MAS1, a gene essential for yeast mitochondrial assembly, encodes a subunit of the mitochondrial processing protease.

We have previously described a yeast mutant (mas1) that accumulates mitochondrial precursor proteins at high temperature and is deficient in the activity of a matrix-localized protease which cleaves presequences from mitochondrial precursor proteins. We have now cloned and sequenced the wild-type MAS1 gene and found that it encodes a subunit of the mitochondrial processing protease, that it is essential for cell viability and that the protein product participates in its own cleavage during import into mitochondria. The MAS1 protein is thus the first genetically defined component of the mitochondrial protein import pathway.     

The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix.

Yeast cytochrome c oxidase subunit IV (an imported mitochondrial protein) is made as a larger precursor with a transient pre-sequence of 25 amino acids. If this pre-sequence is fused to the amino terminus of mouse dihydrofolate reductase (a cytosolic protein) the resulting fusion protein is imported into the matrix space, and cleaved to a smaller size, by isolated yeast mitochondria. We have now fused progressively shorter amino-terminal segments of the subunit IV pre-sequence to dihydrofolate reductase and tested each fusion protein for import into the matrix space and cleavage by the matrix-located processing protease. The first 12 amino acids of the subunit IV pre-sequence were sufficient to direct dihydrofolate reductase into the mitochondrial matrix, both in vitro and in vivo. However, import of the corresponding fusion protein into the matrix was no longer accompanied by proteolytic processing. Fusion proteins containing fewer than nine amino-terminal residues from the subunit IV pre-piece were not imported into isolated mitochondria. The information for transporting attached mouse dihydrofolate reductase into mitochondria is thus contained within the first 12 amino acids of the subunit IV pre-sequence.     

Homologous and heterologous protein import into mitochondria isolated from the green alga Chlamydomonas reinhardtii

We have established a homologous system for studying mitochondrial protein import in Chlamydomonas reinhardtii, using C. reinhardtii precursor proteins and mitochondria isolated from C. reinhardtii. The precursors of the F₁ ATP synthase subunit and the Rieske FeS protein were imported into mitochondria with high efficiency, while the F₁ subunit precursor was imported with much lower efficiency. The import of heterologous precursor proteins from higher plants was also less efficient. The precursor of the C. reinhardtii PsaF chloroplast protein was converted into a protease-protected form upon incubation with mitochondria. In vitro processing studies revealed that in contrast to the situation in higher plants, the processing of the precursors was catalysed by a soluble, matrix-located peptidase.     

MAS6 encodes an essential inner membrane component of the yeast mitochondrial protein import pathway

To identify new components that mediate mitochondrial protein import, we analyzed mas6, an import mutant in the yeast Saccharomyces cerevisiae. mas6 mutants are temperature sensitive for viability, and accumulate mitochondrial precursor proteins at the restrictive temperature. We show that mas6 does not correspond to any of the presently identified import mutants, and we find that mitochondria isolated from mas6 mutants are defective at an early stage of the mitochondrial protein import pathway. MAS6 encodes a 23-kD protein that contains several potential membrane spanning domains, and yeast strains disrupted for MAS6 are inviable at all temperatures and on all carbon sources. The Mas6 protein is located in the mitochondrial inner membrane and cannot be extracted from the membrane by alkali treatment. Antibodies to the Mas6 protein inhibit import into isolated mitochondria, but only when the outer membrane has been disrupted by osmotic shock. Mas6p therefore represents an essential import component located in the mitochondrial inner membrane.     

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.     

Fluorescence-mediated analysis of mitochondrial preprotein import in vitro

Mitochondrial biogenesis is a crucial element of the functional maintenance of a eukaryotic cell. The organelle must import the majority of its proteins from the cytosol where they are synthesized as precursors. In vitro import assays have been developed in which isolated mitochondria are incubated with precursor proteins, that are generated either by in vitro translation systems or by expression and purification as recombinant proteins. The detection of imported proteins is performed by autoradiography or by Western blot. We have now established a novel detection system for imported precursor proteins that is based on fluorescent labeling. We constructed a mitochondrial preprotein containing a C-terminal SNAP-tag that can label itself with a single fluorescein molecule in an enzymatic reaction. The fluorescent preproteins were efficiently imported into isolated mitochondria and showed kinetic behavior similar to that of standard preproteins. The fluorescence detection was sensitive and significantly faster than other comparable procedures. We also showed that precursor proteins containing a SNAP-tag domain could be successfully labeled in a postimport reaction in intact mitochondria. In summary, the use of a reporter domain modified with a fluorescent dye provides a novel, sensitive, and fast detection method to analyze the properties of the mitochondrial import reaction in vitro.     

Precursor proteins are transported into mitochondria in the absence of proteolytic cleavage of the additional sequences

Many nuclear-coded mitochondrial proteins are synthesized as larger precursor polypeptides that are proteolytically processed during import into the mitochondrion. This processing appears to be catalyzed by a soluble, metal-dependent protease localized in the mitochondrial matrix. In this report we employ an in vitro system to investigate the role of processing in protein import. Intact Neurospora crassa mitochondria were incubated with radiolabeled precursors in the presence of the chelator o-phenanthroline. Under these conditions, the processing of the precursors of the beta-subunit of F1-ATPase (F1 beta) and subunit 9 of the F0F1-ATPase was strongly inhibited. Protease-mapping studies indicated that import of the precursor proteins into the mitochondria continued in the absence of processing. Upon readdition of divalent metal to the treated mitochondria, the imported precursors were quantitatively converted to their mature forms. This processing of imported precursors occurred in the absence of a mitochondrial membrane potential and was extremely rapid even at 0 degrees C. This suggests that all or part of the polypeptide chain of the imported precursors had been translocated into the matrix location of the processing enzyme. Localization experiments suggested that the precursor to F1 beta is peripherally associated with the mitochondrial membrane while the precursor to subunit 9 appeared to be tightly bound to the membrane. We conclude that proteolytic processing is not necessary for the translocation of precursor proteins across mitochondrial membranes, but rather occurs subsequent to this event. On the basis of these and other results, a hypothetical pathway for the import of F1 beta and subunit 9 is proposed.     

Mutant alcohol dehydrogenase (ADH III) presequences that affect both in vitro mitochondrial import and in vitro processing by the matrix protease.

Point mutations in the presequence of the mitochondrial alcohol dehydrogerase isoenzyme (ADH III) have been shown to affect either the import of the precursor protein into yeast mitochondria in vivo or its processing within the organelle. In the present work, the behavior of these mutants during in vitro import into isolated mitochondria was investigated. All point mutants tested were imported with a slower initial rate than that of the wild-type precursor. This defect was corrected when the precursors were treated with urea prior to import. Once imported, the extent of processing to the mature form of mutant precursors varied greatly and correlated well with the defects observed in vivo. This result was not affected by prior urea treatment. When matrix extracts enriched for the processing protease were used, this defect was shown to be due to failure of the protease to efficiently recognize or cleave the presequence, rather than to a lack of access to the precursor. The rate of import of two ADH III precursors bearing internal deletions in the leader sequence was similar to those of the point mutants, whereas a deletion leading to the removal of the 15 amino-terminal amino acids was poorly imported. The mature amino terminus of wild-type ADH III was determined to be Gln-25. Mutant m01 (Ser-26 to Phe), which reduced the efficiency of cleavage in vitro by 80%, was cleaved at the correct site.     

A matrix-located processing peptidase of plant mitochondria

Nuclear-encoded mitochondrial precursor proteins are proteolytically processed inside the mitochondrion after import. The general mitochondrial processing activity in plant mitochondria has been shown to be integrated into the cytochrome bc₁ complex of the respiratory chain. Here we investigate the occurrence of an additional, matrix-located processing activity by incubation of the precursors of the soybean mitochondrial proteins, alternative oxidase, the F_Ad subunit of the ATP synthetase and the tobacco F₁ subunit of the ATP synthase, with the membrane and soluble components of mitochondria isolated from soybean cotyledons and spinach leaves. A matrix-located peptidase specifically processed the precursors to the predicted mature form in a reaction which was sensitive to orthophenanthroline, a characteristic inhibitor of mitochondrial processing peptidase (MPP). The specificity of the matrix peptidase was illustrated by the inhibition of processing of the alternative oxidase precursor in both soybean and spinach matrix extracts upon altering a single amino acid residue in the targeting presequence (-2 Arg to Gly). Additionally, there was no evidence for general proteolysis of precursor proteins incubated with the matrix. The purity of the matrix fractions was ascertained by spectrophotometric and immunological analyses. The results demonstrate that there is a specific processing activity in the matrix of soybean and spinach in addition to the previously well characterized membrane-bound MPP integrated into the cytochrome bc₁ complex of the respiratory chain.     

Import of proteins into mitochondria. Import and maturation of the mitochondrial intermembrane space enzymes cytochrome b2 and cytochrome c peroxidase in intact yeast cells

The import of cytochrome b2 and cytochrome c peroxidase into mitochondria was investigated by pulse-chase experiments with intact yeast cells combined with subcellular fractionation. Import and processing of the precursors of these intermembrane space proteins is blocked by uncouplers of oxidative phosphorylation, indicating that an "energized" inner membrane is required. Cytochrome b2 is processed in two steps. The first step involves energy-dependent transport across both mitochondrial membranes and cleavage by a matrix-located protease to yield an intermediate which is smaller than the precursor, but larger than the mature protein. The second step involves conversion of the intermediate to the mature form. Whereas the precursor and the mature form are soluble, the intermediate is membrane-bound and exposed to the intermembrane space. The maturation of cytochrome c peroxidase is much slower than that of cytochrome b2. Proteolytic processing rather than import is rate-limiting since cytochrome c peroxidase precursor labeled during a 3-min pulse is already found attached to the outer face of the mitochondrial inner membrane. Import of cytochrome b2 and probably also of cytochrome c peroxidase thus involves energy-dependent transport to the matrix and cleavage by a matrix-localized protease. Maturation of cytochrome b2 proceeds in the sequence: soluble precursor leads to membrane-bound intermediate form leads to soluble mature form.     

Sequences distal to the mitochondrial targeting sequences are necessary for the maturation of the F1-ATPase beta-subunit precursor in mitochondria

The beta-subunit of the mitochondrial F1-ATPase is synthesized as a precursor in the cytoplasm which is delivered through two bilayers bounding the mitochondria prior to its assembly with other proteins into a functional complex. In order to determine the role of the amino-terminal 50 residues of the precursor on its localization, maturation, and assembly, a set of deletions within this region of the ATP2 gene encoding the beta-subunit has been analyzed. These studies reveal that deletions between residue 10 of the F1 beta-presequence and residue 36 can still direct in vivo mitochondrial import and assembly of the mutant subunit into a functional complex. Deletions within ATP2 which contain less than the first 10 residues of the precursor are not imported. Thus, the extreme amino terminus (about half of the transient presequence) of the F1 beta-subunit can direct its mitochondrial import. The wild-type F1 beta-subunit precursor is matured by the matrix-located metalloprotease at Lys19-Gln20; however, small in-frame deletions up to 17 residues distal to this site fail to be matured either in vitro or in vivo. This nonmatured F1 beta-subunit is also assembled into a functional enzyme and supports growth of its host on a nonfermentable carbon source. These data indicate that maturation of the F1 beta-subunit precursor is dependent on a protein sequence located distal to the proteolytic maturation site which is distinct from the mitochondrial targeting sequence.     

Polypeptides traverse the mitochondrial envelope in an extended state

Most mitochondrial proteins are synthesized as precursors in the cytosol and imported through contact sites between outer and inner mitochondrial membranes. The molecular mechanism of membrane translocation of precursor proteins is largely unclear. For this report, various hybrid proteins between portions of the precursor of cytochrome b2 and the entire dihydrofolate reductase (DHFR) were accumulated in mitochondrial contact sites. We unexpectedly found that about 50 amino acid residues of the polypeptide chain in transit were sufficient to span both membranes. This suggests a linear translocation of the polypeptide chain and presents evidence for a high degree of unfolding of polypeptides traversing the mitochondrial membranes.     

A metalloprotease involved in the processing of mitochondrial precursor proteins

A protease that cleaves the precursor of ornithine carbamoyltransferase (EC 2.1.3.3), a mitochondrial matrix enzyme, has been partially purified from the matrix fraction of rat liver mitochondria. The protease cleaved the precursors of several other matrix proteins at apparently correct sites. The protease was inhibited by 1,10-phenanthroline and EDTA, was reactivated by excess Mn2+ or Co2+, and did not cleave the alkali-denatured precursor proteins. These and other results indicate that this protease is responsible for the processing of at least several matrix protein precursors, and that the enzyme recognizes some three-dimensional conformation of the precursors as well as the amino acid sequences around the cleavage sites.     

Protein import into yeast mitochondria is accelerated by the outer membrane protein MAS70.

The yeast mitochondrial outer membrane contains a major 70 kd protein with an amino-terminal hydrophobic membrane anchor and a hydrophilic 60 kd domain exposed to the cytosol. We now show that this protein (which we term MAS70) accelerates the mitochondrial import of many (but not all) precursor proteins. Anti-MAS70 IgGs or removal of MAS70 from the mitochondria by either mild trypsin treatment or by disrupting the nuclear MAS70 gene inhibits import of the F1-ATPase beta-subunit, the ADP/ATP translocator, and of several other precursors into isolated mitochondria by up to 75%, but has little effect on the import of porin. Intact cells of a mas70 null mutant import the F1-ATPase alpha-subunit and beta-subunits, cytochrome c1 and other precursors at least several fold more slowly than wild-type cells. Removal of MAS70 from wild-type mitochondria inhibits binding of the ADP/ATP translocator to the mitochondrial surface, indicating that MAS70 mediates one of the earliest import steps. Several precursors are thus imported by a pathway in which MAS70 functions as a receptor-like component. MAS70 is not essential for import of these precursors, but only accelerates this process.     

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.     

蛋白质跨线粒体膜运送的研究进展

线粒体拥有约1000种蛋白质,其中98％以上系由细胞核编码,在细胞质核糖体上以前体形式合成,之后再运至线粒体,经跨膜运送并分选定位于各部分。现对定位于外膜、基质和内膜的蛋白质的运送途径的研究进展作一扼要介绍。脱血红素细胞色素c是细胞色素c的前体,它不含导肽,对其转运的研究概况也作了评述。     

Import into chloroplasts of a yeast mitochondrial protein directed by ferredoxin and plastocyanin transit peptides

Many chloroplast proteins are synthesized in the cytoplasm as precursors which contain an amino terminal transit peptide. These precursors are subsequently imported into chloroplast and targeted to one of several organellar locations. This import is mediated by the transit peptide, which is cleaved off during import. We have used the transit peptides of ferredoxin (chloroplast stroma) and plastocyanin (thylakoid lumen) to study chloroplast protein import and intra-organellar routing toward different compartments. Chimeric genes were constructed that encode precursor proteins in which the transit peptides are linked to yeast mitochondrial manganese superoxide dismutase. Chloroplast protein import and localization experiments show that both chimeric proteins are imported into the chloroplast stroma and processed. The plastocyanin transit sequence did not direct superoxide dismutase to the thylakoids; this protein was found in the stroma as an intermediate that still contains part of the plastocyanin transit peptide. The organelle specificity of these chimeric precursors reflected the transit peptide parts of the molecules, because neither the ferredoxin and plastocyanin precursors nor the chimeric proteins were imported into isolated yeast mitochondria.     

A Highlights from MBoC Selection: More than just a ticket canceller: the mitochondrial processing peptidase tailors complex precursor proteins at internal cleavage sites

Most mitochondrial proteins are synthesized as precursors that carry N-terminal presequences. After they are imported into mitochondria, these targeting signals are cleaved off by the mitochondrial processing peptidase (MPP). Using the mitochondrial tandem protein Arg5,6 as a model substrate, we demonstrate that MPP has an additional role in preprotein maturation, beyond the removal of presequences. Arg5,6 is synthesized as a polyprotein precursor that is imported into mitochondria and subsequently separated into two distinct enzymes. This internal processing is performed by MPP, which cleaves the Arg5,6 precursor at its N-terminus and at an internal site. The peculiar organization of Arg5,6 is conserved across fungi and reflects the polycistronic arginine operon in prokaryotes. MPP cleavage sites are also present in other mitochondrial fusion proteins from fungi, plants, and animals. Hence, besides its role as a “ticket canceller” for removal of presequences, MPP exhibits a second conserved activity as an internal processing peptidase for complex mitochondrial precursor proteins.     

Dissecting membrane insertion of mitochondrial beta-barrel proteins

Communication of mitochondria with the rest of the cell requires beta-barrel proteins of the outer membrane. All beta-barrel proteins are synthesized as precursors in the cytosol and imported into mitochondria by the general translocase TOM and the sorting machinery SAM. The SAM complex contains two proteins essential for cell viability, the channel-forming Sam50 and Sam35. We have identified the sorting signal of mitochondrial beta-barrel proteins that is universal in all eukaryotic kingdoms. The beta-signal initiates precursor insertion into a hydrophilic, proteinaceous membrane environment by forming a ternary complex with Sam35 and Sam50. Sam35 recognizes the beta-signal, inducing a major conductance increase of the Sam50 channel. Subsequent precursor release from SAM is coupled to integration into the lipid phase. We propose that a two-stage mechanism of signal-driven insertion into a membrane protein complex and subsequent integration into the lipid phase may represent a general mechanism for biogenesis of beta-barrel proteins.     

Import of proteins into yeast mitochondria: the nuclear MAS2 gene encodes a component of the processing protease that is homologous to the MAS1-encoded subunit.

The mas2 mutant of Saccharomyces cerevisiae is temperature sensitive for import of proteins into mitochondria. To identify the lesion in this mutant, we have cloned and sequenced the wild-type MAS2 gene and determined the intracellular location of its protein product. MAS2 encodes an essential 53-kd protein that is located in the mitochondrial matrix and is homologous to the MAS1 protein, a previously identified subunit of the protease that cleaves presequences from mitochondrial precursor proteins. The activity of this enzyme is temperature sensitive in mas2 cells. Together with the results of the accompanying study these results show that MAS2 and MAS1 encode the two subunits of the processing protease.