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.     

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.     

Biogenesis of mitochondrial porin: the import pathway.

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 cleavable '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 which is characterized by its extreme protease resistance.     

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.     

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 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.     

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.     

Biogenesis of prokaryotic pores.

The prokaryotic pore-forming proteins are synthesized in the cytoplasm, and are assembled in their functional form in the outer membrane. They begin to traverse the cytoplasmic membrane via the SecY/SecA export pathway, which is shared also by periplasmic proteins. The sorting signals that direct these proteins to the outer membrane could be present in the three-dimensional conformations of the proteins, but some results suggest that they may be present in short, contiguous sequences. Outer membrane proteins share a rather hydrophilic amino acid composition, and appear to be rich in beta-sheets (with the exception of lipoproteins). This observation as well as the demonstration of periplasmic export intermediates favor the secretion pathway through the periplasm, as opposed to export through fusion sites between the inner and the outer membrane, but such intermediates have not yet been observed with the wild type proteins under physiological conditions.     

Influence of reactive oxygen species production by monoamine oxidase activity on aluminum-induced mitochondrial permeability transition

Treatment of Ca2+-loaded mitochondria with both aluminum and tyramine results in a swelling of higher amplitude than with aluminum alone, while tyramine alone is ineffective. The phenomenon is accompanied by H2O2 production and thiol and pyridine nucleotide oxidation. Cyclosporin A, N-ethylmaleimide or dithioerythritol completely prevent these effects, while catalase exhibits a lower inhibition, pointing to the induction of the permeability transition (MPT) by an oxidative stress. Reactive oxygen species are generated by the interaction of aluminum with the inner membrane and the oxidation of tyramine by monoamine oxidase on the outer membrane. This different localization determines the oxidation of critical thiol groups located on both internal and external sides of pore-forming structures, resulting in MPT induction. The reduced effect by aluminum or the inefficacy by tyramine, when implied alone, can be attributable to the oxidation of thiol groups located only on the internal or external side, respectively. Ultrastructural observations show that aluminum plus tyramine induce the typical configuration of mitochondria that have undergone the MPT. Instead, with aluminum alone, the sensitive subpopulation, although swollen, preserves the outer membrane and shows an apparently orthodox configuration.     

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.     

Spore germination

Despite being relatively insensitive to environmental insult, the spore is responsive to low concentrations of chemical germinants, which induce germination. The process of bacterial spore germination involves membrane permeability changes, ion fluxes and the activation of enzymes that degrade the outer layers of the spore. A number of components in the spore that are required for the germination response have been identified, including a spore-specific family of receptor proteins (the GerA family), an ion transporter and cortex lytic enzymes. The germinant traverses the outer layers of the spore and interacts with its receptor in the inner membrane to initiate the cascade of germination events, but the molecular details of this signal transduction process remain to be identified.     

Permeability coefficients of the egg-case membrane of Scyliorhinus canicula L

The cleidoic egg-case of the dogfish appears to have a highly porous and permeable outer membrane, the pore radius being computed to be 13.6 A. It does not present any physiological barrier to small molecules and therefore constitutes an open ionic and osmotic system for the embryo. Being a porous protein membrane it may be of value as a model for molecular transport mechanisms.     

Pathophysiology of mitochondrial cell death control

Mitochondria have been recently recognized to play a major role in the control of apoptosis or programmed cell death. Permeabilization of mitochondrial membranes, a decisive feature of early cell death, is regulated by members of the Bcl-2 family which interact with the permeability transition pore complex (PTPC). Thus, the cytoprotective oncoprotein Bcl-2 stabilizes the mitochondrial membrane barrier function, whereas the tumor suppressor protein Bax permeabilizes mitochondrial membranes. The regulation of membrane permeabilization is intertwined with that of the bioenergetic and redox functions of mitochondria. The implications of alterations in the composition of the PTPC and in mitochondrial function for the pathophysiology of cancer (reduced apoptosis) and neurodegeneration (enhanced apoptosis) are discussed.     

Permeability coefficients of the egg-case membrane ofScyliorhinus canicula L.

Summary The cleidoic egg-case of the dogfish appears to have a highly porous and permeable outer membrane, the pore radius being computed to be 13.6 Å. It does not present any physiological barrier to small molecules and therefore constitutes an open ionic and osmotic system for the embryo. Being a porous protein membrane it may be of value as a model for molecular transport mechanisms.     

Mitochondrial bioenergetics decay in aging: beneficial effect of melatonin

Aging is a biological process characterized by progressive decline in physiological functions, increased oxidative stress, reduced capacity to respond to stresses, and increased risk of contracting age-associated disorders. Mitochondria are referred to as the powerhouse of the cell through their role in the oxidative phosphorylation to generate ATP. These organelles contribute to the aging process, mainly through impairment of electron transport chain activity, opening of the mitochondrial permeability transition pore and increased oxidative stress. These events lead to damage to proteins, lipids and mitochondrial DNA. Cardiolipin, a phospholipid of the inner mitochondrial membrane, plays a pivotal role in several mitochondrial bioenergetic processes as well as in mitochondrial-dependent steps of apoptosis and in mitochondrial membrane stability and dynamics. Cardiolipin alterations are associated with mitochondrial bienergetics decline in multiple tissues in a variety of physiopathological conditions, as well as in the aging process. Melatonin, the major product of the pineal gland, is considered an effective protector of mitochondrial bioenergetic function. Melatonin preserves mitochondrial function by preventing cardiolipin oxidation and this may explain, at least in part, the protective role of this compound in mitochondrial physiopathology and aging. Here, mechanisms through which melatonin exerts its protective role against mitochondrial dysfunction associated with aging and age-associated disorders are discussed.     

Colchicine inhibition of ADH effect on frog skin permeability

ADH and AMPc enhance both thiourea unidirectional fluxes in frog skin. This effect is completely abolished by colchicine pretreatment. The ADH increase of thiourea discharge with or without colchicine led us to suppose that colchicine does not directly affect ADH action on outer membrane permeability, but exerts its effects on a site which is limiting for the ADH action on transepithelial permeability.     

Structure of the gap junction channel and its implications for its biological functions

Gap junctions consist of arrays of intercellular channels composed of integral membrane proteins called connexin in vertebrates. Gap junction channels regulate the passage of ions and biological molecules between adjacent cells and, therefore, are critically important in many biological activities, including development, differentiation, neural activity, and immune response. Mutations in connexin genes are associated with several human diseases, such as neurodegenerative disease, skin disease, deafness, and developmental abnormalities. The activity of gap junction channels is regulated by the membrane voltage, intracellular microenvironment, interaction with other proteins, and phosphorylation. Each connexin channel has its own property for conductance and molecular permeability. A number of studies have tried to reveal the molecular architecture of the channel pore that should confer the connexin-specific permeability/selectivity properties and molecular basis for the gating and regulation. In this review, we give an overview of structural studies and describe the structural and functional relationship of gap junction channels.     

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.     

Colchicine inhibition of ADH effect on frog skin permeability

Summary ADH and AMPc enhance both thiourea unidirectional fluxes in frog skin. This effect is completely abolished by colchicine pretreatment. The ADH increase of thiourea discharge with or without colchicine led us to suppose that colchicine does not directly affect ADH action on outer membrane permeability, but exerts its effects on a site which is limiting for the ADH action on transepithelial permeability.The present work has been supported by C.N.R., Rome.     

Mitochondrial permeability transition in cardiac ischemia–reperfusion: whether cyclophilin D is a viable target for cardioprotection?

Growing number of studies provide strong evidence that the mitochondrial permeability transition pore (PTP), a non-selective channel in the inner mitochondrial membrane, is involved in the pathogenesis of cardiac ischemia–reperfusion and can be targeted to attenuate reperfusion-induced damage to the myocardium. The molecular identity of the PTP remains unknown and cyclophilin D is the only protein commonly accepted as a major regulator of the PTP opening. Therefore, cyclophilin D is an attractive target for pharmacological or genetic therapies to reduce ischemia–reperfusion injury in various animal models and humans. Most animal studies demonstrated cardioprotective effects of PTP inhibition; however, a recent large clinical trial conducted by international groups demonstrated that cyclosporine A, a cyclophilin D inhibitor, failed to protect the heart in patients with myocardial infarction. These studies, among others, raise the question of whether cyclophilin D, which plays an important physiological role in the regulation of cell metabolism and mitochondrial bioenergetics, is a viable target for cardioprotection. This review discusses previous studies to provide comprehensive information on the physiological role of cyclophilin D as well as PTP opening in the cell that can be taken into consideration for the development of new PTP inhibitors.