The four cytoplasmically made subunits of yeast mitochondrial cytochrome c oxidase are synthesized individually and not as a polyprotein.

Subunit-specific antisera prepared against each of the four cytoplasmically made subunits (IV, V, VI, and VII) of yeast mitochondrial cytochrome c oxidase (EC 1.9.3.1) were used to precipitate immunoreactive polypeptides that were synthesized either in vitro, in a cell-free protein-synthesizing system programmed with total yeast mRNA, or in vivo, in intact cells and in spheroplasts, under conditions of pulse labeling, pulse-chase labeling, and continuous labeling. Using N-formyl-[35S]Met-rTNA as the only radioactively labeled component in the cell-free system, we demonstrated (i) that each of the four cytoplasmically made subunits is synthesized as a separate entity and not as part of a polyprotein as was claimed by others; (ii) that subunits IV, V, and VI are synthesized as precursors, larger by 1500-3000 daltons than their mature counterparts; in contrast, subunit VII is not synthesized as a larger precursor. Precursor forms of subunits IV, V, and VI identical to those synthesized in vitro were also detected in vivo by pulse-labeling of spheroplasts. The observed disappearance of these larger forms after a chase is compatible with the notion that they represent short-lived precursors that are rapidly converted to their mature counterparts during or shortly after import into mitochondria. Furthermore, using N-formyl-[35S]Met-tRNA, we provide definitive evidence that two of the cytoplasmically made subunits (beta and gamma) of another oligomeric inner mitochondrial membrane protein (F1-ATPase, EC 3.6.1.3) are not synthesized as part of a polyprotein but as individual precursors.     

Energy-dependent processing of cytoplasmically made precursors to mitochondrial proteins.

Earlier work has shown that mitochondrial proteins synthesized in the cytosol are initially made as larger precursors which are then transferred into the organelles and processed to their mature size in the absence of protein synthesis. It is now demonstrated that depletion of the mitochondrial matrix ATP in intact yeast spheroplasts by various combinations of inhibitors and mutations prevents the processing of precursors to the three largest subunits of the mitochondrial F1-ATPase and two subunits of the cytochrome bc1 complex. These polypeptides are all synthesized outside the mitochondria and transported to the mitochondrial matrix or inserted into the mitochondrial inner membrane. In contrast, depletion of the matrix ATP does not inhibit processing of the precursor to cytochrome c peroxidase; this enzyme is located in the mitochondrial intermembrane space which is freely accessible to ATP made in the cytosol. The processing of extramitochondrially made precursors or the transfer of these precursors across the mitochondrial inner membrane is thus dependent on ATP.     

Cytoplasmically made subunits of yeast mitochondrial F1-ATPase and cytochrome c oxidase are synthesized as individual precursors, not as polyproteins.

At least three subunits of yeast mitochondrial F1-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and at least two subunits of cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) are synthesized outside the mitochondria and imported into the organelles as individual precursors that are between 2000 and 6000 daltons larger than the mature subunits. These precursors were shown to be primary translation products. Therefore, neither the five F1 subunits nor the four small cytochrome c oxidase subunits are synthesized as a single polyprotein.     

Mitochondrial membrane biogenesis: identification of a precursor to yeast cytochrome c oxidase subunit II, an integral polypeptide.

Many of the polypeptides made on endogenous ribosomes inside of yeast mitochondria are hydrophobic "integral polypeptides" which are subunits of at least three oligomeric enzyme complexes (cytochrome c oxidase, rutamycin-sensitive ATPase, and coenzyme QH2-cytochrome c reductase) of the inner mitochondrial membrane. In order to elucidate the pathway(s) followed by these polypeptides into the inner membrane we have used an in vitro mitochondrial translation system from yeast. By inhibiting this system with aurintricarboxylic acid, we have been able to demonstrate and accumulate a transient precursor to subunit II of cytochrome c oxidase. This precursor, designated II', is approximately 1,500 daltons larger than mature subunit II and most likely is a form of subunit II with an NH2-terminal extension. Although this precursor appears to be processed cotranslationally under normal conditions, it does associate in unprocessed form with mitochondrial membranes when allowed to accumulate in the presence of aurintricarboxylic acid, and it can be processed postranslationally upon removal of the drug. None of the other mitochondrial translation products made in this system exhibits larger precursors. These results indicate that at least one mitochondrial translation product has a transient "leader sequence" a,d is inserted into the inner mitochondrial membrane and processed cotranslationally, but they suggest that other pathways may be followed by the other translation products.     

Imported mitochondrial proteins cytochrome b2 and cytochrome c1 are processed in two steps.

Cytochrome b2 of yeast is located in the space between the inner and outer mitochondrial membranes whereas cytochrome c1 is bound to the outer face of the mitochondrial inner membrane. Both proteins are made outside the mitochondria as larger precursors that are processed to their mature forms in two steps. In the first step, at least a segment of the precursor polypeptide chain penetrates into the mitochondrial matrix and is cleaved to an intermediate form by a matrix-localized soluble protease. This step requires an electrochemical gradient across the mitochondrial inner membrane. In the second step, the intermediate form is converted to the mature form. For cytochrome c1, this second step requires heme. Import of these two cytochromes thus involves a "detour" across the inner membrane.     

Two nuclear mutations that block mitochondrial protein import in yeast.

We isolated two yeast mutants that are temperature-sensitive for import of mitochondrial proteins. Each strain contains a single mutation that results in arrest of growth and accumulation of precursor to the beta subunit of the mitochondrial F1-ATPase after incubation at 37 degrees C. These lesions (mas1 and mas2) are nonallelic and recessive. Cells harboring either mutation stop growing only after 2-3 generations at 37 degrees C. Import of the F1 beta subunit at 37 degrees C is more than 250 times slower in mas1 and 15 times slower in mas2 than in wild-type cells. At 23 degrees C, import occurs with similar rates in mutant and wild-type cells. The two mutations also reduce the rate of import of other proteins; however, import of different precursors is affected to different degrees in the two strains. The temperature-sensitive step in import in both mas1 and mas2 occurs before arrival of precursors in the mitochondrial matrix.     

Cyclophilin catalyzes protein folding in yeast mitochondria.

Cyclophilins are a family of ubiquitous proteins that are the intracellular target of the immunosuppressant drug cyclosporin A. Although cyclophilins catalyze peptidylprolyl cis-trans isomerization in vitro, it has remained open whether they also perform this function in vivo. Here we show that Cpr3p, a cyclophilin in the matrix of yeast mitochondria, accelerates the refolding of a fusion protein that was synthesized in a reticulocyte lysate and imported into the matrix of isolated yeast mitochondria. The fusion protein consisted of the matrix-targeting sequence of subunit 9 of F1F0-ATPase fused to mouse dihydrofolate reductase. Refolding of the dihydrofolate reductase moiety in the matrix was monitored by acquisition of resistance to proteinase K. The rate of refolding was reduced by a factor of 2-6 by 2.5 microM cyclosporin A. This reduced rate of folding was also observed with mitochondria lacking Cpr3p. In these mitochondria, protein folding was insensitive to cyclosporin A. The rate of protein import was not affected by cyclosporin A or by deletion of Cpr3p.     

Import and processing of human ornithine transcarbamoylase precursor by mitochondria from Saccharomyces cerevisiae.

Expression of the subunit precursor of the human mitochondrial matrix enzyme ornithine transcarbamoylase (OTCase; EC 2.1.3.3) was programmed in Saccharomyces cerevisiae from a 2-micron plasmid by using an inducible galactose operon promoter. In the presence of the inducing sugar (galactose), two polypeptides were specifically precipitable with anti-OTCase antiserum: the human OTCase precursor (40 kDa); and the mature OTCase subunit (36 kDa). When yeast cells containing these species were lysed and fractionated, the OTCase precursor was found to be associated with mitochondrial membranes, while the mature subunit was found partly with mitochondrial membranes and partly in the soluble mitochondrial matrix-containing fraction. When OTCase enzymatic activity was assayed in fractions similarly derived from an S. cerevisiae strain devoid of yeast OTCase activity (an arg3 mutant) but expressing human OTCase, activity was detected specifically in the mitochondrial matrix fraction. A mutant human OTCase precursor containing an artificial mutation in the NH2-terminal leader peptide (arginine-23 to glycine) was similarly examined. As was previously observed with mammalian mitochondria, this precursor failed both to reach the matrix compartment and to be proteolytically processed; it also failed to exhibit OTCase enzymatic activity. Presence of OTCase enzymatic activity in an arg3 strain expressing wild-type precursor was utilized to obtain selective growth in a medium devoid of arginine but supplemented with the OTCase substrate ornithine. We conclude that, during evolution, the pathway of mitochondrial import utilized by the human OTCase precursor is conserved between yeast and humans, and that, by using selective growth conditions, it may be possible to examine genetically this pathway in S. cerevisiae.     

Import of proteins into mitochondria: A novel pathomechanism for progressive neurodegeneration

The vast majority of mitochondrial proteins are encoded as precursors by the nuclear genome. A major aspect of mitochondrial biogenesis is therefore the transfer of nuclear-encoded, cytosplasmically synthesized precursor proteins across and into the mitochondrial membranes. During the past years the use of simple model organisms such as the yeasts S. cerevisiae and N. crassa has helped considerably to identify and unravel the structure and function of a substantial number of components involved in targeting of nuclear-encoded preproteins to mitochondria. Several pathways and a number of components were characterized that are involved in guiding mitochondrial preproteins to their specific sites of function. In particular, import of nuclear-encoded precursor proteins into and across the mitochondrial inner membrane is mediated by two distinct translocases, the TIM23 complex and the TIM22 complex. Both TIM complexes cooperate with the general preprotein translocase of the outer membrane, TOM complex. The TIM complexes differ in the their substrate specificity. While the TIM23 complex mediates import of preproteins with a positively charged matrix targeting signal, the TIM22 complex facilitates the insertion of a class of hydrophobic proteins with internal targeting signals into the inner membrane. Most recently the rapid progress of research has allowed elucidation of a new mitochondrial disease on the molecular level. This rare X-linked progressive neurodegenerative disorder, named Mohr-Tranebjaerg (MT syndrome), is caused by mutations in the DDP1 gene and includes sensorineural deafness, blindness, mental retardation and a complex movement disorder. The analysis of the novel pathomechanism is based on the homology of the affected DDP1 protein to a family of conserved yeast components acting along the TIM22 pathway. This contribution briefly summarizes the current knowledge of the pathways of protein import and proposes a mechanism to explain how defective import leads to neurodegeneration.     

In vitro transport of F1-ATPase beta-subunit into mitochondria of Zajdela hepatoma and rat liver

Transport of precursor of F1-ATPase beta-subunit into isolated mitochondria of Zajdela hepatoma and rat liver was examined. The hepatoma mitochondria were more active in the process than the liver organelles indicating that the relative F1-ATPase deficiency in the tumor mitochondria does not result from an impaired transport of F1-ATPase subunits into the tumor organelles. Similar results were obtained using digitonin-treated rat hepatocytes and Zajdela hepatoma cells instead of isolated mitochondria. The suitability of the digitonin-treated cells in the study of protein transport into mitochondria in vitro is demonstrated and the advantages of this system over isolated mitochondria are discussed.     

Identification of a 45-kDa protein at the protein import site of the yeast mitochondrial inner membrane.

Import of proteins into mitochondria involves the cooperation of protein translocation systems in the outer and inner membranes. We have identified a 45-kDa protein at the protein import site of the yeast mitochondrial inner membrane. This 45-kDa protein could be crosslinked to a partly translocated precursor, which cannot be imported across the inner membrane when the matrix is depleted of ATP. In addition, an antibody against this protein strongly inhibited protein import into right-side-out inner-membrane vesicles. The 45-kDa protein accounts for only 0.1% of mitochondrial protein and appears peripherally attached to the outer face of the inner membrane. The properties of this protein suggest that it is a component of the protein import system of the mitochondrial inner membrane.     

Carboxyl-terminal sequences influence the import of mitochondrial protein precursors in vivo.

The large subunit of carbamoyl phosphate synthase A [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5] from Neurospora crassa is encoded by a nuclear gene but is localized in the mitochondrial matrix. We have utilized N. crassa strains that produce both normal and carboxyl-terminal-truncated forms of carbamoyl phosphate synthase A to ask whether the carboxyl terminus affects import of the carbamoyl phosphate synthase A precursor. We found that carboxyl-terminal-truncated precursors were directed to mitochondria but that they were imported less efficiently than full-length proteins that were synthesized in the same cytoplasm. Our results suggest that effective import of proteins into mitochondria requires appropriate combinations of targeting sequences and three-dimensional structure.     

Uptake of labeled alloxan in mouse organs and mitochondria in vivo and in vitro

[14C]2-Alloxan was administered in vivo and in vitro for study of the uptake of alloxan in different organs and their mitochondia of mice. After in vivo administration, radioactivity was demonstrated in all organs investigated, with quantitative differences: endocrine pancreas greater than liver greater than exocrine pancreas and heart. No significant difference was found between the iv and ip routes of injection. An in vivo uptake of alloxan was also found in mitochondria, with significant quantitative differences as to the origin of the organelles: endocrine pancreas greater than liver greater than exocrine pancreas and heart. Pretreatment with D-glucose caused significantly decreased uptake in liver, exocrine pancreas, and heart, but significantly increased uptake in endocrine pancreas, whereas the uptake was significantly decreased in the mitochondria from all of these organs. In vitro uptake was observed in all kinds of mitochondria studied. This uptake was higher than the in vivo uptake in mitochondria from liver, exocrine pancreas, and heart, whereas the uptake in vivo was higher than the in vitro uptake in islet mitochondria. The presence of D-glucose did not affect the in vitro uptake of alloxan in mitochondria. The findings show that in vivo, alloxan passes across plasma membranes and is taken up by mitochondria, and data obtained with mitochondrial subfractions may also indicate a passage across mitochondrial membranes. D-Glucose protection against alloxan diabetogenicity may be associated with prevention of mitochondrial uptake of alloxan. This prevention seems to be dependent on the metabolism of glucose.     

Posttranslational uptake and processing of in vitro synthesized ornithine transcarbamoylase precursor by isolated rat liver mitochondria.

The mitochondrial matrix enzyme ornithine transcarbamoylase (OTCase; ornithine carbamoyltransferase; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) is encoded by a nuclear gene on the X chromosome, synthesized on cytoplasmic ribosomes, and translocated across both mitochondrial membranes. Using specific immunoprecipitation, we presented evidence previously that the primary in vitro translation product of OTCase in rat liver is a polypeptide about 4000 daltons larger than the "mature" OTCase augment subunit purified from homologous mitochondria. In this report we augment the immunological identification of this cell-free translation product (pOTCase) with structural information and show, by electrophoresis of proteolysis products, that pOTCase is structurally similar to mitochondrial OTCase. Moreover, we now demonstrate that, when pOTCase is incubated posttranslationally with isolated rat liver mitochondria, it is converted to the size of mature OTCase and is sequestered within the mitochondria in such a way that it becomes resistant to externally added proteases. Such posttranslational processing is catalyzed specifically by the mitochondrial fraction of rat liver cells and is dependent both on the duration of incubation with mitochondria and on the amount of mitochondrial protein added. We conclude that pOTCase is indeed the bona fide precursor of mitochondrial OTCase and that use of this simplified cell-free system will facilitate analysis of OTCase biogenesis at both the cellular and the molecular level.     

In vivo zippering of inner and outer mitochondrial membranes by a stable translocation intermediate

It was previously assumed that the import of cytoplasmically synthesized precursor proteins into mitochondria occurs through a single structure spanning both outer and inner membranes at contact sites. Based on recent findings, however, the two membranes appear to contain independent translocation elements that reversibly cooperate during protein import. This feature makes it difficult to generate a means of isolating a fully integrated and functional translocation complex. To study these independent translocases in vitro and in vivo, we have constructed a chimeric protein consisting of an N-terminal authentic mitochondrial precursor (delta¹-pyrroline-5-carboxylate dehydrogenase) linked, through glutathione S-transferase, to IgG binding domains derived from staphylococcal protein A. This construct becomes trapped en route to the matrix, spanning both outer and inner membranes in such a way that the entire signal-less delta¹-pyrroline-5-carboxylate dehydrogenase moiety reaches the matrix, while only the folded protein A domain remains outside. During in vivo import of this precursor, outer and inner membranes of yeast mitochondria become progressively “zippered” together, forming long stretches of close contact. Using this novel intermediate, the outer and inner mitochondrial membrane channels, which normally interact only transiently, can be tightly joined (both in vitro and in vivo), forming a stable association. This suggests a method for isolating the functional translocation complex as a single entity.     

The general mitochondrial matrix processing protease from rat liver: structural characterization of the catalytic subunit.

A critical step in the import of nuclear-encoded precursor proteins into mitochondria involves proteolytic cleavage of their amino-terminal leader peptides by processing proteases found in the mitochondrial matrix. We report here the characterization of the general matrix processing protease from rat liver mitochondria. The final enzyme preparation consisted of two polypeptides, a catalytically active 55-kDa subunit and a 52-kDa one. To deduce the complete primary structure of the 55-kDa subunit, we first sequenced its mature amino terminus and several tryptic peptides derived from the pure protein. Next, using mixed oligonucleotide primers that had sequences based on two of these peptides, we synthesized a partial cDNA probe by selective amplification of liver RNA with the polymerase chain reaction. The amplified probe was then used to obtain a nearly full-length clone from a rat liver cDNA library. This cDNA codes for 508 amino acid residues, including 16 residues of an amino-terminal leader peptide, the cleavage site of which is located two polypeptide bonds downstream from an arginine residue. The mature portion has a predicted molecular mass of 55.2 kDa; it shows 36% identity with the mitochondrial processing peptidases of Saccharomyces cerevisiae and Neurospora crassa. A conserved structural feature is a putative, negatively charged alpha-helix, located in the amino-terminal half of the subunit; this element might be important for the recognition of positively charged leader peptides characteristic of mitochondrial precursor proteins.     

Functional reconstitution in Escherichia coli of the yeast mitochondrial matrix peptidase from its two inactive subunits.

The matrix processing peptidase from yeast (Saccharomyces cerevisiae) mitochondria was expressed in Escherichia coli via a plasmid-borne operon encoding the mature forms of the alpha and beta subunits of the enzyme. The subunits assembled into a fully active, soluble enzyme. The mature subunits were also expressed individually. The alpha subunit accumulated in large amounts and was obtained at a purity of 80% after a single chromatographic step. The beta-subunit-producing strain expressed an intact and a degraded form of the beta subunit, both of them soluble in the cytoplasm. Extract from either the alpha- or the beta-subunit-producing strain (S-alpha or S-beta extract, respectively), as well as the purified alpha subunit, was enzymatically inactive. However, precursor cleavage activity was restored by mixing either the S-alpha extract or the purified alpha subunit with the S-beta extract. The reconstituted processing activity was indistinguishable from the authentic holopeptidase.     

Effects of Chronic Ethanol Treatment on Rat Liver Mitochondrial Protein Synthesis

Chronic ethanol treatment of male Sprague-Dawley rats resulted in a 50% decrease in the rate of incorporation of precursor leucine into isolated mitochondria. This decrease is manifest in a decreased labeling of three polypeptides of inner mitochondrial membranes that are the major products of in vitro mitochondrial protein synthesis under the conditions employed. Immunoprecipitation of cytochrome-c oxidase revealed that these three polypeptides are subunits 1, 2, and 3 of cytochrome-c oxidase and have apparent molecular weights of 33,000, 25,000, and 20,000. Sixty percent of the total incorporated radioactivity is associated with these polypeptides. A decrease in the contents of subunit 2 and of a second polypeptide with an apparent molecular weight of 22,000 was also noted as an effect of chronic ethanol treatment.     

In vivo protein-interaction mapping of a mitochondrial translocator protein Tom22 at work

Mitochondrial protein import requires cooperation of the machineries called translocators in the outer and inner mitochondrial membranes. Here we analyze the interactions of Tom22, a multifunctional subunit of the outer membrane translocator TOM40 complex, with other translocator subunits such as Tom20, Tom40, and Tim50 and with substrate precursor proteins at a spatial resolution of the amino acid residue by in vivo and in organello site-specific photocross-linking. Changes in cross-linking patterns caused by excess substrate precursor proteins or presequence peptides indicate how the cytosolic receptor domain of Tom22 accepts substrate proteins and how the intermembrane space domain of Tom22 transfers them to Tim50 of the inner-membrane translocator.