Gamma-secretase-regulated proteolysis of the Notch receptor by mitochondrial intermediate peptidase

Notch is a transmembrane receptor that controls a diverse array of cellular processes including cell proliferation, differentiation, survival, and migration. The cellular outcome of Notch signaling is dependent on extracellular and intracellular signals, but the complexities of its regulation are not well understood. Canonical Notch signaling involves ligand association that triggers sequential and regulated proteolysis of Notch at several sites. Ligand-dependent proteolysis at the S2 site removes the bulk of the extracellular domain of Notch. Subsequent γ-secretase-mediated intramembrane proteolysis of the remaining membrane-tethered Notch fragment at the S3 site produces a nuclear-destined Notch intracellular domain (NICD). Here we show that following γ-secretase cleavage, Notch is proteolyzed at a novel S5 site. We have identified this S5 site to be eight amino acids downstream of the S3 site. Biochemical fractionation and purification resulted in the identification of the S5 site protease as the mitochondrial intermediate peptidase (MIPEP). Expression of the MIPEP-cleaved NICD (ΔNICD) results in a decrease in cell viability and mitochondria membrane potential. The sequential and regulated proteolysis by γ-secretase and MIPEP suggests a new means by which Notch function can be modulated.     

Mitochondrial proteolysis: Its emerging roles in stress responses

Background

Mitochondria are multifunctional organelles that not only serve as cellular energy stores but are also actively involved in several cellular stress responses, including apoptosis. In addition, mitochondria themselves are also continuously challenged by stresses such as reactive oxygen species (ROS), an inevitable by-product of oxidative phosphorylation. To exert various functions against these stresses, mitochondria must be equipped with appropriate stress responses that monitor and maintain their quality.

Scope of review

Interestingly, increasing evidence indicates that mitochondrial proteolysis has important roles in mitochondrial and cellular stress responses. In this review, we summarize current advances in mitochondrial proteolysis-mediated stress responses.

Major conclusions

Mitochondrial proteases do not only function as surveillance systems of protein quality control by degrading unfolded proteins but also regulate mitochondrial stress responses by processing specific mitochondrial proteins.

General significance

Studies on the regulation of mitochondrial proteolysis-mediated stress responses will provide the novel mechanistic insights into the stress response research fields.     

Regulation of CIRL-1 proteolysis and trafficking

Calcium-independent receptor of α-latrotoxin (CIRL-1) is an adhesion G protein-coupled receptor implicated in the regulation of exocytosis. CIRL-1 biosynthesis involves constitutive proteolytic processing that takes place in the endoplasmic reticulum, requires the receptor's GPS domain, and yields heterologous two-subunit receptor complexes. It was proposed that the GPS-directed cleavage is based on cis-autoproteolysis. In this study, we demonstrate that activators of protein kinase C - PMA and ionomycin, can inhibit the cleavage of CIRL-1 precursor in transfected cells. Both reagents also downregulate trafficking of CIRL-1 to the cell surface that results in accumulation of the uncleaved receptor precursor inside the cells. Experiments with a non-cleavable soluble mutant of CIRL-1 showed that the downregulation of the receptor trafficking is independent of its cleavage. Our data suggest that the GPS proteolysis of CIRL-1 is not a purely autocatalytic process and may involve auxiliary proteins or factors that become available in the course of CIRL-1 trafficking.     

Modulation of inner mitochondrial membrane channel activity

Three classes of inner mitochondrial membrane (IMM) channel activities have been defined by direct measurement of conductance levels in membranes with patch clamp techniques in 150 mM K Cl. The 107 pS activity is slightly anion selective and voltage dependent (open with matrix positive potentials). Multiple conductance channel (MCC) activity includes several levels from about 40 to over 1000 pS and can be activated by voltage or Ca²⁺. MCC may be responsible for the Ca²⁺-induced permeability transition observed with mitochondrial suspensions. A low conductance channel (LCC) is activated by alkaline pH and inhibited by Mg²⁺. LCC has a unit conductance of about 15 pS and may correspond to the inner membrane anion channel, IMAC, which was proposed from results obtained from suspension studies. All of the IMM channels defined thus far appear to be highly regulated and have a low open probability under physiological conditions. A summary of what is known about IMM channel regulation and pharmacology is presented and possible physiological roles of these channels are discussed.     

Regulation of the inner membrane mitochondrial permeability transition by the outer membrane translocator protein (peripheral benzodiazepine receptor)

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca(2+) uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.     

Protein translocase of mitochondrial inner membrane in Trypanosoma brucei

Translocases of mitochondrial inner membrane (TIMs) are multiprotein complexes. The only Tim component so far characterized in kinetoplastid parasites such as Trypanosoma brucei is Tim17 (TbTim17), which is essential for cell survival and mitochondrial protein import. Here, we report that TbTim17 is present in a protein complex of about 1,100 kDa, which is much larger than the TIM complexes found in fungi and mammals. Depletion of TbTim17 in T. brucei impairs the mitochondrial import of cytochrome oxidase subunit IV, an N-terminal signal-containing protein. Pretreatment of isolated mitoplasts with the anti-TbTim17 antibody inhibited import of cytochrome oxidase subunit IV, indicating a direct involvement of the TbTim17 in the import process. Purification of the TbTim17-containing protein complex from the mitochondrial membrane of T. brucei by tandem affinity chromatography revealed that TbTim17 associates with seven unique as well as a few known T. brucei mitochondrial proteins. Depletion of three of these novel proteins, i.e. TbTim47, TbTim54, and TbTim62, significantly decreased mitochondrial protein import in vitro. In vivo targeting of a newly synthesized mitochondrial matrix protein, MRP2, was also inhibited due to depletion of TbTim17, TbTim54, and TbTim62. Co-precipitation analysis confirmed the interaction of TbTim54 and TbTim62 with TbTim17 in vivo. Overall, our data reveal that TbTim17, the single homolog of Tim17/22/23 family proteins, is present in a unique TIM complex consisting of novel proteins in T. brucei and is critical for mitochondrial protein import.     

Thyroid hormone regulation of nuclear-encoded mitochondrial inner membrane polypeptides of the liver

The effects of thyroid hormone on nuclear-encoded mitochondrial inner membrane proteins were investigated by in vitro translation of the endogenous mRNA present in a postmitochondrial fraction from the livers of rats treated in vivo with hormone. The levels of the mRNAs were estimated by quantitative immunoabsorption of the translation mixture. Total protein synthesis was increased 2.6-fold after 4 days of in vivo hormone treatment, but only 10-15% of the polypeptides were dramatically altered (greater than 5-fold). Among the most highly elevated were cytochrome c1 (greater than 10-fold increase) and the Rieske iron-sulfur protein of the cytochrome bc1 complex. Other inner membrane proteins (core protein 1, beta subunit of F1 ATPase, subunit IV of cytochrome oxidase, 3-hydroxybutyrate dehydrogenase) and non-mitochondrial proteins (rat serum albumin, beta 2-microglobulin) were not altered significantly by hormone treatment. Cytochrome c1 and the Rieske protein increased after 12 h of hormone treatment, a relatively early response in mammalian mitochondrial biogenesis. The possible significance of this response for the regulation of mitochondrial synthesis and assembly is discussed.     

Regulation of proteolysis by human deubiquitinating enzymes

The post-translational attachment of one or several ubiquitin molecules to a protein generates a variety of targeting signals that are used in many different ways in the cell. Ubiquitination can alter the activity, localization, protein–protein interactions or stability of the targeted protein. Further, a very large number of proteins are subject to regulation by ubiquitin-dependent processes, meaning that virtually all cellular functions are impacted by these pathways. Nearly a hundred enzymes from five different gene families (the deubiquitinating enzymes or DUBs), reverse this modification by hydrolyzing the (iso)peptide bond tethering ubiquitin to itself or the target protein. Four of these families are thiol proteases and one is a metalloprotease. DUBs of the Ubiquitin C-terminal Hydrolase (UCH) family act on small molecule adducts of ubiquitin, process the ubiquitin proprotein, and trim ubiquitin from the distal end of a polyubiquitin chain. Ubiquitin Specific Proteases (USPs) tend to recognize and encounter their substrates by interaction of the variable regions of their sequence with the substrate protein directly, or with scaffolds or substrate adapters in multiprotein complexes. Ovarian Tumor (OTU) domain DUBs show remarkable specificity for different Ub chain linkages and may have evolved to recognize substrates on the basis of those linkages. The Josephin family of DUBs may specialize in distinguishing between polyubiquitin chains of different lengths. Finally, the JAB1/MPN +/MOV34 (JAMM) domain metalloproteases cleave the isopeptide bond near the attachment point of polyubiquitin and substrate, as well as being highly specific for the K63 poly-Ub linkage. These DUBs regulate proteolysis by: directly interacting with and co-regulating E3 ligases; altering the level of substrate ubiquitination; hydrolyzing or remodeling ubiquitinated and poly-ubiquitinated substrates; acting in specific locations in the cell and altering the localization of the target protein; and acting on proteasome bound substrates to facilitate or inhibit proteolysis. Thus, the scope and regulation of the ubiquitin pathway is very similar to that of phosphorylation, with the DUBs serving the same functions as the phosphatase. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

The organization of NADH dehydrogenase polypeptides in the inner mitochondrial membrane.

The organization of the constituent polypeptides of mitochondrial NADH dehydrogenase was studied by using two membrane-impermeable probes, diazobenzene[35S]sulphonate and lactoperoxidase-catalysed radioiodination. The incorporation of label into the subunits of the isolated enzyme was compared with that obtained with enzyme immunoprecipitated from labelled mitochondria or inverted submitochondrial particles. On the basis of accessibility to these two labels, we divide the polypeptides of Complex I into five groups: those that are apparently buried in the enzyme, those that are accessible to labelling in the isolated enzyme but not in the membrane, those that are exposed on the cytoplasmic face of the membrane, those that are exposed on the matrix face and finally those that are exposed on both faces and are therefore transmembranous. We conclude that NADH dehydrogenase is asymmetrically organized across the inner mitochondrial membrane.     

Intracellular proteolysis

Our present understanding of intracellular protein degradation developed from an attempt to understand the metabolic stability of proteins in cells. With the unravelling of its complex biochemical machinery has come a realization that protein degradation plays an important role in the most crucial events in the cell cycle, in signal transduction and in maintaining the integrity of the proper folded state of proteins. In this review, I attempt to trace the curious history of intracellular protein degradation, its novel and elegant biochemistry and to extract the features that might be important for the next era of studies in cell physiology.     

Intracellular proteolysis

Our present understanding of intracellular protein degradation developed from an attempt to understand the metabolic stability of proteins in cells. With the unravelling of its complex biochemical machinery has come a realization that protein degradation plays an important role in the most crucial events in the cell cycle, in signal transduction and in maintaining the integrity of the proper folded state of proteins. In this review, I attempt to trace the curious history of intracellular protein degradation, its novel and elegant biochemistry and to extract the features that might be important for the next era of studies in cell physiology.     

Selective effect of inhibitors on inner mitochondrial membrane channels.

The effect of amphiphilic cationic drugs on the channel activity of the mitochondrial inner membrane was examined with patch-clamp techniques. The therapeutic drugs amiodarone, propranolol and quinine reduced the probability of being open for the multiconductance channel (MCC) activity (levels from 30 pS to over 1 nS). While amiodarone decreased the probability of being open for the voltage dependent approximately 100 pS channel, it increased the conductance 42 +/- 20% (mean +/- SD, n = 6) with no significant change in mean open time. Similar results were obtained with propranolol. These data indicate that the approximately 100 pS channel is distinct from MCC activity.     

Effect of cyclosporine A and oligomycin on non-specific permeability of the inner mitochondrial membrane

The effect of oligomycin and cyclosporine A on the induction of non-specific permeability of the inner mitochondrial membrane by Ca²⁺ was under study. Both oligomycin and cyclosporine A were able to prevent the activation of non-specific permeability, but cyclosporine A was the only agent which could restore initial permeability of the inner mitochondrial membrane. The effect of cyclosporine A was shown not to be mediated through redistribution of Ca²⁺ between different mitochondrial subpopulations     

Stilbene derivatives inhibit the activity of the inner mitochondrial membrane chloride channels

Ion channels selective for chloride ions are present in all biological membranes, where they regulate the cell volume or membrane potential. Various chloride channels from mitochondrial membranes have been described in recent years. The aim of our study was to characterize the effect of stilbene derivatives on single-chloride channel activity in the inner mitochondrial membrane. The measurements were performed after the reconstitution into a planar lipid bilayer of the inner mitochondrial membranes from rat skeletal muscle (SMM), rat brain (BM) and heart (HM) mitochondria. After incorporation in a symmetric 450/450 mM KCl solution (cis/trans), the chloride channels were recorded with a mean conductance of 155 ± 5 pS (rat skeletal muscle) and 120 ± 16 pS (rat brain). The conductances of the chloride channels from the rat heart mitochondria in 250/50 mM KCl (cis/trans) gradient solutions were within the 70–130 pS range. The chloride channels were inhibited by these two stilbene derivatives: 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS). The skeletal muscle mitochondrial chloride channel was blocked after the addition of 1 mM DIDS or SITS, whereas the brain mitochondrial channel was blocked by 300 μM DIDS or SITS. The chloride channel from the rat heart mitochondria was inhibited by 50–100 μM DIDS. The inhibitory effect of DIDS was irreversible. Our results confirm the presence of chloride channels sensitive to stilbene derivatives in the inner mitochondrial membrane from rat skeletal muscle, brain and heart cells.     

Differential protein distributions define two sub-compartments of the mitochondrial inner membrane in yeast

The mitochondrial inner membrane exhibits a complex topology. Its infolds, the cristae membranes, are contiguous with the inner boundary membrane (IBM), which runs parallel to the outer membrane. Using live cells co-expressing functional fluorescent fusion proteins, we report on the distribution of inner membrane proteins in budding yeast. To this end we introduce the enlarged mitochondria of Deltamdm10, Deltamdm31, Deltamdm32, and Deltammm1 cells as a versatile model system to study sub-mitochondrial protein localizations. Proteins of the F(1)F(0) ATP synthase and of the respiratory chain complexes III and IV were visualized in the cristae-containing interior of the mitochondria. In contrast, proteins of the TIM23 complex and of the presequence translocase-associated motor were strongly enriched at the IBM. The different protein distributions shown here demonstrate that the cristae membranes and the IBM are functionally distinct sub-compartments.     

UCP2 is a mitochondrial transporter with an unusual very short half-life

This study focused on the stability of UCP2 (uncoupling protein 2), a mitochondrial carrier located in the inner membrane of mitochondrion. UCP2 is very unstable, with a half-life close to 30min, compared to 30h for its homologue UCP1, a difference that may highlight different physiological functions. Heat production by UCP1 in brown adipocytes is generally a long and adaptive phenomenon, whereas control of mitochondrial ROS by UCP2 needs more subtle regulation. We show that a mutation in UCP2 shown to modify its activity, actually decreases its stability.     

Selective solubilization of nicotinamide nucleotide transhydrogenase from the mitochondrial inner membrane

Nicotinamide nucleotide transhydrogenase was solubilized from beef heart submitochondrial particles employing Triton X-100 or lysolecithin. Lysolecithin was considerably more efficient and selective and released over 80 % of the transhydrogenase acdtivity from the membrane together with succinate dehydrogenase. Solubilization of NADH dehydrogenase and cytochrome oxidase was more efficiently accomplished with Triton than with lysolecithin. Both detergents released ATPase to various extents. Transhydrogenase remaining bound to particles after treatment with lysolecithin still catalyzed energy-linked transhydrogenation.     

Selective solubilization of the components of the mitochondrial inner membrane by lysolecithin.

1. Of various phospholipids tested, lysolecithin was the most efficient in the solubilization of the components of beef heart submitochondrial particles. Lysolecithin solubilized selectively nicotinamide nucleotide transhydrogenase, succinate dehydrogenase, NADH dehydrogenase and oligomycin-sensitive ATPase. Various cytochromes other than cytochrome c were only slightly solubilized. 2. The effect of various parameters, e.g. ionic strength, pH, time of centrifugation, and concentrations of lysolecithin and protein was investigated. Increasing times of centrifugation led to a partial sedimentation of NADH dehydrogenase, and a complete sedimentation of oligomycin-sensitive ATPase and cytochrome oxidase. 3. Further fractionation of the lysolecithin extract by centrifugation in the presence of low concentrations of cholate gave a complete separation of NADH dehydrogenase and transhydrogenase, indicating that these enzymes are not related functionally. 4. With the lysolecithin fractionation procedure a more than 10-fold purification of transhydrogenase was achieved. Polyacrylamide gel electrophoresis of the partially purified transhydrogenase in the presence of sodium dodecyl sulphate showed major increases in protein-stained bands corresponding to between 70 000 and 54 000 daltons. 5. A possible mechanism for the detergent action of lysolecithin involving a specific exchange of bound phospholipids for lysolecithin is discussed.     

Parkin mediates proteasome-dependent protein degradation and rupture of the outer mitochondrial membrane

Upon mitochondrial depolarization, Parkin, a Parkinson disease-related E3 ubiquitin ligase, translocates from the cytosol to mitochondria and promotes their degradation by mitophagy, a selective type of autophagy. Here, we report that in addition to mitophagy, Parkin mediates proteasome-dependent degradation of outer membrane proteins such as Tom20, Tom40, Tom70, and Omp25 of depolarized mitochondria. By contrast, degradation of the inner membrane and matrix proteins largely depends on mitophagy. Furthermore, Parkin induces rupture of the outer membrane of depolarized mitochondria, which also depends on proteasomal activity. Upon induction of mitochondrial depolarization, proteasomes are recruited to mitochondria in the perinuclear region. Neither proteasome-dependent degradation of outer membrane proteins nor outer membrane rupture is required for mitophagy. These results suggest that Parkin regulates degradation of outer and inner mitochondrial membrane proteins differently through proteasome- and mitophagy-dependent pathways.