Insertion into the mitochondrial inner membrane of a polytopic protein, the nuclear-encoded Oxa1p.

Oxa1p, a nuclear-encoded protein of the mitochondrial inner membrane with five predicted transmembrane (TM) segments is synthesized as a precursor (pOxa1p) with an N-terminal presequence. It becomes imported in a process requiring the membrane potential, matrix ATP, mt-Hsp70 and the mitochondrial processing peptidase (MPP). After processing, the negatively charged N-terminus of Oxa1p (approximately 90 amino acid residues) is translocated back across the inner membrane into the intermembrane space and thereby attains its native N(out)-C(in) orientation. This export event is dependent on the membrane potential. Chimeric preproteins containing N-terminal stretches of increasing lengths of Oxa1p fused on mouse dehydrofolate reductase (DHFR) were imported into isolated mitochondria. In each case, their DHFR moieties crossed the inner membrane into the matrix. Thus Oxa1p apparently does not contain a stop transfer signal. Instead the TM segments are inserted into the membrane from the matrix side in a pairwise fashion. The sorting pathway of pOxa1p is suggested to combine the pathways of general import into the matrix with a bacterial-type export process. We postulate that at least two different sorting pathways exist in mitochondria for polytopic inner membrane proteins, the evolutionarily novel pathway for members of the ADP/ATP carrier family and a conserved Oxa1p-type pathway.     

Conserved mechanism of Oxa1 insertion into the mitochondrial inner membrane

Oxa1 is the mitochondrial representative of a family of related proteins that mediate the insertion of substrate proteins into the membranes of bacteria, chloroplasts, and mitochondria. Several studies have demonstrated that the bacterial homologue YidC participates both in the direct uptake of proteins from the bacterial cytosol, and in the uptake of nascent proteins from the Sec translocase. Studies on the biogenesis of membrane proteins in mitochondria established that Oxa1 has the capability to receive substrates at the inner surface of the inner membrane. In this study, we asked if Oxa1 may similarly cooperate with a protein translocase within the membrane. Since Oxa1 is involved in its own biogenesis, we used the precursor of Oxa1 as a model protein and investigated its import pathway. We found that immediately after import into mitochondria, Oxa1 initially accumulates at Tim23 that forms the inner membrane protein translocase. Cleavage of the Oxa1 presequence is dependent on mtHsp70, a heat shock protein of the mitochondrial matrix. However, mutant mtHsp70 showing a defect in the release of bound substrate proteins does not interfere with subsequent membrane insertion, indicating that membrane insertion of the mature protein is essentially mtHsp70-independent. We conclude that Oxa1 has the ability to accept preproteins within the membrane.     

Insertion of bitopic membrane proteins into the inner membrane of mitochondria involves an export step from the matrix

The mitochondrial inner membrane contains a large number of polytopic proteins that are derived from prokaryotic ancestors of mitochondria. Little is known about the intramitochondrial sorting of these proteins. We chose two proteins of known topology as examples to study the pathway of insertion into the inner membrane; Mrs2 and Yta10 are bitopic proteins that expose negatively charged loops of different complexity into the intermembrane space. Here we show that both Mrs2 and Yta10 transiently accumulate as sorting intermediates in the matrix before they integrate into the inner membrane. The sorting pathway of both proteins can be separated into two sequential reactions: (i) import into the matrix and (ii) insertion from the matrix into the inner membrane. The latter process was found to depend on the membrane potential and, in this respect, is similar to the insertion of membrane proteins in bacteria. A comparison of the charge distribution of intermembrane space loops in a variety of mitochondrial inner membrane proteins suggests that this mode of "conservative sorting" might be the typical insertion route for polytopic inner membrane proteins that originated from bacterial ancestors.     

Insertion of hydrophobic membrane proteins into the inner mitochondrial membrane--a guided tour

Only a few mitochondrial proteins are encoded by the organellar genome. The majority of mitochondrial proteins are nuclear encoded and thus have to be transported into the organelle from the cytosol. Within the mitochondrion proteins have to be sorted into one of the four sub-compartments: the outer or inner membranes, the intermembrane space or the matrix. These processes are mediated by complex protein machineries within the different compartments that act alone or in concert with each other. The translocation machinery of the outer membrane is formed by a multi-subunit protein complex (TOM complex), that is built up by signal receptors and the general import pore (GIP). The inner membrane houses two multi-subunit protein complexes that each handles special subsets of mitochondrial proteins on their way to their final destination. According to their primary function these two complexes have been termed the pre-sequence translocase (or TIM23 complex) and the protein insertion complex (or TIM22 complex). The identification of components of these complexes and the analysis of the molecular mechanisms underlying their function are currently an exciting and fast developing field of molecular cell biology.     

The ER membrane complex (EMC) can functionally replace the Oxa1 insertase in mitochondria

Two multisubunit protein complexes for membrane protein insertion were recently identified in the endoplasmic reticulum (ER): the guided entry of tail anchor proteins (GET) complex and ER membrane complex (EMC). The structures of both of their hydrophobic core subunits, which are required for the insertion reaction, revealed an overall similarity to the YidC/Oxa1/Alb3 family members found in bacteria, mitochondria, and chloroplasts. This suggests that these membrane insertion machineries all share a common ancestry. To test whether these ER proteins can functionally replace Oxa1 in yeast mitochondria, we generated strains that express mitochondria-targeted Get2–Get1 and Emc6–Emc3 fusion proteins in Oxa1 deletion mutants. Interestingly, the Emc6–Emc3 fusion was able to complement an Δoxa1 mutant and restored its respiratory competence. The Emc6–Emc3 fusion promoted the insertion of the mitochondrially encoded protein Cox2, as well as of nuclear encoded inner membrane proteins, although was not able to facilitate the assembly of the Atp9 ring. Our observations indicate that protein insertion into the ER is functionally conserved to the insertion mechanism in bacteria and mitochondria and adheres to similar topological principles.

Redirecting the core subunits of the protein membrane insertion complex EMC into mitochondria rescues cells deficient for the mitochondrial Oxa1 system; this supports the hypothesis that the machinery for protein insertion into the ER membrane is functionally analogous to the YidC/Oxa1/Alb3 family of bacteria, mitochondria and chloroplasts.     

Protein insertion into the inner membrane of mitochondria

The inner membrane of mitochondria harbours a large number of polypeptides, many of which have evolved from proteins of the prokaryotic progenitors of mitochondria. The sorting routes on which these proteins are integrated into the mitochondrial inner membrane reflect their phylogenetic origin: Proteins of eukaryotic descent typically reach their destination following arrest of import at the level of the inner membrane. In contrast, many proteins inherited from the prokaryotic progenitor cell are inserted into the inner membrane in an export step following translocation into the matrix. Recently, three different insertion pathways from the matrix into the inner membrane were identified which show considerable parallels to the protein insertion processes in bacteria and chloroplasts. Two of these pathways depend on the related inner membrane proteins Oxa1 and Cox18. A third route is less well defined and depends on the membrane-associated matrix protein Mba1.     

Protein export across the inner membrane of mitochondria: the nature of translocated domains determines the dependence on the Oxa1 translocase

The biogenesis of mitochondria requires the insertion of both nuclear and mitochondrially encoded proteins into the inner membrane. The inner membrane protein Oxa1 plays an important role in this process. Translocation of the terminal intermembrane space domains of subunit 2 of the cytochrome oxidase complex, Cox2, strictly depends on Oxa1. In contrast, other Oxa1 substrates can be inserted independently of Oxa1 function, although at reduced efficiency. A Saccharomyces cerevisiae mutant containing a large deletion in its mitochondrial genome allowed us to analyze the insertion process of a fusion protein of cytochrome b and Cox2. In this mutant, the N-terminal domain of Cox2 is synthesized as a hairpin loop that is flanked by hydrophobic transmembrane segments on both sides. Both genetic and biochemical evidences indicate that translocation of this region across the inner membrane still requires Oxa1 function. Thus, the position of intermembrane space domains within protein sequences does not appear to determine their dependence on the Oxa1 translocase. Our observations rather suggest that the dependence on Oxa1 correlates with the net charge of the domain that has to be translocated across the lipid bilayer.     

Lateral release of proteins from the TOM complex into the outer membrane of mitochondria

The TOM complex of the outer membrane of mitochondria is the entry gate for the vast majority of precursor proteins that are imported into the mitochondria. It is made up by receptors and a protein conducting channel. Although precursor proteins of all subcompartments of mitochondria use the TOM complex, it is not known whether its channel can only mediate passage across the outer membrane or also lateral release into the outer membrane. To study this, we have generated fusion proteins of GFP and Tim23 which are inserted into the inner membrane and, at the same time, are spanning either the TOM complex or are integrated into the outer membrane. Our results demonstrate that the TOM complex, depending on sequence determinants in the precursors, can act both as a protein conducting pore and as an insertase mediating lateral release into the outer membrane.     

Ribosome binding to the Oxa1 complex facilitates co-translational protein insertion in mitochondria

The Oxa1 translocase of the mitochondrial inner membrane facilitates the insertion of both mitochondrially and nuclear-encoded proteins from the matrix into the inner membrane. Most mitochondrially encoded proteins are hydrophobic membrane proteins which are integrated into the lipid bilayer during their synthesis on mitochondrial ribosomes. The molecular mechanism of this co-translational insertion process is unknown. Here we show that the matrix-exposed C-terminus of Oxa1 forms an alpha-helical domain that has the ability to bind to mitochondrial ribosomes. Deletion of this Oxa1 domain strongly diminished the efficiency of membrane insertion of subunit 2 of cytochrome oxidase, a mitochondrially encoded substrate of the Oxa1 translocase. This suggests that co-translational membrane insertion of mitochondrial translation products is facilitated by a physical interaction of translation complexes with the membrane-bound translocase.     

Proline residues of transmembrane domains determine the sorting of inner membrane proteins in mitochondria

Most inner membrane proteins of mitochondria are synthesized in the cytosol and reach the inner membrane using one of two alternative sorting pathways. On the stop transfer route, proteins are arrested during import at the level of the inner membrane. The conservative sorting pathway involves translocation through the inner membrane and insertion from the matrix. It is unclear how the translocase of the inner membrane 23 protein translocation machinery differentiates between the two classes of proteins. Here we show that proline residues in hydrophobic stretches strongly disfavor the translocation arrest of transmembrane domains (TMDs) and favor the transfer of preproteins to the matrix. We propose that proline residues, together with the hydrophobicity of the TMD and the presence of charged residues COOH-terminally flanking the TMD, are determinants of the intramitochondrial sorting of inner membrane proteins.     

Oxa1/Alb3/YidC system for insertion of membrane proteins in mitochondria, chloroplasts and bacteria (review)

Recent studies have shown that there is a pathway that is evolutionarily conserved for the insertion of proteins into the membrane in mitochondria, chloroplasts, and bacteria. In this pathway, the Oxa1/Alb3/YidC proteins are believed to function as membrane insertases that play an important role in the membrane protein biogenesis of respiratory and energy transduction proteins. Additional roles of the Oxa1/Alb3/YidC members may be in the lateral integration of proteins into the lipid bilayer, and in the folding and assembly of proteins into membrane protein complexes.     

Inhibition of the transport of citrate during ADP-ribosylation of inner membrane proteins of the mitochondria

The ability of rat liver submitochondrial particles to catalyze NAD+ hydrolysis with a transfer of ADP-ribose residues to protein membranes has been demonstrated ADP-ribosylation is directly dependent on NAD+ concentration upon saturation with 1 mM NAD+ and is inhibited by physiological compounds (e.g., ATP, 10 mM; nicotinamide, 10 mM); besides, it is an artificial acceptor of ADP-ribose, arginine methyl ester. It was found that ADP-ribose is accepted by inner mitochondrial membrane protein, whose molecular masses amount to 25-30 kDa. The fact that 5'-AMP is a product of ADP-ribose degradation by snake venom phosphodiesterase suggests that the inner membrane vesiculate proteins are modified by mono(ADP-ribose). Covalent modification of membrane proteins by ADP-ribose leads to citrate transport inhibition in inner membrane vesicles the [14C]citrate uptake is significantly decreased thereby. The ability of ADP-ribosylation inhibitors to restore the citrate transport rate is suggestive of a direct regulatory effect of NAD+-dependent ADP-ribosylation on the activity of citrate-translocating system of inner mitochondrial membranes.     

Disrupted yeast mitochondria can import precursor proteins directly through their inner membrane

Import of precursor proteins into the yeast mitochondrial matrix can occur directly across the inner membrane. First, disruption of the outer membrane restores protein import to mitochondria whose normal import sites have been blocked by an antibody against the outer membrane or by a chimeric, incompletely translocated precursor protein. Second, a potential- and ATP-dependent import of authentic or artificial precursor proteins is observed with purified inner membrane vesicles virtually free of outer membrane components. Third, import into purified inner membrane vesicles is insensitive to antibody against the outer membrane. Thus, while outer membrane components are clearly required in vivo, the inner membrane contains a complete protein translocation system that can operate by itself if the outer membrane barrier is removed.     

Insertion of newly synthesized proteins into the outer membrane of Escherichia coli.

The insertion of newly synthesized proteins into the outer membrane of Escherichia coli has been examined. The results show that there is no precurser pool of outer membrane proteins in the cytoplasmic membrane because first, the incorporation of a [35S]methionine pulse into outer membrane proteins completely parallels its incorporation into cytoplasmic membrane proteins, and second, under optimal isolation conditions, no outer membrane proteins are found in the cytoplasmic membrane, even when the membranes are analysed after being labeled for only 15 s. The [35S]methionine present in the outer membrane after a pulse of 15 s was found in protein fragments of varying sizes rather than in specific outer membrane proteins. This label could however be chased into specific proteins within 30--120 s, depending on the size of the protein, indicating that although unfinished protein fragments were present in the outer membrane, they were completed by subsequent chain elongation. Thus, outer membrane proteins are inserted into the outer membrane while still attached to ribosomes. Since ribosomes which are linked to the cell envelope by nascent polypeptide chains are stationary, the mRNA which is being translated by these ribosomes moves along the inner cell surface.     

Mitochondrial protein import: Mia40 facilitates Tim22 translocation into the inner membrane of mitochondria

The mitochondrial intermembrane space assembly (MIA) pathway is generally considered to be dedicated to the redox-dependent import and biogenesis of proteins localized to the intermembrane space of mitochondria. The oxidoreductase Mia40 is a central component of the pathway responsible for the transfer of disulfide bonds to intermembrane space precursor proteins, causing their oxidative folding. Here we present the first evidence that the function of Mia40 is not restricted to the transport and oxidative folding of intermembrane space proteins. We identify Tim22, a multispanning membrane protein and core component of the TIM22 translocase of inner membrane, as a protein with cysteine residues undergoing oxidation during Tim22 biogenesis. We show that Mia40 is involved in the biogenesis and complex assembly of Tim22. Tim22 forms a disulfide-bonded intermediate with Mia40 upon import into mitochondria. Of interest, Mia40 binds the Tim22 precursor also via noncovalent interactions. We propose that Mia40 not only is responsible for disulfide bond formation, but also assists the Tim22 protein in its integration into the inner membrane of mitochondria.     

Protein import into yeast mitochondria: the inner membrane import site protein ISP45 is the MPI1 gene product.

Protein import across both mitochondrial membranes is mediated by the cooperation of two distinct protein transport systems, one in the outer and the other in the inner membrane. Previously we described a 45 kDa yeast mitochondrial inner membrane protein (ISP45) that can be cross-linked to a partially translocated precursor protein (Scherer et al., 1992). We have now purified ISP45 to homogeneity and identified it as the product of the nuclear MPI1 gene. Identity of ISP45 with the MPI1 gene product was shown by microsequencing of three tryptic ISP45 peptides and by demonstrating that an antibody against an Mpi1p-beta-galactosidase fusion protein specifically recognizes ISP45. Antibodies monospecific for ISP45 inhibited protein import into right-side-out mitochondrial inner membrane vesicles, but not into intact mitochondria. On solubilizing mitochondria, ISP45 was rapidly converted to a 40 kDa proteolytic fragment unless mitochondria were first denatured with trichloroacetic acid. The combined genetic and biochemical evidence identifies ISP45/Mpi1p as a component of the protein import system of the yeast mitochondrial inner membrane.     

Insertion of newly synthesized proteins into the outer membrane of Escherichia coli

The insertion of newly synthesized proteins into the outer membrane of Escherichia coli has been examined. The results show that there is no precursor pool of outer membrane proteins in the cytoplasmic membrane because first, the incorporation of a [³⁵S]methionine pulse into outer membrane proteins completely parallels its incorporation into cytoplasmic membrane proteins, and second, under optimal isolation conditions, no outer membrane proteins are found in the cytoplasmic membrane, even when the membranes are analysed after being labeled for only 15 s.The [³⁵S]methionine present in the outer membrane after a pulse of 15 s was found in protein fragments of varying sizes rather than in specific outer membrane proteins. This label could however be chased into specific proteins within 30–120 s, depending on the size of the protein, indicating that although unfinished protein fragments were present in the outer membrane, they were completed by subsequent chain elongation.Thus, outer membrane proteins are inserted into the outer membrane while still attached to ribosomes. Since ribosomes which are linked to the cell envelope by nascent polypeptide chains are stationary, the mRNA which is being translated by these ribosomes moves along the inner cell surface.     

Requirement of a membrane potential for the posttranslational transfer of proteins into mitochondria

Posttranslational transfer of most precursor proteins into mitochondria is dependent on energization of the mitochondria. Experiments were carried out to determine whether the membrane potential or the intramitochondrial ATP is the immediate energy source. Transfer in vitro of precursors to the ADP/ATP carrier and to ATPase subunit 9 into isolated Neurospora mitochondria was investigated. Under conditions where the level of intramitochondrial ATP was high and the membrane potential was dissipated, import and processing of these precursor proteins did not take place. On the other hand, precursors were taken up and processed when the intramitochondrial ATP level was low, but the membrane potential was not dissipated. We conclude that a membrane potential is involved in the import of those mitochondrial precursor proteins which require energy for intracellular translocation.     

Transport of proteins into mitochondria: translocational intermediates spanning contact sites between outer and inner membranes

Translocational intermediates of precursor proteins of ATPase F1 beta subunit and cytochrome c1 across mitochondrial membranes were analyzed using two different approaches, transport at low temperature and transport after binding of precursor proteins to antibodies. Under both conditions precursors were partially transported into mitochondria in an energy-dependent manner. They were processed by the metalloprotease in the matrix but a major proportion of the polypeptide chains was still present at the outer face of the outer mitochondrial membrane. We conclude that transfer of precursors into the inner membrane or matrix space occurs through "translocation contact sites"; precursor polypeptides to F1 beta and cytochrome c1 enter the matrix space with the amino terminus first; and a membrane potential is required for the transmembrane movement on an amino-terminal "domain-like" structure but not for completing translocation of the major part of the polypeptides.