degrees C, and the processed mature portions and the presequence portion were rapidly degraded. Sucrose gradient centrifugation analysis followed by import assay showed that pOTC synthesized in rabbit reticulocyte lysate forms an import-competent complex of about 11S in an hsc70-dependent manner. S values of import-competent forms of aspartate aminotransferase precursor, serine:pyruvate aminotransferase precursor, and 3-oxoacyl coenzyme A thiolase were 9S, 9S, and 4S, respectively. Thus, the S value decreased as the hsc70 dependency decreased. Precursor proteins were coimmunoprecipitated from the reticulocyte lysate containing the newly synthesized precursor proteins with an hsc70 antibody. The amount of coimmunoprecipitated proteins was much larger in the absence of ATP than in its presence. Among the four precursor proteins, the amount of coimmunoprecipitated protein decreased as the hsc70 dependency decreased.     

Sequence of the precursor to rat ornithine aminotransferase deduced from a cDNA clone

The nucleotide sequence of ornithine aminotransferase mRNA from rat liver, including the entire coding and 3' untranslated regions, was determined from two overlapping cDNA clones. The mRNA encodes a precursor polypeptide of 439 amino acid residues with a molecular weight of 48,332. The deduced amino acid composition of the proposed mature enzyme sequence (residues 35 through 439) was in good agreement with that reported for the purified protein. The amino-terminal segment of the precursor corresponding to residues 1 through 34 has an overall positive charge, containing 6 basic residues and only a single acidic residue, and is postulated to be the mitochondrial leader sequence. The first 22 amino acid residues of the proposed leader sequences share 54% homology with the leader peptide of rat ornithine transcarbamylase precursor and more limited homology to the leader peptides of other nuclear-encoded mitochondrial matrix proteins. Homology was also observed between residues 286 through 362 ornithine aminotransferase precursor and a region containing the pyridoxyl phosphate binding domain of mitochondrial aspartate aminotransferase.     

Spermidine and spermine stimulate the transport of the precursor of ornithine carbamoyltransferase into rat liver mitochondria

We have examined the effect of low molecular weight components of the transport mixture generally used for the import of rat liver pre-ornithine carbamoyltransferase by isolated rat liver mitochondria. These studies revealed that spermidine and spermine, at physiological concentrations, stimulate the transport of the precursor of ornithine carbamoyltransferase into mitochondria. This stimulatory effect of spermidine and spermine is concentration-dependent and is completely inhibited at higher than physiological concentrations (20 mM for spermidine and 4 mM for spermine). Magnesium ions, which also have a stimulatory effect, inhibit the stimulatory effect of spermidine.     

The human mitochondrial import receptor, hTom20p, prevents a cryptic matrix targeting sequence from gaining access to the protein translocation machinery

Yeast Mas70p and NADH cytochrome b5 reductase are bitopic integral proteins of the mitochondrial outer membrane and are inserted into the lipid-bilayer in an Nin-Ccyto orientation via an NH2-terminal signal- anchor sequence. The signal anchor of both proteins is comprised of a short, positively charged domain followed by the predicted transmembrane segment. The positively charged domain is capable of functioning independently as a matrix-targeting signal in yeast mitochondria in vitro but does not support import into mammalian mitochondria (rat or human). Rather, this domain represents a cryptic signal that can direct import into mammalian mitochondria only if proximal components of the outer membrane import machinery are removed. This can be accomplished either by treating the surface of the intact mitochondria with trypsin or by generating mitoplasts. The import receptor Tom20p (Mas20p/MOM19) is responsible for excluding the cryptic matrix-targeting signal from mammalian mitochondria since replacement of yeast Tom20p with the human receptor confers this property to the yeast organelle while at the same time maintaining import of other proteins. In addition to contributing to positive recognition of precursor proteins, therefore, the results suggest that hTom20p may also have the ability to screen potential matrix-targeting sequences and exclude certain proteins that would otherwise be recognized and imported by distal components of the outer and inner membrane protein- translocation machinery. These findings also indicate, however, that cryptic signals, if they exist within otherwise native precursor proteins, may remain topogenically silent until the precursor successfully clears hTom20p, at which time the activity of the cryptic signal is manifested and can contribute to subsequent translocation and sorting of the polypeptide.     

A synthetic presequence reversibly inhibits protein import into yeast mitochondria

We show that a synthetic peptide corresponding to the N-terminal 22 residues of the cytochrome c oxidase subunit IV presequence blocked import of pre-subunit IV into yeast mitochondria. The 22-residue peptide pL4-(1-22) did not alter the electrical potential across the mitochondrial inner membrane (the delta psi). Inhibition of import was reversible and could be overcome by the addition of increased amounts of precursor. Two other peptides, pL4-(1-16) and pL4-(1-23), which correspond to, respectively, the N-terminal 16 and 23 residues of the same presequence, also blocked import of pre-subunit IV. However, pL4-(1-16) was a much weaker inhibitor of import, while the inhibitory effect of pL4-(1-23) was due to its ability to completely collapse the delta psi. pL4-(1-22) seems to be a general inhibitor of mitochondrial import, in that it also blocked uptake of several other proteins. These included the precursors of the yeast proteins cytochrome c oxidase subunit Va, the F1-ATPase beta subunit, mitochondrial malate dehydrogenase, and the ATP/ADP carrier. In addition, uptake of two non-yeast precursor proteins (human ornithine transcarbamylase and a cytochrome oxidase subunit IV-dihydrofolate reductase fusion), was also blocked by the peptide. Subsequent studies revealed that pL4-(1-22) did not block the initial recognition or binding of proteins to mitochondria. Rather, our results suggest that the peptide acts at a subsequent translocation step which is common to the import pathways of many different precursor proteins.     

Transport of proteins to the mitochondrial intermembrane space: the ''sorting'' domain of the cytochrome c1 presequence is a stop-transfer sequence specific for the mitochondrial inner membrane.

The presequence of yeast cytochrome c1 (an inner membrane protein protruding into the intermembrane space) contains a matrix-targeting domain and an intramitochondrial sorting domain. This presequence transports attached subunit IV of cytochrome c oxidase into the intermembrane space (van Loon et al. (1987) EMBO J., 6, 2433-2439). In order to determine how this fusion protein reaches the intermembrane space, we studied the kinetics of its import into isolated mitochondria or mitoplasts and its accumulation in the various submitochondrial compartments. The imported, uncleaved fusion precursor and a cleavage intermediate were bound to the inner membrane and were always exposed to the intermembrane space; they were never found at the matrix side of the inner membrane. In contrast, analogous import experiments with the authentic subunit IV precursor, or the precursor of the iron-sulphur protein of the cytochrome bc1 complex also an inner membrane protein exposed to the intermembrane space), readily showed that these precursors were initially transported across both mitochondrial membranes. We conclude that the intramitochondrial sorting domain within the cytochrome c1 presequence prevents transport of attached proteins across the inner, but not the outer membrane: it is a stop-transfer sequence for the inner membrane. Since the presequence of the iron-sulphur protein lacks such 'stop-transfer' domain, it acts by a different mechanism.     

Presequence binding factor-dependent and -independent import of proteins into mitochondria.

A cytosolic protein factor(s) is involved in the import of precursor proteins into mitochondria. PBF (presequence binding factor) is a protein factor which binds to the precursor form (pOTC) of rat ornithine carbamoyltransferase (OTC) but not to the mature OTC, and is required for the mitochondrial import of pOTC. The precursors for aspartate aminotransferase and malate dehydrogenase as well as pOTC synthesized in a reticulocyte lysate were efficiently imported into the mitochondria. However, the precursors synthesized in the lysate depleted for PBF by treatment with pOTC-Sepharose were not imported. Readdition of the purified PBF to the depleted lysate fully restored the import. pOTC synthesized in the untreated lysate sedimented as a complex with a broad peak of around 9 S, whereas pOTC synthesized in the PBF-depleted lysate sedimented at an expected position of monomer (2.5 S). When the purified PBF was readded to the depleted lysate, pOTC sedimented as a complex of about 7 S. In contrast to most mitochondrial proteins, rat 3-oxoacyl-CoA thiolase is synthesized with no cleavable presequence and an NH2-terminal portion of the mature protein functions as a mitochondrial import signal. The thiolase synthesized in the PBF-depleted lysate could be efficiently imported into the mitochondria, and readdition of PBF had little effect on the import. The thiolase synthesized in the untreated, the PBF-depleted, or the PBF-readded lysate sedimented at an expected position of monomer (2.5 S). These observations provide support for the existence of PBF-dependent and -independent pathways of mitochondrial protein import.     

Identification of an outer mitochondrial membrane protein that interacts with a synthetic signal peptide

Chemical cross-linking procedures have been employed to study possible interactions between components of the mitochondrial outer membrane and NH2-terminal signal sequences located in proteins destined for import into the organelle. A synthetic peptide comprising amino acids 1-27 of pre-ornithine carbamyltransferase (pOCT) was found to interact specifically with a mitochondrial polypeptide of apparent molecular size 30 kDa. Membrane fractionation and protease accessibility analyses indicated that the polypeptide, designated p30, is located in the outer membrane. Binding of the synthetic peptide to p30 was saturable and reversible; Scatchard analysis of the binding data revealed a dissociation constant of 2 X 10(-6) M and predicts that p30 constitutes 4-10% of the outer mitochondrial membrane protein. Mild trypsin digestion of the mitochondrial surface destroyed both the ability of p30 to cross-link to the signal peptide and the ability of the organelle to import pOCT. Neither parameter was affected, however, by pretreatment of mitochondria with 1 M KCl.