Biogenesis of mitochondrial porin: The import pathway

Summary We review here the present knowledge about the pathway of import and assembly of porin into mitochondria and compare it to those of other mitochondrial proteins. Porin, like all outer mitochondrial membrane proteins studied so far is made as a precursor without a cleavble signal sequence; thus targeting information must reside in the mature sequence. At least part of this information appears to be located at the amino-terminal end of the molecule. Transport into mitochondria can occur post-translationally. In a first step, the porin precursor is specifically recognized on the mitochondrial surface by a protease sensitive receptor. In a second step, porin precursor inserts partially into the outer membrane. This step is mediated by a component of the import machinery common to the import pathways of precursor proteins destined for other mitochondrial subcompartments. Finally, porin is assembled to produce the functional oligomeric form of an integral membrane protein wich is characterized by its extreme protease resistance.     

The biogenesis and function of eukaryotic porins.

Like most other mitochondrial proteins porin is synthesized in the cytosol and imported posttranslationally into the outer mitochondrial membrane. This transport follows the general rules for mitochondrial protein import with a few aberrations: a) porin contains an uncleaved NH2-terminal signal sequence, b) also its carboxyterminus might be involved in the import process, and c) this transport does not seem to require a membrane potential delta psi, although it is ATP-dependent. Most likely the actual import step occurs at contact sites between the outer and the inner mitochondrial membrane and involves at least one receptor protein. Although porin is known to be the major gate through the outer mitochondrial membrane, its absence only causes transient respiratory problems in yeast cells. This could mean a) that there is a bypass for some mitochondrial functions in the cytosol and/or b) that there are alternative channel proteins in the outer membrane. The first idea is supported by the overexpression of cytosolic virus-like particles in yeast cells lacking porin and the second by the occurrence of residual pore activity in mitochondrial outer membrane purified from porinless mutant cells.     

The biogenesis and function of eukaryotic porins

Summary Like most other mitochondrial proteins porin is synthesized in the cytosol and imported posttranslationally into the outer mitochondrial membrane. This transport follows the general rules for mitochondrial, protein import with a few aberrations: a) porin contains an,uncleaved NH₂-terminal signal sequence, b) also its carboxyterminus might be involved in the import process, and c) this transport does not seem to require a membrane potential , although it is ATP-dependent. Most likely the actual import step occurs at contact sites between the outer and the inner mitochondrial membrane and involved at least one receptor protein.Although porin is known to be the major gate through the outer mitochondrial membrane, its absence only causes transient respiratory problems in yeast cells. This could mean a) that there is a bypass for some mitochondrial functions in the cytosol and/or b) that there are alternative channel proteins in the outer membrane. The first idea is supported by the overexpression of cytosolic virus-like particles in yeast cells lacking porin and the second by the occurrence of residual pore activity in mitochondrial outer membrane purified from porinless mutant cells.     

Cytosolic factors in mitochondrial protein import

In vitro import studies have confirmed the participation of cytosolic protein factors in the import of various precursor proteins into mitochondria. The requirement for extramitochondrial adenosine triphosphate for the import of a group of precursor proteins seems to be correlated with the chaperone activity of the cytosolic protein factors. One of the cytosolic protein factors is hsp70, which generally recognizes and binds unfolded proteins in the cytoplasm. Hsp70 keeps the newly synthesized mitochondrial precursor proteins in import-competent unfolded conformations. Another cytosolic protein factor that has been characterized is mitochondrial import stimulation factor (MSF), which seems to be specific to mitochondrial precursor proteins. MSF recognizes the mitochondrial precursor proteins, forms a complex with them and targets them to the receptors on the outer surface of mitochondria.     

Biophysical properties of porin pores from mitochondrial outer membrane of eukaryotic cells

Summary The matrix space of mitochondria is surrounded by two membranes. The mitochondrial inner membrane contains the respiration chain and a large number of highly specific carriers for the mostly anionic substrates of mitochondrial metabolism. In contrast to this the permeability properties of the mitochondrial outer membrane are by far less specific. It acts as a molecular sieve for hydrophilic molecules with a defined exclusion limit around 3000 Da. Responsible for the extremely high permeability of the mitochondrial outer membrane is the presence of a pore-forming protein termed mitochondrial porin. Mitochondrial porins have been isolated from a variety of eukaryotic cells. They are basic proteins with molecular masses between 30 and 35 kDa. Reconstitution experiments define their function as pore-forming components with a single-channel conductance of about 0.40 nS (nano Siemens) in 0.1 M KCl at low voltages. In the open state mitochondrial porin behaves as a general diffusion pore with an effective diameter of 1.7 nm. Eukaryotic porins are slightly anion-selective in the open state but become cation-selective after voltage-dependent closure.     

Biophysical properties of porin pores from mitochondrial outer membrane of eukaryotic cells.

The matrix space of mitochondria is surrounded by two membranes. The mitochondrial inner membrane contains the respiration chain and a large number of highly specific carriers for the mostly anionic substrates of mitochondrial metabolism. In contrast to this the permeability properties of the mitochondrial outer membrane are by far less specific. It acts as a molecular sieve for hydrophilic molecules with a defined exclusion limit around 3000 Da. Responsible for the extremely high permeability of the mitochondrial outer membrane is the presence of a pore-forming protein termed mitochondrial porin. Mitochondrial porins have been isolated from a variety of eukaryotic cells. They are basic proteins with molecular masses between 30 and 35 kDa. Reconstitution experiments define their function as pore-forming components with a single-channel conductance of about 0.40 nS (nano Siemens) in 0.1 M KCl at low voltages. In the open state mitochondrial porin behaves as a general diffusion pore with an effective diameter of 1.7 nm. Eukaryotic porins are slightly anion-selective in the open state but become cation-selective after voltage-dependent closure.     

Bacterial signal peptide recognizes HeLa cell mitochondrial import receptors and functions as a mitochondrial leader sequence

Phage display was used to identify new components of the mammalian mitochondrial receptor complex using Tom20 as a binding partner. Two peptides were identified. One had partial identity (SMLTVMA) with a bacterial signal peptide from Toho-1, a periplasmic protein. The other had partial identity with a mitochondrial inner membrane glutamate carrier. The bacterial signal peptide could carry a protein into mitochondria both in vivo and in vitro. The first six residues of the sequence, SMLTVM, were necessary for import but the two adjacent arginine residues in the 30-amino-acid leader were not critical for import. The signal peptides of Escherichia coli β-lactamase and Bacillsus subtilis lipase could not carry proteins into mitochondria. Presumably, the Toho-1 leader can adopt a structure compatible for recognition by the import apparatus.Received 29 April 2005; received after revision 8 June 2005; accepted 17 June 2005     

The preprotein translocase of the outer mitochondrial membrane: receptors and a general import pore

Cytosol-synthesized preproteins destined for the mitochondria are transported across the outer membrane by the translocase of the mitochondrial outer membrane (TOM complex). This dynamic transport machinery can be divided into receptors that recognize preprotein targeting signals and components of the general import pore complex that mediate preprotein transport across the outer membrane. This review focuses on recent studies dealing with the central questions regarding the pore-forming subunits, and architecture and gating of the translocation channel of the outer membrane.     

Regulated protein degradation in mitochondria

Various adenosine triphosphate (ATP)-dependent proteases were identified within mitochondria which mediate selective mitochondrial protein degradation and fulfill crucial functions in mitochondrial biogenesis. The matrix-localized PIM1 protease, a homologue of theEscherichia coli Lon protease, is required for respiration and maintenance of mitochondrial genome integrity. Degradation of non-native polypeptides by PIM1 protease depends on the chaperone activity of the mitochondrial Hsp70 system, posing intriguing questions about the relation between the proteolytic system and the folding machinery in mitochondria. The mitochondrial inner membrane harbors two ATP-dependent metallopeptidases, them- and thei-AAA protease, which expose their catalytic sites to opposite membrane surfaces and cooperate in the degradation of inner membrane proteins. In addition to its proteolytic activity, them-AAA protease has chaperone-like activity during the assembly of respiratory and ATP-synthase complexes. It constitutes a quality control system in the inner membrane for membrane-embedded protein complexes.     

ATP-dependent proteases controlling mitochondrial function in the yeast Saccharomyces cerevisiae

Regulated protein degradation by ATP-dependent proteases plays a fundamental role in the biogenesis of mitochondria. Membrane-bound and soluble ATP-dependent proteases have been identified in various subcompartments of this organelle. Subunits composing these proteases are evolutionarily conserved from yeast to humans and, in support of an endosymbiotic origin of mitochondria, evolved from prokaryotic ancestors: the PIM1/Lon protease is active in the matrix of mitochondria, while the i-AAA protease and the m-AAA protease mediate the turnover of inner membrane proteins. Most of the knowledge concerning the biogenesis and the physiological role of ATP-dependent proteases comes from studies in the yeast Saccharomyces cerevisiae. Proteases were found to be required for mitochondrial stasis, for the maintenance of the morphology of the organelle and for mitochondrial genome integrity. ATP-dependent proteolysis is crucial for the expression of mitochondrially encoded subunits of respiratory chain complexes and for the assembly of these complexes. Hence, mitochondrial ATP-dependent proteases exert multiple roles which are essential for the maintenance of cellular respiratory competence.     

The ever-growing complexity of the mitochondrial fission machinery

The mitochondrial network constantly changes and remodels its shape to face the cellular energy demand. In human cells, mitochondrial fusion is regulated by the large, evolutionarily conserved GTPases Mfn1 and Mfn2, which are embedded in the mitochondrial outer membrane, and by OPA1, embedded in the mitochondrial inner membrane. In contrast, the soluble dynamin-related GTPase Drp1 is recruited from the cytosol to mitochondria and is key to mitochondrial fission. A number of new players have been recently involved in Drp1-dependent mitochondrial fission, ranging from large cellular structures such as the ER and the cytoskeleton to the surprising involvement of the endocytic dynamin 2 in the terminal abscission step. Here we review the recent findings that have expanded the mechanistic model for the mitochondrial fission process in human cells and highlight open questions.     

Regulation of mitochondrial dynamics: convergences and divergences between yeast and vertebrates

In eukaryotic cells, the shape of mitochondria can be tuned to various physiological conditions by a balance of fusion and fission processes termed mitochondrial dynamics. Mitochondrial dynamics controls not only the morphology but also the function of mitochondria, and therefore is crucial in many aspects of a cell’s life. Consequently, dysfunction of mitochondrial dynamics has been implicated in a variety of human diseases including cancer. Several proteins important for mitochondrial fusion and fission have been discovered over the past decade. However, there is emerging evidence that there are as yet unidentified proteins important for these processes and that the fusion/fission machinery is not completely conserved between yeast and vertebrates. The recent characterization of several mammalian proteins important for the process that were not conserved in yeast, may indicate that the molecular mechanisms regulating and controlling the morphology and function of mitochondria are more elaborate and complex in vertebrates. This difference could possibly be a consequence of different needs in the different cell types of multicellular organisms. Here, we review recent advances in the field of mitochondrial dynamics. We highlight and discuss the mechanisms regulating recruitment of cytosolic Drp1 to the mitochondrial outer membrane by Fis1, Mff, and MIEF1 in mammals and the divergences in regulation of mitochondrial dynamics between yeast and vertebrates.     

The exportomer: the peroxisomal receptor export machinery

Peroxisomes constitute a dynamic compartment of almost all eukaryotic cells. Depending on environmental changes and cellular demands peroxisomes can acquire diverse metabolic roles. The compartmentalization of peroxisomal matrix enzymes is a prerequisite to carry out their physiologic function. The matrix proteins are synthesized on free ribosomes in the cytosol and are ferried to the peroxisomal membrane by specific soluble receptors. Subsequent to cargo release into the peroxisomal matrix, the receptors are exported back to the cytosol to facilitate further rounds of matrix protein import. This dislocation step is accomplished by a remarkable machinery, which comprises enzymes required for the ubiquitination as well as the ATP-dependent extraction of the receptor from the membrane. Interestingly, receptor ubiquitination and dislocation are the only known energy-dependent steps in the peroxisomal matrix protein import process. The current view is that the export machinery of the receptors might function as molecular motor not only in the dislocation of the receptors but also in the import step of peroxisomal matrix protein by coupling ATP-dependent removal of the peroxisomal import receptor with cargo translocation into the organelle. In this review we will focus on the architecture and function of the peroxisomal receptor export machinery, the peroxisomal exportomer.     

Translational control of endogenous and recoded nuclear genes in yeast mitochondria: Regulation and membrane targeting

Mitochondrial gene expression in yeast,Saccharomyces cerevisiae, depends on translational activation of individual mRNAs by distinct proteins encoded in the nucleus. These nuclearly coded mRNA-specific translational activators are bound to the inner membrane and function to mediate the interaction between mRNAs and mitochondrial ribosomes. This complex system, found to date only in organelles, appears to be an adaptation for targeting the synthesis of mitochondrially coded integral membrane proteins to the membrane. In addition, mRNA-specific translational activation is a rate-limiting step used to modulate expression of at least one mitochondrial gene in response to environmental conditions. Direct study of mitochondrial gene regulation and the targeting of mitochondrially coded proteins in vivo will now be possible using synthetic genes inserted into mtDNA that encode soluble reporter/passenger proteins.     

Biogenesis of β-barrel membrane proteins in bacteria and eukaryotes: evolutionary conservation and divergence

Membrane-embedded β-barrel proteins span the membrane via multiple amphipathic β-strands arranged in a cylindrical shape. These proteins are found in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. This situation is thought to reflect the evolutionary origin of mitochondria and chloroplasts from Gram-negative bacterial endosymbionts. β-barrel proteins fulfil a variety of functions; among them are pore-forming proteins that allow the flux of metabolites across the membrane by passive diffusion, active transporters of siderophores, enzymes, structural proteins, and proteins that mediate protein translocation across or insertion into membranes. The biogenesis process of these proteins combines evolutionary conservation of the central elements with some noticeable differences in signals and machineries. This review summarizes our current knowledge of the functions and biogenesis of this special family of proteins.     

Plant mitochondrial carriers: an overview

In the two last decades, biochemical studies using mitochondrial swelling experiments or direct solute uptake in isolated mitochondria have lead to the identification of different transport systems at the level of the plant mitochondrial inner membrane. Although most of them have been found to have similar features to those identified in animal mitochondria, some differences have been observed between plant and animal transporters. More recently, molecular biology studies have revealed that most of the mitochondrial exchanges are performed by nuclear encoded proteins, which form a superfamily. Members of this family have been reported in animals, yeast as well as plants. This review attempts to give an overview of the present knowledge concerning the biochemical and molecular characterisation of plant members of the mitochondrial carrier family and, when possible, a comparison with carriers from other organisms.     

Optic atrophy 3 as a protein of the mitochondrial outer membrane induces mitochondrial fragmentation

The optic atrophy 3 (OPA3) gene, which has no known homolog or biological function, is mutated in patients with hereditary optic neuropathies. Here, we identified OPA3 as an integral protein of the mitochondrial outer membrane (MOM), with a C-terminus exposed to the cytosol and an N-terminal mitochondrial targeting domain. By quantitative analysis, we demonstrated that overexpression of OPA3 significantly induced mitochondrial fragmentation, whereas OPA3 knockdown resulted in highly elongated mitochondria. Cells with mitochondria fragmented by OPA3 did not undergo spontaneous apoptotic cell death, but were significantly sensitized to staurosporine- and TRAIL-induced apoptosis. In contrast, overexpression of a familial OPA3 mutant (G93S) induced mitochondrial fragmentation and spontaneous apoptosis, suggesting that OPA3 mutations may cause optic atrophy via a gain-of-function mechanism. Together, these results indicate that OPA3, as an integral MOM protein, has a crucial role in mitochondrial fission, and provides a direct link between mitochondrial morphology and optic atrophy.     

New mitochondrial carriers: an overview

The transport of metabolites, nucleotides and cofactors across the mitochondrial inner membrane is performed by members of mitochondrial carrier family (MCF). These proteins share marked structural features that have made feasible the functional characterization of numerous MCs in the last years. The MCs responsible for transport activities in mitochondria known for decades such as glutamate uptake or ATP-Mg/Pi exchange have recently been identified as well as novel carriers such as those involved in S-adenosylmethionine or thiamine pyrophosphate uptake. Here, after a brief review of the novel data on structural characteristics and import mechanisms of MCF members, we present an exhaustive compilation of human MC sequences, including previously characterized carriers, together with their respective Saccharomyces cerevisiae orthologues, ordered according to the phylogenetic analysis of el Moualij and co-workers [Yeast (1997) 13: 573-581]. We have detected the existence of at least 49 human MC sequences, including those of yet unknown function. An overview of novel MCF members functionally characterized in recent years in mammals and in yeast genomes is presented.     

Structural analysis of mitochondrial pores.

Structural information about the channel in the mitochondrial outer membrane, derived from sequence analysis and electron microscopy of two-dimensional crystals, is summarized. A model for the channel is presented, consisting of a cylindrical beta-barrel that is formed by one or two 30-kDa polypeptides, with an alpha-carbon backbone diameter of 3.8 nm. The radial distributions of basic amino acids and lipid-contact regions on the projected cylinder are mapped relative to interchannel bonding sites inferred from channel packing in the arrays. Speculation on the kinds of conformational changes that the channel might undergo is also presented.