Complement attack of altered outer membrane areas synthesized after inhibition of the 3-deoxy-D-manno-octulosonate pathway leads to cell death

Salmonella typhimurium containing specific genes coding for either temperature-sensitive (TS) 3-deoxy-D-manno-octulosonate (KDO) 8-phosphate synthetase or TS cytidine monophosphate-KDO synthetase grow normally when incubated at 30 degrees C and are resistant to C-mediated killing. However, bacteria become avirulent and sensitive to C-mediated killing upon thermal inhibition of TS KDO-8-phosphate synthetase (incubation at 38 degrees C) or TS cytidine monophosphate-KDO synthetase (incubation at 42 degrees C). Such thermal inhibition concurrently causes synthesis of an altered outer membrane which we now show is the site that renders cells susceptible to C-mediated killing. After incubation of cells in serum, the altered outer membrane area contains C9 in a trypsin-resistant state and membrane attack complex (MAC) lesions observable by electron microscopy. Trypsin-resistant C9 and MAC lesions were also observed in the inner membrane fraction from such serum-treated cells. In contrast, little C9 and few MAC lesions were associated with unaltered outer membrane areas present on these same serum treated cells. Control cells, grown at 30 degrees C and treated with serum (1) bound one-fifth as much C9 as was bound to cells incubated at 42 degrees C, (2) contained only a rare MAC lesion in the outer membrane, and (3) no observable MAC lesions in the inner membrane. We conclude that the altered outer membrane area is the site that renders cells susceptible to insertion of the MAC into both the outer and inner membrane resulting in cell death.     

How the Membrane Attack Complex Damages the Bacterial Cell Envelope and Kills Gram‐Negative Bacteria

The human immune system can directly lyse invading micro‐organisms and aberrant host cells by generating pores in the cell envelope, called membrane attack complexes (MACs). Recent studies using single‐particle cryoelectron microscopy have revealed that the MAC is an asymmetric, flexible pore and have provided a structural basis on how the MAC ruptures single lipid membranes. Despite these insights, it remains unclear how the MAC ruptures the composite cell envelope of Gram‐negative bacteria. Recent functional studies on Gram‐negative bacteria elucidate that local assembly of MAC pores by surface‐bound C5 convertase enzymes is essential to stably insert these pores into the bacterial outer membrane (OM). These convertase‐generated MAC pores can subsequently efficiently damage the bacterial inner membrane (IM), which is essential for bacterial killing. This review summarizes these recent insights of MAC assembly and discusses how MAC pores kill Gram‐negative bacteria. Furthermore, this review elaborates on how MAC‐dependent OM damage could lead to IM destabilization, which is currently not well understood. A better understanding on how MAC pores kill bacteria could facilitate the future development of novel strategies to treat infections with Gram‐negative bacteria.     

Bacterial killing and inhibition of inner membrane activity by C5b-9 complexes as a function of the sequential addition of C9 to C5b-8 sites

The assembly of the C5b-9 complex on the outer membrane of C-sensitive cells of Escherichia coli results in a rapid inhibition of inner membrane function and ultimately a loss of cell viability. Cells bearing C5b-8 sites suffer no deleterious effects; however, the addition of C9 results in a rapid inhibition of inner membrane function and cell death. An attempt was made to examine the relationship between the toxic effects of the C5b-9 complex and the number of C9 molecules per C5b-8 site. Cells bearing C5b-8 sites were exposed to excess C9 at 0 degrees C and washed three times at 4 degrees C. The number of C9 molecules bound to each cell was equivalent to the number of C5b-8 sites present on each cell, and no additional C9 molecules could be bound when the cells were maintained at 4 degrees C. These cells were then incubated at 37 degrees C for 3 min and returned to 0 degrees C, a technique which exposed additional C9-binding sites equivalent to the number of C9 molecules previously bound to the cells. This technique was repeated and demonstrated that the sequential build-up of a C5b-9 site with two C9 molecules per C5b-8 site was capable of inhibiting both inner membrane function (respiration and amino acid transport) and cell viability. Three C9 molecules per complex had effects that approached the inhibitory effects of complexes formed in the presence of excess C9.     

Complement-mediated killing of Escherichia coli: dissipation of membrane potential by a C9-derived peptide

The molecular mechanism of complement-mediated killing of Gram-negative bacteria has yet to be resolved, but it is generally accepted that assembly of the membrane attack complex (MAC) of complement on the outer bacterial membrane is a required step. We have now investigated the effect of the MAC and its precursor complex, C5b-8, on the membrane potential (delta Em) across the inner bacterial membrane. Delta Em of whole cells was measured directly by using a lipophilic cation (tetraphenylphosphonium) that equilibrates with the potential or indirectly by measuring transport of solutes (proline and galactoside), which is dependent on delta Em. Our results indicate that the C5b-8 complex caused a transient collapse of delta Em in the absence of cell killing. Addition of C9 to allow formation of the MAC dissipated delta Em irreversibly, and the cells were killed. Since delta Em is generated across the inner membrane in Gram-negative bacteria, inner membrane vesicles were prepared and membrane potentials were generated either by adding D-lactate to energize the electron-transport chain or by creating a K+ diffusion potential with valinomycin. C9 added in the absence of earlier acting complement proteins had no effect on delta Em of isolated, actively respiring vesicles or on K+ diffusion potentials. In contrast, its C-terminal thrombin fragment (C9b), which has been shown earlier to contain the membrane-active domain of C9, efficiently collapsed delta Em in such vesicles. C9b did not require a specific receptor since it was effective on "right-side-out" and "inside-out" vesicles. These results are interpreted to indicate that a C9-derived fragment deenergizes cells and may be the causative agent for cell death.     

Structure of human complement C8, a precursor to membrane attack

Complement component C8 plays a pivotal role in the formation of the membrane attack complex (MAC), an important antibacterial immune effector. C8 initiates membrane penetration and coordinates MAC pore formation. High-resolution structures of C8 subunits have provided some insight into the function of the C8 heterotrimer; however, there is no structural information describing how the intersubunit organization facilitates MAC assembly. We have determined the structure of C8 by electron microscopy and fitted the C8α-MACPF (membrane attack complex/perforin)-C8γ co-crystal structure and a homology model for C8β-MACPF into the density. Here, we demonstrate that both the C8γ protrusion and the C8α-MACPF region that inserts into the membrane upon activation are accessible.     

Serum-resistant strains of Borrelia burgdorferi evade complement-mediated killing by expressing a CD59-like complement inhibitory molecule

Borrelia burgdorferi, the etiological agent of Lyme disease, comprises three genospecies, Borrelia garinii, afzelii, and burgdorferi sensu strictu, that exhibit different pathogenicity and differ in the susceptibility to C-mediated killing. We examined C-sensitive and C-resistant strains of B. burgdorferi for deposition of C3 and late C components by fluorescence microscope and flow cytometry. Despite comparable deposition of C3 on the two strains, the resistant strain exhibited reduced staining for C6 and C7, barely detectable C9, and undetectable poly C9. Based on these findings, we searched for a protein that inhibits assembly of C membrane attack complex and documented an anti-human CD59-reactive molecule on the surface of C-resistant spirochetes by flow cytometry and electron microscopy. A molecule of 80 kDa recognized by polyclonal and monoclonal anti-CD59 Abs was identified in the membrane extract of C-resistant strains by SDS-PAGE and Western blot analysis. The molecule was released from the bacterial wall using deoxycholate and trypsin, suggesting its insertion into the bacterial membrane. The CD59-like molecule acts as C inhibitor on Borrelia because incubation with F(ab')(2) anti-CD59 renders the serum-resistant strain exquisitely susceptible to C-mediated killing and guinea pig erythrocytes bearing C5b-8, unlike the RBC coated with C5b-7, are protected from reactive lysis by the bacterial extract. Western blot analysis revealed preferential binding of the C inhibitory molecule to C9 and weak interaction with C8 beta.     

Polymerization of C9 enhances bacterial cell envelope damage and killing by membrane attack complex pores

Complement proteins can form membrane attack complex (MAC) pores that directly kill Gram-negative bacteria. MAC pores assemble by stepwise binding of C5b, C6, C7, C8 and finally C9, which can polymerize into a transmembrane ring of up to 18 C9 monomers. It is still unclear if the assembly of a polymeric-C9 ring is necessary to sufficiently damage the bacterial cell envelope to kill bacteria. In this paper, polymerization of C9 was prevented without affecting binding of C9 to C5b-8, by locking the first transmembrane helix domain of C9. Using this system, we show that polymerization of C9 strongly enhanced damage to both the bacterial outer and inner membrane, resulting in more rapid killing of several Escherichia coli and Klebsiella strains in serum. By comparing binding of wildtype and ‘locked’ C9 by flow cytometry, we also show that polymerization of C9 is impaired when the amount of available C9 per C5b-8 is limited. This suggests that an excess of C9 is required to efficiently form polymeric-C9. Finally, we show that polymerization of C9 was impaired on complement-resistant E. coli strains that survive killing by MAC pores. This suggests that these bacteria can specifically block polymerization of C9. All tested complement-resistant E. coli expressed LPS O-antigen (O-Ag), compared to only one out of four complement-sensitive E. coli. By restoring O-Ag expression in an O-Ag negative strain, we show that the O-Ag impairs polymerization of C9 and results in complement-resistance. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.     

Complement-mediated killing of the Lyme disease spirochete Borrelia burgdorferi. Role of antibody in formation of an effective membrane attack complex

Lyme disease is a multisystemic illness caused by the spirochete Borrelia burgdorferi. In the absence of specific antibody, the spirochete is resistant to the bactericidal activity of C, despite the capacity of B. burgdorferi to activate both C pathways. We examined the mechanism of serum resistance by measuring the deposition of C3 and terminal C components on B. burgdorferi in the presence and absence of immune IgG. In normal human serum antibody-sensitized borreliae bound similar amounts of C3, and similar or increased amounts of C8 and C9, in comparison to unsensitized bacteria. However, at comparable levels of C3, C8, or C9 uptake, only sensitized bacteria were killed. The requirement of antibody for killing could not be explained by differences in the rate of C deposition or by differences in the C9 to C8 ratio in the membrane attack complex (MAC). We found that bacteria incubated in C5-depleted human serum, but not in C6-depleted serum, were killed when this treatment was followed by antibody and the missing C components. Bacteria were also killed by reactive lysis (C5b-9) provided that antibody was present. Therefore, the effect of bactericidal IgG occurred at the stage of C5b binding to the bacterial surface. Elution studies of bound C9 indicated that the MAC was stably bound to the outer membrane of B. burgdorferi, whether or not the bacteria were treated with antibody. However, treatment with 0.1% trypsin released 48% of 125I-C9 from the surface of unsensitized borreliae and 24% from IgG-sensitized cells, demonstrating that the presence of the antibody changed the accessibility to trypsin of C9 in the MAC. These results indicate that the effect of antibody in the killing process is not to enhance the rate or extent of initial or terminal component binding, but rather to alter the bacterial outer membrane to allow effective MAC formation.     

Outer membrane permeabilization by the membrane attack complex sensitizes Gram-negative bacteria to antimicrobial proteins in serum and phagocytes

Infections with Gram-negative bacteria form an increasing risk for human health due to antibiotic resistance. Our immune system contains various antimicrobial proteins that can degrade the bacterial cell envelope. However, many of these proteins do not function on Gram-negative bacteria, because the impermeable outer membrane of these bacteria prevents such components from reaching their targets. Here we show that complement-dependent formation of Membrane Attack Complex (MAC) pores permeabilizes this barrier, allowing antimicrobial proteins to cross the outer membrane and exert their antimicrobial function. Specifically, we demonstrate that MAC-dependent outer membrane damage enables human lysozyme to degrade the cell wall of E. coli. Using flow cytometry and confocal microscopy, we show that the combination of MAC pores and lysozyme triggers effective E. coli cell wall degradation in human serum, thereby altering the bacterial cell morphology from rod-shaped to spherical. Completely assembled MAC pores are required to sensitize E. coli to the antimicrobial actions of lysozyme and other immune factors, such as Human Group IIA-secreted Phospholipase A2. Next to these effects in a serum environment, we observed that the MAC also sensitizes E. coli to more efficient degradation and killing inside human neutrophils. Altogether, this study serves as a proof of principle on how different players of the human immune system can work together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate or work synergistically with the immune system.     

Resistance of spheroplasts and whole cells of Pseudomonas aeruginosa to bactericidal activity of various biocides: evidence of the membrane implication

To emphasise the role of outer and inner membranes in the resistance of Pseudomonas aeruginosa to bactericidal activity of various disinfectants, spheroplasts and whole cells were compared. Spheroplasts are more sensitive than whole cells to quaternary ammonium compounds such as didecyl dimethyl ammonium bromide (DDAB) and C16-benzalkonium chloride. The outer membrane acts as a barrier to prevent these disinfectants from entering the cell. It seems to have no influence on activities of smaller molecules such as C12, C14-benzalkonium chlorides and sodium dichloroisocyanurate. For tri-sodium phosphate, the presence of outer membrane emphasized the action of the molecule. Moreover, resistance of DDAB-adapted spheroplasts to bactericidal activity of DDAB is higher than the resistance of non-adapted spheroplasts. This suggests that the inner membrane could also play a role in resistance to DDAB.     

Metaxin deficiency alters mitochondrial membrane permeability and leads to resistance to TNF-induced cell killing

Metaxin, a mitochondrial outer membrane protein, is critical for TNF-induced cell death in L929 cells. Its deficiency, caused by retroviral insertion-mediated mutagenesis, renders L929 cells resistance to TNF killing. In this study, we further characterized metaxin deficiency-caused TNF resistance in parallel with Bcl-X_L overexpression-mediated death resistance. We did not find obvious change in mitochondria membrane potential in metaxin-deficient (Met^mut) and Bcl-X_L-overexpressing cells, but we did find an increase in the release rate of the mitochondrial membrane potential probe rhodamine 123 (Rh123) that was preloaded into mitochondria. In addition, overexpression of a function-interfering mutant of metaxin (MetaDTM/C) or Bcl-X_L in MCF-7.3.28 cells also resulted in an acquired resistance to TNF killing and a faster rate of Rh123 release, indicating a close correlation between TNF resistance and higher rates of the dye release from the mitochondria. The release of Rh123 can be controlled by the mitochondrial membrane permeability transition (PT) pore, as targeting an inner membrane component of the PT pore by cyclosporin A (CsA) inhibited Rh123 release. However, metaxin deficiency and Bcl-X_L overexpression apparently affect Rh123 release from a site(s) different from that of CsA, as CsA can overcome their effect. Though both metaxin and Bcl-X_L appear to function on the outer mitochondrial membrane, they do not interact with each other. They may use different mechanisms to increase the permeability of Rh123, since previous studies have suggested that metaxin may influence certain outer membrane porins while Bcl-X_L may form pores on the outer membrane. The alteration of the mitochondrial outer membrane properties by metaxin deficiency and Bcl-X_L overespression, as indicated by a quicker Rh123 release, may be helpful in maintaining mitochondrial integrity.     

The type IV pilus assembly complex: biogenic interactions among the bundle-forming pilus proteins of enteropathogenic Escherichia coli

Production of type IV bundle-forming pili (BFP) by enteropathogenic Escherichia coli (EPEC) requires the protein products of 12 genes of the 14-gene bfp operon. Antisera against each of these proteins were used to demonstrate that in-frame deletion of individual genes within the operon reduces the abundance of other bfp operon-encoded proteins. This result was demonstrated not to be due to downstream polar effects of the mutations but rather was taken as evidence for protein-protein interactions and their role in the stabilization of the BFP assembly complex. These data, combined with the results of cell compartment localization studies, suggest that pilus formation requires the presence of a topographically discrete assembly complex that is composed of BFP proteins in stoichiometric amounts. The assembly complex appears to consist of an inner membrane component containing three processed, pilin-like proteins, BfpI, -J, and -K, that localize with BfpE, -L, and -A (the major pilin subunit); an outer membrane, secretin-like component, BfpB and -G; and a periplasmic component composed of BfpU. Of these, only BfpL consistently localizes with both the inner and outer membranes and thus, together with BfpU, may articulate between the Bfp proteins in the inner membrane and outer membrane compartments.     

Membrane vesiculation protects erythrocytes from destruction by complement

Nucleated cells can resist attack by C by exocytosis or endocytosis of the terminal C components C5b-9 (membrane attack complex) (MAC), but it is generally accepted that formation of a single MAC channel on E leads to lysis (one-hit theory). We find that human and guinea pig E, but not SRBC, can eliminate the MAC from the membrane in the form of microvesicles and escape destruction. When guinea pig or human E are incubated with C5b-9, vesiculation proceeds without a lag and is detected at nonlytic doses of C9. Continuous Ca2+ influx is required for vesiculation. The amount of released vesicles is in direct relation to Ca2+ concentration, and the increase in vesiculation is associated with a parallel decrease in lysis. SRBC, which do not vesiculate when Ca2+ loaded, are lysed by C5b-9 with the same efficiency in the presence or absence of Ca2+. Vesicles released from guinea pig RBC under C5b-9 attack are enriched in C9 by a factor of 10, compared with the unlysed cells, and by a factor of 3 to 4, compared with ghosts. We conclude that E are protected from lysis not only by CD59 and C8bp/HRF, which prevent MAC assembly, but also by selective elimination of the MAC.     

Disruption of the gene for Hsp30, an alpha-crystallin-related heat shock protein of Neurospora crassa, causes defects in import of proteins into mitochondria

The gene for Hsp30, the only known alpha-crystallin-related heat shock protein of Neurospora crassa, was disrupted by repeat-induced point mutagenesis, leading to loss of cell survival at high temperature. Hsp30, which is not synthesized at 30 degrees C, associates reversibly with the mitochondria at high temperature (45 degrees C). In this study, we found that import of selected proteins into internal compartments of mitochondria, following their synthesis in the cytosol, was severely impaired at high temperature in a strain mutant in Hsp30. After 70 min of cell incubation at 45 degrees C, most matrix, inner membrane, and intermembrane-space proteins tested were reduced in import by about 50-70% in the mutant, as compared to wild-type cells. In contrast, assembly of selected proteins into the outer mitochondrial membrane was not reduced, except for one component of the preprotein translocase complex of the mitochondrial outer membrane. Three proteins of this complex co-immunoprecipitated with Hsp30 of wild-type cells incubated at 45 degrees C. We propose that Hsp30 interacts with the preprotein translocase of the mitochondrial outer membrane and that it chaperones the activity of one or more components of this translocase complex at high temperature.     

人补体调节蛋白MCP、CD59共表达体系的构建及其功能研究

利用双启动子构建含人补体调节蛋白MCP和CD59 cDNA的双顺反子重组表达载体pcDNA3-MCPCD59-DP,以磷酸钙沉淀法转染NIH3T3细胞,用G418筛选获得NIH3T3pcDNA3-MCPCD59-DP转化细胞。PCR实验结果显示人MCP和CD59整合在转化的NIH3T3细胞的染色体上,RT-PCR和Western印迹实验分别从RNA水平和蛋白质水平证实了人MCP和CD59在转化细胞中的共表达。 检测连续传代30次的NIH3T3pcDNA3-MCPCD59-DP结果表明人MCP和CD59基因仍稳定整合在细胞基因组中,并未随着传代而丢失,为稳定的转双基因细胞系。 补体溶破实验表明, pcDNA3-MCPCD59-DP转染细胞由于人MCP和CD59的共表达获得了较MCP或CD59单一表达时更好的保护功效,能有效地保护NIH3T3细胞免受人补体的攻击,从而抑制补体依赖的细胞毒反应的发生。 以上结果表明本研究所构建的双基因重组表达载体实现了人补体调节蛋白基因高效转移和高水平共表达,在克服超急性排斥反应的基因治疗中有潜在的应用价值。     

Streptococcus pneumoniae Phosphoglycerate Kinase Is a Novel Complement Inhibitor Affecting the Membrane Attack Complex Formation

The Gram-positive bacterium Streptococcus pneumoniae is a major human pathogen that causes infections ranging from acute otitis media to life-threatening invasive disease. Pneumococci have evolved several strategies to circumvent the host immune response, in particular the complement attack. The pneumococcal glycolytic enzyme phosphoglycerate kinase (PGK) is both secreted and bound to the bacterial surface and simultaneously binds plasminogen and its tissue plasminogen activator tPA. In the present study we demonstrate that PGK has an additional role in modulating the complement attack. PGK interacted with the membrane attack complex (MAC) components C5, C7, and C9, thereby blocking the assembly and membrane insertion of MAC resulting in significant inhibition of the hemolytic activity of human serum. Recombinant PGK interacted in a dose-dependent manner with these terminal pathway proteins, and the interactions were ionic in nature. In addition, PGK inhibited C9 polymerization both in the fluid phase and on the surface of sheep erythrocytes. Interestingly, PGK bound several MAC proteins simultaneously. Although C5 and C7 had partially overlapping binding sites on PGK, C9 did not compete with either one for PGK binding. Moreover, PGK significantly inhibited MAC deposition via both the classical and alternative pathway at the pneumococcal surface. Additionally, upon activation plasmin(ogen) bound to PGK cleaved the central complement protein C3b thereby further modifying the complement attack. In conclusion, our data demonstrate for the first time to our knowledge a novel pneumococcal inhibitor of the terminal complement cascade aiding complement evasion by this important pathogen.     

Heavy metal transport by the CusCFBA efflux system

It is widely accepted that the increased use of antibiotics has resulted in bacteria with developed resistance to such treatments. These organisms are capable of forming multi‐protein structures that bridge both the inner and outer membrane to expel diverse toxic compounds directly from the cell. Proteins of the resistance nodulation cell division (RND) superfamily typically assemble as tripartite efflux pumps, composed of an inner membrane transporter, a periplasmic membrane fusion protein, and an outer membrane factor channel protein. These machines are the most powerful antimicrobial efflux machinery available to bacteria. In Escherichia coli, the CusCFBA complex is the only known RND transporter with a specificity for heavy metals, detoxifying both Cu⁺ and Ag⁺ ions. In this review, we discuss the known structural information for the CusCFBA proteins, with an emphasis on their assembly, interaction, and the relationship between structure and function.     

Attenuation of experimental allergic encephalomyelitis in complement component 6-deficient rats is associated with reduced complement C9 deposition, P-selectin expression, and cellular infiltrate in spinal cords

The role of Ab deposition and complement activation, especially the membrane attack complex (MAC), in the mediation of injury in experimental allergic encephalomyelitis (EAE) is not resolved. The course of active EAE in normal PVG rats was compared with that in PVG rats deficient in the C6 component of complement (PVG/C6(-)) that are unable to form MAC. Following immunization with myelin basic protein, PVG/C6(-) rats developed significantly milder EAE than PVG/C rats. The anti-myelin basic protein response was similar in both strains, as was deposition of C3 in spinal cord. C9 was detected in PVG/C rats but not in PVG/C6(-), consistent with their lack of C6 and inability to form MAC. In PVG/C6(-) rats, the T cell and macrophage infiltrate in the spinal cord was also significantly less than in normal PVG/C rats. There was also reduced expression of P-selectin on endothelial cells, which may have contributed to the reduced cellular infiltrate by limiting migration from the circulation. Assay of cytokine mRNA by RT-PCR in the spinal cords showed no differences in the profile of Th1 or Th2 cytokines between PVG/C and PVG/C6(-) rats. PVG/C rats also had a greater increase in peripheral blood white blood cell, neutrophil, and basophil counts than was observed in the PVG/C6(-). These findings suggest that the MAC may have a role in the pathogenesis of EAE, not only by Ig-activated MAC injury but also via induction of P-selectin on vascular endothelium to promote infiltration of T cells and macrophages into the spinal cord.     

YaeT (Omp85) affects the assembly of lipid-dependent and lipid-independent outer membrane proteins of Escherichia coli

The Omp85 family of proteins has been found in all Gram-negative bacteria and even several eukaryotic organisms. The previously uncharacterized Escherichia coli member of this family is YaeT. The results of this study, consistent with previous Omp85 studies, show that the yaeT gene encodes for an essential cellular function. Direct examinations of the outer membrane fraction and protein assembly revealed that cells depleted for YaeT are severely defective in the biogenesis of outer membrane proteins (OMPs). Interestingly, assemblies of the two distinct groups of OMPs that follow either SurA- and lipopolysaccharide-dependent (OmpF/C) or -independent (TolC) folding pathways were affected, suggesting that YaeT may act as a general OMP assembly factor. Depletion of cells for YaeT led to the accumulation of OMPs in the fraction enriched for periplasm, thus indicating that YaeT facilitates the insertion of soluble assembly intermediates from the periplasm to the outer membrane. Our data suggest that YaeT's role in the assembly of OMPs is not mediated through a role in lipid biogenesis, as debated for Omp85 in Neisseria, thus advocating a conserved OMP assembly function of Omp85 homologues.     

Complex formation and turnover of mitochondrial transporters and ion channels

Mitochondria are responsible for many vital cellular functions in eukaryotic cells, such as ATP production, steroid synthesis and prosthetic group biogenesis. The vital functions of mitochondria are possible due to the compartmental nature of this organelle. Mitochondria form a dynamic network that can exist as a network throughout a cell or as distinct individual structures. Mitochondria are also composed of two membranes, an inner and outer membrane. The inner mitochondrial membrane (IMM) is significantly larger than the outer membrane and must fold upon itself to be contained within the outer mitochondrial membrane (OMM). These folds are known as cristae. Altogether these different membrane compartments specialize in different functions of the mitochondria. The OMM is responsible for passage of small metabolites into and out of the mitochondria while excluding macromolecules. The IMM is a highly selective barrier between the solutes of the cytosol and those within the mitochondrial matrix. Cristae specialize in oxidative phosphorylation. The functions of these membranes are afforded by membrane proteins that are able to transport specific solutes. The appropriate localization, assembly into multi-subunit protein complexes, and wild-type function of these membrane proteins therefore is vital for mitochondria to maintain appropriate function and support cellular survival. This review will address the composition and functions of mitochondrial membrane localized multi-subunit protein complexes along with how these proteins undergo degradation to maintain homeostatic functions of mitochondria in the context of mitochondria specific transporters and ion channels. Due to the large number of known mitochondrial membrane transporters and ion channels this review will focus on the topics presented at the Mitochondrial Ion Channels and Transporters Symposium hosted by the New York University College of Dentistry in September 2015 in honor of Casey Kinnally.