ChChd3, an inner mitochondrial membrane protein, is essential for maintaining crista integrity and mitochondrial function

The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952-14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of β-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function.     

The protein kinase C-mediated MAP kinase pathway involved in the maintenance of cellular integrity in Saccharomyces cerevisiae

Signal transduction mediated by the single yeast isozyme of protein kinase C (Pkc1p) is essential for the maintenance of cellular integrity in this model eukaryote. The past few years have seen a dramatic increase in our knowledge of the upstream regulatory factors that modulate Pkc1p activity (e.g. Tor2p, Rom1p, Rom2p, Rho1p, Slg1p, Mid2p) and of the downstream targets of the MAP kinase cascade triggered by it (e.g. Rlm1p, SBF complex). The picture that has emerged connects this pathway to a variety of other cellular processes, such as cell cycle progression (Cdc28p, Swi4p), mating (Ste20p), nutrient sensing (Ira1p), calcium homeostasis (calcineurin, Mid2p, Fks2p) and the structural dynamics of the cytoskeleton (Spa1p, Bni1p).     

Targeting of PKA, PKC and protein phosphatases to cellular microdomains

The intracellular responses to many distinct extracellular signals involve the direction of broad-based protein kinases and protein phosphatases to catalyse quite specific protein phosphorylation/dephosphorylation events. It is now clear that such specificity is often achieved through subcellular targeting of distinct pools of kinase or phosphatase towards particular substrates at specific subcellular locations. Given the dynamic nature of protein phosphorylation reactions, coordinated control of both kinase and phosphatases is often required and complexes formed by common scaffold or targeting proteins exist to direct both kinase and phosphatase to the same subcellular location. In many cases more than one kinase or phosphatase is required and binding proteins which target more than one kinase or phosphatase have now been identified. This review summarizes recent findings relating to the concept of targeting PKA, PKC and the major serine/threonine phosphatases, PP1, PP2A and PP2B, through the formation of multi-enzyme signalling complexes.     

CH-ILKBP regulates cell survival by facilitating the membrane translocation of protein kinase B/Akt

Cell survival depends on proper propagation of protective signals through intracellular signaling intermediates. We report here that calponin homology domain-containing integrin-linked kinase (ILK)-binding protein (CH-ILKBP), a widely expressed adaptor protein localized at plasma membrane-actin junctions, is essential for transmission of survival signals. Cells that are depleted of CH-ILKBP undergo extensive apoptosis despite the presence of cell-extracellular matrix contacts and soluble growth factors. The activating phosphorylation of protein kinase B (PKB/Akt), a key regulator of apoptosis, is impaired in the absence of CH-ILKBP. Importantly, loss of CH-ILKBP prevents the membrane translocation of PKB/Akt. Furthermore, forced membrane targeting of PKB/Akt bypasses the requirement of CH-ILKBP for the activating phosphorylation of PKB/Akt, suggesting that CH-ILKBP is required for the membrane translocation but not the subsequent phosphorylation of PKB/Akt. Finally, we show that loss of CH-ILKBP is also required for the full activation of extracellular signal-regulated kinase (ERK)1/2. However, restoration of the PKB/Akt activation is sufficient for protection of cells from apoptosis induced by the depletion of CH-ILKBP despite the persistent suppression of the ERK1/2 activation. Thus, CH-ILKBP is an important component of the prosurvival signaling pathway functioning primarily by facilitating the membrane translocation of PKB/Akt and consequently the activation of PKB/Akt in response to extracellular survival signals.     

Lipoic acid affects cellular migration into the central nervous system and stabilizes blood-brain barrier integrity

Reactive oxygen species (ROS) play an important role in various events underlying multiple sclerosis (MS) pathology. In the initial phase of lesion formation, ROS are known to mediate the transendothelial migration of monocytes and induce a dysfunction of the blood-brain barrier (BBB). In this study, we describe the beneficial effect of the antioxidant alpha-lipoic acid (LA) on these phenomena. In vivo, LA dose-dependently prevented the development of clinical signs in a rat model for MS, acute experimental allergic encephalomyelitis (EAE). Clinical improvement was coupled to a decrease in leukocyte infiltration into the CNS, in particular monocytes. Monocytes isolated from the circulation of LA-treated rats revealed a reduced migratory capacity to cross a monolayer of rat brain endothelial cells in vitro compared with monocytes isolated from untreated EAE controls. Using live cell imaging techniques, we visualized and quantitatively assessed that ROS are produced within minutes upon the interaction of monocytes with brain endothelium. Monocyte adhesion to an in vitro model of the BBB subsequently induced enhanced permeability, which could be inhibited by LA. Moreover, administration of exogenous ROS to brain endothelial cells induced cytoskeletal rearrangements, which was inhibited by LA. In conclusion, we show that LA has a protective effect on EAE development not only by affecting the migratory capacity of monocytes, but also by stabilization of the BBB, making LA an attractive therapeutic agent for the treatment of MS.     

Mammalian Oxa1 protein is useful for assessment of submitochondrial protein localization and mitochondrial membrane integrity

Taking advantage of the unique topology of oxidase assembly 1 (Oxa1) protein, a mitochondrial inner membrane protein with N (intermembrane space)-C (matrix) orientation, we explored the usefulness of the protein as a marker for submitochondrial protein localization. Mammalian Oxa1 protein exhibited different proteolytic patterns depending on mitochondrial membrane integrity, and in mitochondria with a disrupted outer membrane and outer and inner membranes, the proteolytic patterns of Oxa1 protein were consistent with those of mitochondrial intermembrane space and matrix marker proteins, respectively, suggesting that Oxa1 protein, a single molecule, can serve as a versatile submitochondrial localization marker that doubles as a membrane integrity marker.     

Regulation of mitochondrial integrity, autophagy and cell survival by BNIP3

Understanding the role of BNIP3 in the systemic response to hypoxia has been complicated by conflicting results that indicate on the one hand that BNIP3 promotes cell death, and other data, including our own that BNIP3 is not sufficient for cell death, but rather plays a critical role in hypoxia-induced autophagy. This work suggests that rather than promoting death, BNIP3 may actually allow survival either by preventing ATP depletion or by eliminating damaged mitochondria. However, the function of BNIP3 may be subverted under unusual conditions associated with acidosis that arise following extended periods of hypoxia and anaerobic glycolysis. Despite this novel insight into BNIP3 function, much remains to be done in terms of pinning down a molecular activity for BNIP3 that explains both its role in autophagy and how this may be subverted to induce cell death. As a target of the RB tumor suppressor, our work also places BNIP3 at the center of efforts to exploit autophagy to better treat human cancers in which tumor hypoxia is implicated as a progression factor.     

The mitochondrial membrane protein FgLetm1 regulates mitochondrial integrity,production of endogenous reactive oxygen species and mycotoxin biosynthesis in Fusarium graminearum

Deoxynivalenol (DON) is a mycotoxin produced in cereal crops infected with Fusarium graminearum. DON poses a serious threat to human and animal health, and is a critical virulence factor. Various environmental factors, including reactive oxygen species (ROS), have been shown to interfere with DON biosynthesis in this pathogen. The regulatory mechanisms of how ROS trigger DON production have been investigated extensively in F. graminearum. However, the role of the endogenous ROS‐generating system in DON biosynthesis is largely unknown. In this study, we genetically analysed the function of leucine zipper‐EF‐hand‐containing transmembrane 1 (LETM1) superfamily proteins and evaluated the role of the mitochondrial‐produced ROS in DON biosynthesis. Our results show that there are two Letm1 orthologues, FgLetm1 and FgLetm2, in F. graminearum. FgLetm1 is localized to the mitochondria and is essential for mitochondrial integrity, whereas FgLetm2 plays a minor role in the maintenance of mitochondrial integrity. The ΔFgLetm1 mutant demonstrated a vegetative growth defect, abnormal conidia and increased sensitivity to various stress agents. More importantly, the ΔFgLetm1 mutant showed significantly reduced levels of endogenous ROS, decreased DON biosynthesis and attenuated virulence in planta. To our knowledge, this is the first report showing that mitochondrial integrity and endogenous ROS production by mitochondria are important for DON production and virulence in Fusarium species.     

A rit GTPase-p38 mitogen-activated protein kinase survival pathway confers resistance to cellular stress

Cells mobilize diverse signaling cascades to protect against stress-mediated injury. Ras family GTPases play a pivotal role in cell fate determination, serving as molecular switches to control the integration of multiple signaling pathways. p38 mitogen-activated protein kinase (MAPK) signaling serves as a critical fulcrum in this process, regulating networks that stimulate cellular apoptosis but also have the capacity to promote cell survival. However, relatively little is known concerning this functional dichotomy, particularly the regulation of p38-dependent survival pathways. Here, we demonstrate that the Rit GTPase promotes cell survival by directing an unexpected p38 MAPK-dependent AKT survival pathway. Following stress exposure, Rit small hairpin RNA interference (shRNAi)-treated cells display increased apoptosis and selective disruption of p38 MAPK signaling, while expression of constitutively activated Rit promotes p38-AKT-dependent cell survival. Rit, but not Ras or Rap GTPases, can associate with, and is critical for, stress-mediated activation of the scaffolded p38-MK2-HSP27-AKT prosurvival signaling complex. Together, our studies establish Rit as a central regulator of a p38 MAPK-dependent signaling cascade that functions as a critical cellular survival mechanism in response to stress.     

Sphingolipids in apoptosis, survival and regeneration in the nervous system

Simple sphingolipids such as ceramide, sphingosine and sphingosine 1-phosphate are key regulators of diverse cellular functions. Their roles in the nervous system are supported by extensive evidence derived primarily from studies in cultured cells. More recently animal studies and studies with human samples have revealed the importance of ceramide and its metabolites in the development and progression of neurodegenerative disorders. The roles of sphingolipids in neurons and glial cells are complex, cell dependent, and many times contradictory. In this review I will summarize the effects elicited by ceramide and ceramide metabolites in cells of the nervous system, in particular those effects related to cell survival and death, emphasizing the molecular mechanisms involved. I also discuss recent evidence for the implication of sphingolipids in the development and progression of certain dementias.     

Sphingolipids in apoptosis, survival and regeneration in the nervous system

Simple sphingolipids such as ceramide, sphingosine and sphingosine 1-phosphate are key regulators of diverse cellular functions. Their roles in the nervous system are supported by extensive evidence derived primarily from studies in cultured cells. More recently animal studies and studies with human samples have revealed the importance of ceramide and its metabolites in the development and progression of neurodegenerative disorders. The roles of sphingolipids in neurons and glial cells are complex, cell dependent, and many times contradictory. In this review I will summarize the effects elicited by ceramide and ceramide metabolites in cells of the nervous system, in particular those effects related to cell survival and death, emphasizing the molecular mechanisms involved. I also discuss recent evidence for the implication of sphingolipids in the development and progression of certain dementias.     

Targeting of a tail-anchored protein to endoplasmic reticulum and mitochondrial outer membrane by independent but competing pathways

Many mitochondrial outer membrane (MOM) proteins have a transmembrane domain near the C terminus and an N-terminal cytosolic moiety. It is not clear how these tail-anchored (TA) proteins posttranslationally select their target, but C-terminal charged residues play an important role. To investigate how discrimination between MOM and endoplasmic reticulum (ER) occurs, we used mammalian cytochrome b(5), a TA protein existing in two, MOM or ER localized, versions. Substitution of the seven C-terminal residues of the ER isoform or of green fluorescent protein reporter constructs with one or two arginines resulted in MOM-targeted proteins, whereas a single C-terminal threonine caused promiscuous localization. To investigate whether targeting to MOM occurs from the cytosol or after transit through the ER, we tagged a MOM-directed construct with a C-terminal N-glycosylation sequence. Although in vitro this construct was efficiently glycosylated by microsomes, the protein expressed in vivo localized almost exclusively to MOM, and was nearly completely unglycosylated. The small fraction of glycosylated protein was in the ER and was not a precursor to the unglycosylated form. Thus, targeting occurs directly from the cytosol. Moreover, ER and MOM compete for the same polypeptide, explaining the dual localization of some TA proteins.     

Management of cellular energy by the AMP-activated protein kinase system

The AMP-activated protein kinase is a sensor of cellular energy status that is found in all eukaryotic cells. It is activated by rising AMP and falling ATP by a complex mechanism that results in an ultrasensitive response. The functions of the different domains on the three subunits of the alphabetagamma heterotrimer are slowly being unravelled, and a recent development has been the identification of a glycogen-binding domain on the beta subunit. Along with findings that high cellular glycogen represses kinase activation, this suggests that the system may be a sensor of glycogen content as well as of AMP and ATP. New insights have been obtained into the sequence and structural features by which the kinase recognises its downstream target proteins, and these are discussed. Once activated by depletion of cellular energy reserves, the kinase switches on ATP-producing catabolic pathways and switches off ATP-consuming processes, both via direct phosphorylation of regulatory proteins and via indirect effects on gene expression. A survey of the range of downstream targets for this important signalling pathway is presented.     

Targeting of the mitochondrial membrane proteins to the cell surface for functional studies

Studying mitochondrial membrane proteins for ion or substrate transport is technically difficult, as the organelles are hidden within the cell interior and thus inaccessible to many conventional nondisruptive techniques. This technical barrier can potentially be overcome if the mitochondrial membrane proteins are targeted to the cell surface, where they can be more readily studied. We undertook experiments presented here to target two related mitochondrial membrane proteins, mitochondrial ATP-binding cassette-1 and -2 protein (mABC1 and mABC2, respectively) to the cell surface for functional studies. These two proteins have an N-terminal mitochondrial targeting signal (MTS), and we hypothesized that removal of this sequence or addition of a cell surface targeting signal would lead to cell membrane targeting of these proteins. When the MTS was removed from mABC1, it localized to intracellular secretory compartments as well as the plasma membrane. However, truncated mABC2 lacking the MTS aggregated inside the cell. Addition of a cell membrane signal sequence or the transmembrane domain from CD8 to the N-terminus of mABC1 or mABC2 resulted in similar subcellular localizations. We then performed patch clamp on cells expressing mABC1 on their surface. These cells exhibited nonselective transport of K(+) and Na(+) ions and resulted in the loss of membrane potential. Our findings open new ways to study mitochondrial membrane proteins in established cell culture systems by targeting them to the cell surface, where they can more reliably be studied using various molecular and cellular techniques.     

Myelin,DIGs, and membrane rafts in the central nervous system

Over the past 40 years our understanding of the organization of cell membranes has changed dramatically. Membranes are no longer viewed as a homogenous sea of phospholipids studded with randomly positioned islands of proteins. Our current view of the membrane involves the formation of small lipid clusters, comprised mainly of cholesterol and sphingolipids, known as membrane rafts. These lipid clusters apparently include and exclude specific proteins leading to the hypothesis that these domains (1) regulate cellular polarity and compartmentalization through trafficking and sorting, (2) provide platforms for cellular signaling and adhesion, and (3) function as cellular gate keepers. Tremendous controversy surrounds the concept of membrane rafts primarily because these small, highly dynamic entities are too small to be observed with traditional microscopic methods and the most utilized approach for raft analysis relies on poorly quantified, inconsistent biochemical extractions. New analytical approaches are being developed and applied to the study of membrane rafts and these techniques provide great promise for furthering our understanding of these enigmatic domains. In this review we will provide a brief summary of the current understanding of membrane rafts, utilizing the CNS myelin literature for illustrative purposes, and present caveats that should be considered when studying these domains.     

Targeting and insertion of nuclear-encoded preproteins into the mitochondrial outer membrane

Most mitochondrial proteins are synthesized in the cytosol as preproteins with a cleavable presequence and are delivered to the import receptors on the mitochondria by cytoplasmic import factors. The proteins are then imported to the intramitochondrial compartments by the import systems of the outer and inner membranes, TOM and TIM. Mitochondrial outer membrane proteins are synthesized without a cleavable presequence and most of them contain hydrophobic transmembrane domains, which, in conjunction with the flanking segments, function as the mitochondria import signals. Some of the proteins are inserted into the outer membrane by the TOM machinery; the import signal probably arrests further translocation and is released from the translocation channel to the lipid bilayer. The other proteins are inserted into the membrane by a novel pathway independent of the TOM machinery. This article reviews recent developments in the biogenesis of mitochondrial outer membrane proteins.     

Localization of subspecies of protein kinase C in the mammalian central nervous system

Activation of protein kinase C (PKC) is regulated by dual second messengers; diacylglycerol (DG) produced by receptor mediated hydrolysis of phosphatidylinositol and Ca²⁺ which is released by inositol 1,4,5-triphosphate (IP3) from intracellular stores in the endoplasmic reticulum. In the mammalian central nervous system, available evidence suggests that PKC plays a prominent role in the processing of neuronal signals and in the short-term or long-term modulation of synaptic transmission. This enzyme is a member of a family consisting of at least eight subspecies, , βI, βII, γ, δ, , ζ and η. The homologous structure of each subspecies makes difficult resolution of the enzymological properties of the enzyme. The distinct functional roles of PKC subspecies in mammalian tissues have been elucidated by defining the localization of each subspecies. We identified -, βI-, βII- and γ-PKC subspecies in the rat brain by in situ hybridization and by light and electron microscopic immunohistochemistry, using antibodies specific for each subspecies. Most immunoreactions of the , βI, βII and γ subspecies were evident in neurons and there were few, if any, in glial cells. In this article, we summarize known cellular and subcellular localizations of PKC subspecies in mammalian CNS and some aspects of current studies in neuronal functions regulated by this enzyme are discussed.