Structure and evolution of mitochondrial outer membrane proteins of β-barrel topology

Gram-negative bacteria are the ancestors of mitochondrial organelles. Consequently, both entities contain two surrounding lipid bilayers known as the inner and outer membranes. While protein synthesis in bacteria is accomplished in the cytoplasm, mitochondria import 90–99% of their protein ensemble from the cytosol in the opposite direction. Three protein families including Sam50, VDAC and Tom40 together with Mdm10 compose the set of integral β-barrel proteins embedded in the mitochondrial outer membrane in S. cerevisiae (MOM). The 16-stranded Sam50 protein forms part of the sorting and assembly machinery (SAM) and shows a clear evolutionary relationship to members of the bacterial Omp85 family. By contrast, the evolution of VDAC and Tom40, both adopting the same fold cannot be traced to any bacterial precursor. This finding is in agreement with the specific function of Tom40 in the TOM complex not existent in the enslaved bacterial precursor cell. Models of Tom40 and Sam50 have been developed using X-ray structures of related proteins. These models are analyzed with respect to properties such as conservation and charge distribution yielding features related to their individual functions.     

Characterization of the insertase for β-barrel proteins of the outer mitochondrial membrane

The TOB–SAM complex is an essential component of the mitochondrial outer membrane that mediates the insertion of β-barrel precursor proteins into the membrane. We report here its isolation and determine its size, composition, and structural organization. The complex from Neurospora crassa was composed of Tob55–Sam50, Tob38–Sam35, and Tob37–Sam37 in a stoichiometry of 1:1:1 and had a molecular mass of 140 kD. A very minor fraction of the purified complex was associated with one Mdm10 protein. Using molecular homology modeling for Tob55 and cryoelectron microscopy reconstructions of the TOB complex, we present a model of the TOB–SAM complex that integrates biochemical and structural data. We discuss our results and the structural model in the context of a possible mechanism of the TOB insertase.     

How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane?

Bcl-2 family members, like the structurally similar translocation domain of diphtheria toxin, can form ion-selective channels and larger-diameter pores in artificial lipid bilayers. Recent studies show how Bcl-2 family members change topology in membranes during apoptosis and that these different states may either promote or inhibit apoptosis. Binding of BH3-only proteins alters the subcellular localization and/or membrane topology and probably affects the channel formation of Bcl-2, Bcl-xL and Bcl-w. However, it remains unclear how the pore-forming activity functions in cells to regulate mitochondrial membrane permeabilization and cell death. Bcl-2 family members in flies and worms regulate apoptosis by mechanisms seemingly unrelated to membrane permeabilization, leaving a unifying model for the biochemical activity of this protein family unknown. Work linking Bcl-2 family members to mitochondrial morphogenesis in worms and mammals suggests some common functions of Bcl-2 family proteins may exist.     

How does the TOM complex mediate insertion of precursor proteins into the mitochondrial outer membrane?

A multisubunit translocase of the outer mitochondrial membrane (TOM complex) mediates both the import of mitochondrial precursor proteins into the internal compartments of the organelle and the insertion of proteins residing in the mitochondrial outer membrane. The proposed beta-barrel structure of Tom40, the pore-forming component of the translocase, raises the question of how the apparent uninterrupted beta-barrel topology can be compatible with a role of Tom40 in releasing membrane proteins into the lipid core of the bilayer. In this review, I discuss insertion mechanisms of proteins into the outer membrane and present alternative models based on the opening of a multisubunit beta-barrel TOM structure or on the interaction of outer membrane precursors with the outer face of the Tom40 beta-barrel structure.     

How do BCL-2 proteins induce mitochondrial outer membrane permeabilization?

The mitochondrial pathway of apoptosis proceeds when molecules sequestered between the outer and inner mitochondrial membranes are released to the cytosol by mitochondrial outer membrane permeabilization (MOMP). This process is controlled by the BCL-2 family, which is composed of both pro- and anti-apoptotic proteins. Although there is no disagreement that BCL-2 proteins regulate apoptosis, the mechanism leading to MOMP remains controversial. Current debate focuses on what interactions within the family are crucial to initiate MOMP. Specifically, do the BH3-only proteins directly engage BAX and/or BAK activation or do these proteins solely promote apoptosis by neutralization of anti-apoptotic BCL-2 proteins? We describe these models and contend that BH3-only proteins must perform both functions to efficiently engage MOMP and apoptosis.     

Ca²+-induced release of mitochondrial m-calpain from outer membrane with binding of calpain small subunit and Grp75

Although mitochondrial μ- and m-calpains play significant roles in apoptotic cell death, their activating mechanisms have not been determined. The purpose of this study was to determine the core factors that are involved in activating mitochondrial outer membrane (OM)-bound calpains. To accomplish this, we solubilized OM-bound calpains and separated them by DEAE-Sepharose column chromatography, and identified them by immunoblots. We also determined the core factors that activated the OM-bound calpains and release them from the OM by calpain assays, immunoprecipitations, and immunoblots. The OM-bound m-calpain large subunit was not associated with the small subunit or with Grp75 chaperone. Free calpain small subunit was located in the IMS and caused the release of the OM-bound m-calpain large subunit from the OM together with Grp75, ATP, and Ca2+. Our results showed that the activating mechanism of mitochondrial OM-bound m-calpain and the release of mitochondrial m-calpain from the OM have important implications in facilitating apoptotic cell death.     

Apparent “mild depolarization of the inner mitochondrial membrane” as a result of a possible generation of the outer membrane potential

Recently reported kinase-linked mild depolarization of mitochondria, which prevents the generation of the reactive oxygen species (ROS) and disappears in various organs of the old mice, has been assumed to represent a crucial component of the mitochondrial anti-aging program. To measure mitochondrial inner membrane potential (IMP), the authors used fluorescent probe safranin O⁺. It is widely accepted that the accumulation of such cationic probes in the mitochondrial matrix depends exclusively on IMP, thus completely ignoring the possibility of the outer membrane potential (OMP) generation. However, computational analysis performed in the presented work suggests that the kinase-linked generation of the positive OMP might take place under the described conditions, because the measured potential includes the algebraic sum of both IMP and OMP. Alternatively to the suggested mild depolarization of mitochondria, the reported experimental data might reflect mainly a change of the positive OMP generated by the VDAC-kinase complexes. We also demonstrate that the reported in the literature mitochondrial hyperpolarization induced by erastin (known to prevent VDAC-tubulin interactions) and the depolarization caused by the mitochondrial VDAC knockdowns in the cancer cells might actually represent a decrease or increase, respectively, of the magnitude of the kinase-linked positive OMP. This is consistent with our hypothesis that VDAC voltage gating by the kinase-linked metabolically-dependent OMP plays a very important physiological role in regulating the cell energy metabolism under normal and pathological conditions, in the maintenance of the cell death resistance and even in the genetic aging program.     

Targeting of Neisserial PorB to the mitochondrial outer membrane: an insight on the evolution of β-barrel protein assembly machines

Mitochondria originated from Gram-negative bacteria through endosymbiosis. In modern day mitochondria, the Sorting and Assembly Machinery (SAM) is responsible for eukaryotic β-barrel protein assembly in the mitochondrial outer membrane. The SAM is the functional equivalent of the β-barrel assembly machinery found in the outer membrane of Gram-negative bacteria. In this study we examined the import pathway of a pathogenic bacterial protein, PorB, which is targeted from pathogenic Neisseria to the host mitochondria. We have developed a new method for measurement of PorB assembly into mitochondria that relies on the mobility shift exhibited by bacterial β-barrel proteins once folded and separated under semi-native electrophoretic conditions. We show that PorB is targeted to the outer mitochondrial membrane with a dependence on the intermembrane space shuttling chaperones and the core component of the SAM, Sam50, which is a functional homologue of BamA that is required for PorB assembly in bacteria. The peripheral subunits of the SAM, Sam35 and Sam37, which are essential for eukaryotic β-barrel protein assembly but do not have distinguishable functional homologues in bacteria, are not required for PorB assembly in eukaryotes. This shows that PorB uses an evolutionary conserved 'bacterial like' mechanism to infiltrate the host mitochondrial outer membrane.     

Deficiency of mitochondrial outer membrane protein 64 confers rice resistance to both piercing‐sucking and chewing insects in rice

The brown planthopper (BPH) and striped stem borer (SSB) are the most devastating insect pests in rice (Oryza sativa ) producing areas. Screening for endogenous resistant genes is the most practical strategy for rice insect‐resistance breeding. Forty‐five mutants showing high resistance against BPH were identified in a rice T‐DNA insertion population (11,000 putative homozygous lines) after 4 years of large‐scale field BPH‐resistance phenotype screening. Detailed analysis showed that deficiency of rice mitochondrial outer membrane protein 64 (OM64 ) gene resulted in increased resistance to BPH. Mitochondrial outer membrane protein 64 protein is located in the outer mitochondrial membrane by subcellular localization and its deficiency constitutively activated hydrogen peroxide (H₂O₂) signaling, which stimulated antibiosis and tolerance to BPH. The om64 mutant also showed enhanced resistance to SSB, a chewing insect, which was due to promotion of Jasmonic acid biosynthesis and related responses. Importantly, om64 plants presented no significant changes in rice yield‐related characters. This study confirmed OM64 as a negative regulator of rice herbivore resistance through regulating H₂O₂ production. Mitochondrial outer membrane protein 64 is a potentially efficient candidate to improve BPH and SSB resistance through gene deletion. Why the om64 mutant was resistant to both piercing‐sucking and chewing insects via a gene deficiency in mitochondria is discussed.     

Biogenesis of β-barrel integral proteins of bacterial outer membrane

Gram-negative bacteria are enveloped by two membranes, the inner (cytoplasmic) (CM) and the outer (OM). The majority of integral outer membrane proteins are arranged in β-barrels of cylindrical shape composed of amphipathic antiparallel β-strands. In bacteria, β-barrel proteins function as water-filled pores, active transporters, enzymes, receptors, and structural proteins. Proteins of bacterial OM are synthesized in the cytoplasm as unfolded polypeptides with an N-terminal sequence that marks them for transport across the CM. Precursors of membrane proteins move through the aqueous medium of the cytosol and periplasm under the protection of chaperones (SecB, Skp, SurA, and DegP), then cross the CM via the Sec system composed of a polypeptide-conducting channel (SecYEG) and ATPase (SecA), the latter providing the energy for the translocation of the pre-protein. Pre-protein folding and incorporation in the OM require the participation of the Bam-complex, probably without the use of energy. This review summarizes current data on the biogenesis of the β-barrel proteins of bacterial OM. Data on the structure of the proteins included in the multicomponent system for delivery of the OM proteins to their destination in the cell and on their complexes with partners, including pre-proteins, are pre-sented. Molecular models constructed on the basis of structural, genetic, and biochemical studies that describe the mechanisms of β-barrel protein assembly by this molecular transport machinery are also considered.     

Incorporation of β-sitosterol into mitochondrial membrane enhances mitochondrial function by promoting inner mitochondrial membrane fluidity

Recent findings suggest that mitochondrial membrane fluidity could influence mitochondrial energy metabolism. β-sitosterol (BS) is a common plant sterol that is prevalent in plant oils, nuts, cereals and plant food products. Its chemical structure is very similar to that of cholesterol. As a cholesterol analog, BS is highly lipid soluble and largely resides in the membranes of cells or organelles where it may have an influence on the membrane fluidity. The present study reports that, with the cholesterol chelator 2-hydroxypropyl-β-cyclodextrin (HPβCD) as its carrier, BS is able to increase the fluidity of the inner mitochondrial membrane (IMM) without affecting the fluidity of the outer mitochondrial membrane (OMM), and consequently to increase the mitochondrial membrane potential (?Ψm) and mitochondrial ATP content. It has been previously proposed that a therapeutical boost in adenosine triphosphate (ATP) levels in mitochondria may be beneficial for neurodegenerative diseases such as Alzheimer’s disease (AD). Given that dietary administration of plant sterols could increase brain BS concentrations, these results may provide a better understanding of the beneficial effects of plant sterol-enriched nutrients on neurodegenerative diseases such as AD.     

Assembly of β-barrel proteins into bacterial outer membranes

Membrane proteins with a β-barrel topology are found in the outer membranes of Gram-negative bacteria and in the plastids and mitochondria of eukaryotic cells. The assembly of these membrane proteins depends on a protein folding reaction (to create the barrel) and an insertion reaction (to integrate the barrel within the outer membrane). Experimental approaches using biophysics and biochemistry are detailing the steps in the assembly pathway, while genetics and bioinformatics have revealed a sophisticated production line of cellular components that catalyze the assembly pathway in vivo. This includes the modular BAM complex, several molecular chaperones and the translocation and assembly module (the TAM). Recent screens also suggest that further components of the pathway might remain to be discovered. We review what is known about the process of β-barrel protein assembly into membranes, and the components of the β-barrel assembly machinery. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Activity of nucleoside diphosphate kinase α (NDPK α) capable of binding to outer mitochondrial membrane accounts for less than 10% of total NDPK activity present in cytoplasm of liver cells

During incubation of a constant volume of rat liver cytosol with an increasing quantity of mitochondrial protein in the presence of 3.3 mM MgCl₂, the binding of nucleoside diphosphate kinase (NDPK) from the cytosol to mitochondrial membranes is described by a saturation curve. The highest bound NDPK activity accounts for less than 9% of the added activity. Analysis of the results suggests that only one NDPK isozyme is bound to the membranes. Western blotting showed it to be NDPK α, a homolog of human NDPK-B. Substrates of NDPK, hexokinase, and glycerol kinase, as well as N,N’-dicyclohexylcarbodiimide and palmitate, did not influence the association of NDPK with mitochondrial membranes. We conclude that the sites of NDPK binding to the outer mitochondrial membrane are not identical to those of hexokinase and glycerol kinase.     

The accessory subunit of mitochondrial DNA polymerase γ determines the DNA content of mitochondrial nucleoids in human cultured cells

The accessory subunit of mitochondrial DNA polymerase γ, POLGβ, functions as a processivity factor in vitro. Here we show POLGβ has additional roles in mitochondrial DNA metabolism. Mitochondrial DNA is arranged in nucleoprotein complexes, or nucleoids, which often contain multiple copies of the mitochondrial genome. Gene-silencing of POLGβ increased nucleoid numbers, whereas over-expression of POLGβ reduced the number and increased the size of mitochondrial nucleoids. Both increased and decreased expression of POLGβ altered nucleoid structure and precipitated a marked decrease in 7S DNA molecules, which form short displacement-loops on mitochondrial DNA. Recombinant POLGβ preferentially bound to plasmids with a short displacement-loop, in contrast to POLGα. These findings support the view that the mitochondrial D-loop acts as a protein recruitment centre, and suggest POLGβ is a key factor in the organization of mitochondrial DNA in multigenomic nucleoprotein complexes.     

Structural biology of membrane-intrinsic β-barrel enzymes: Sentinels of the bacterial outer membrane

The outer membranes of Gram-negative bacteria are replete with integral membrane proteins that exhibit antiparallel β-barrel structures, but very few of these proteins function as enzymes. In Escherichia coli, only three β-barrel enzymes are known to exist in the outer membrane; these are the phospholipase OMPLA, the protease OmpT, and the phospholipid∷lipid A palmitoyltransferase PagP, all of which have been characterized at the structural level. Structural details have also emerged for the outer membrane β-barrel enzyme PagL, a lipid A 3-O-deacylase from Pseudomonas aeruginosa. Lipid A can be further modified in the outer membrane by two β-barrel enzymes of unknown structure; namely, the Salmonella enterica 3′-acyloxyacyl hydrolase LpxR, and the Rhizobium leguminosarum oxidase LpxQ, which employs O₂ to convert the proximal glucosamine unit of lipid A into 2-aminogluconate. Structural biology now indicates how β-barrel enzymes can function as sentinels that remain dormant when the outer membrane permeability barrier is intact. Host immune defenses and antibiotics that perturb this barrier can directly trigger β-barrel enzymes in the outer membrane. The ensuing adaptive responses occur instantaneously and rapidly outpace other signal transduction mechanisms that similarly function to restore the outer membrane permeability barrier.     

Contribution of Müller cells toward the regulation of photoreceptor outer segment assembly

The assembly of photoreceptor outer segments into stacked discs is a complicated process, the precise regulation of which remains a mystery. It is known that the integrity of the outer segment is heavily dependent upon surrounding cell types including the retinal pigment epithelium and Müller cells; however the role played by Müller cells within this photoreceptor-specific process has not been fully explored. Using an RPE-deprived but otherwise intact Xenopus laevis eye rudiment preparation, we reveal that Müller cell involvement in outer segment assembly is dependent upon the stimulus provided to the retina. Pigment epithelium-derived factor is able to support proper membrane folding after inhibition of Müller cell metabolism by alpha-aminoadipic acid, while isopropyl beta-D-thiogalactoside, a permissive glycan, requires intact Müller cell function. These results demonstrate that both intrinsic and extrinsic redundant mechanisms exist to support the ability of photoreceptors to properly assemble their outer segments. Our study further suggests that the receptor for pigment epithelium-derived factor resides in photoreceptors themselves while that for permissive glycans is likely localized to Müller cells, which in turn communicate with photoreceptors to promote proper membrane assembly.