The 30-kDa mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumor cells are processed from larger precursors

Acute regulation of steroidogenesis in steroidogenic tissue is controlled by the transfer of cholesterol from the outer to the inner mitochondrial membrane where cleavage to produce pregnenolone occurs. Hormonal stimulation of MA-10 mouse Leydig tumor cells results in a large increase in steroidogenesis and the concomitant appearance of a series of 30-kDa proteins which have been localized to the mitochondria. In the present study we have shown that the appearance of these proteins occurs in a dose-responsive manner with both human chorionic gonadotropin and cyclic AMP analog. We have also shown that while steroidogenesis is inhibited rapidly in response to a cessation of protein synthesis, the 30-kDa mitochondrial proteins remain in the mitochondria, posing a potential dilemma for arguments favoring their role in the acute regulation of steroidogenesis. We report that the 30-kDa mitochondrial proteins arise from two precursor proteins with molecular masses of 37 and 32 kDa which are also found to be associated with the mitochondria. The use of pulse-chase experiments and the inhibitors ortho-phenanthroline and carbonyl cyanide m-chlorophenylhydrazone demonstrated the precursor-product relationship between the 37-, 32-, and 30-kDa proteins. We have also demonstrated that, as shown for a number of other mitochondrial proteins, the 30-kDa proteins are transferred to the inner mitochondrial membrane by a process requiring both proteolytic removal of the targeting sequences and an electrical potential across the inner mitochondrial membrane. We propose that during this transfer contact sites form between the two mitochondrial membranes and may offer an ideal situation for the transfer of cholesterol from the outer membrane to the inner membrane by an as yet unknown mechanism. Following transfer, the 30-kDa proteins remain in the inner membrane no longer able to function in the further transfer of cholesterol, and it is the continuing synthesis and processing of more precursor proteins which provides additional substrate for steroidogenesis.     

Cholesterol transport in steroid biosynthesis: Role of protein–protein interactions and implications in disease states

The transfer of cholesterol from the outer to the inner mitochondrial membrane is the rate-limiting step in hormone-induced steroid formation. To ensure that this step is achieved efficiently, free cholesterol must accumulate in excess at the outer mitochondrial membrane and then be transferred to the inner membrane. This is accomplished through a series of steps that involve various intracellular organelles, including lysosomes and lipid droplets, and proteins such as the translocator protein (18 kDa, TSPO) and steroidogenic acute regulatory (StAR) proteins. TSPO, previously known as the peripheral-type benzodiazepine receptor, is a high-affinity drug- and cholesterol-binding mitochondrial protein. StAR is a hormone-induced mitochondria-targeted protein that has been shown to initiate cholesterol transfer into mitochondria. Through the assistance of proteins such as the cAMP-dependent protein kinase regulatory subunit Iα (PKA-RIα) and the PKA-RIα- and TSPO-associated acyl-coenzyme A binding domain containing 3 (ACBD3) protein, PAP7, cholesterol is transferred to and docked at the outer mitochondrial membrane. The TSPO-dependent import of StAR into mitochondria, and the association of TSPO with the outer/inner mitochondrial membrane contact sites, drives the intramitochondrial cholesterol transfer and subsequent steroid formation. The focus of this review is on (i) the intracellular pathways and protein–protein interactions involved in cholesterol transport and steroid biosynthesis and (ii) the roles and interactions of these proteins in endocrine pathologies and neurological diseases where steroid synthesis plays a critical role.     

Protein import into yeast mitochondria: the inner membrane import site protein ISP45 is the MPI1 gene product.

Protein import across both mitochondrial membranes is mediated by the cooperation of two distinct protein transport systems, one in the outer and the other in the inner membrane. Previously we described a 45 kDa yeast mitochondrial inner membrane protein (ISP45) that can be cross-linked to a partially translocated precursor protein (Scherer et al., 1992). We have now purified ISP45 to homogeneity and identified it as the product of the nuclear MPI1 gene. Identity of ISP45 with the MPI1 gene product was shown by microsequencing of three tryptic ISP45 peptides and by demonstrating that an antibody against an Mpi1p-beta-galactosidase fusion protein specifically recognizes ISP45. Antibodies monospecific for ISP45 inhibited protein import into right-side-out mitochondrial inner membrane vesicles, but not into intact mitochondria. On solubilizing mitochondria, ISP45 was rapidly converted to a 40 kDa proteolytic fragment unless mitochondria were first denatured with trichloroacetic acid. The combined genetic and biochemical evidence identifies ISP45/Mpi1p as a component of the protein import system of the yeast mitochondrial inner membrane.     

Disrupting mitochondrial function with surfactants inhibits MA-10 Leydig cell steroidogenesis

It is well established that surfactants can elicit cytotoxic effects at threshold concentrations by changing the permeability and solubilizing components of cell membranes. The purpose of this study was to characterize the relationship between perturbation of the mitochondrial membrane resulting from treatment with representative cationic, nonionic, and anionic surfactants and the extent to which this perturbation affects steroid formation and StAR protein expression and activity in MA-10 Leydig cells. The StAR protein is synthesized as an active 37 kDa extramitochondrial form, which is processed into a 30 kDa intramitochondrial form after cholesterol transfer and mitochondrial import and processing. It has been shown in several in vitro studies that the mitochondrial electrochemical gradient is required for the StAR protein to transfer cholesterol to the inner mitochondrial membrane. Each substance that was tested produced a concentration-dependent decrease in steroid formation in hCG-stimulated MA-10 cells. Decreases in progesterone production were accompanied by loss of mitochondrial membrane potential and by a decrease in the levels of the 30 kDa form of the StAR protein. However, levels of the 37 kDa form of the StAR protein did not decrease, indicating no effect on StAR protein expression. These results demonstrate how perturbation of the mitochondrial membrane by surfactants inhibits import, processing, and cholesterol transfer activity and underscore the importance of including sensitive assays that evaluate mitochondrial function when screening for potential effects on steroidogenesis with in vitro test systems.     

Synergistic stimulation of pregnenolone synthesis in rat adrenal mitochondria by n-hexane and cardiolipin

n-Hexane and cardiolipin each stimulate pregnenolone production by isolated rat adrenal mitochondria. Following corticotropin (ACTH) stimulation, mitochondrial cholesterol metabolism exhibits a fast phase lasting 2 min, followed by a 10-fold slower metabolism. ACTH suppression by dexamethazone or cycloheximide (CX) treatment removes this fast phase. n-Hexane, at concentrations approaching 80% of the aqueous solubility limit (approximately 0.08 mM), selectively stimulates the slow phase of metabolism, while cardiolipin (100 microM) stimulates only the fast phase. Other alkanes and ethers are effective. The effect of n-hexane is dependent on mitochondrial integrity, as evidenced by decreased effects in hypoosmotically shocked mitochondria (outer membrane disrupted) and ineffectiveness in sonicated mitochondria (both membranes disrupted). n-Hexane apparently enhances the transfer of outer membrane cholesterol to inner membrane P-450scc. Stimulation by cardiolipin is retained by disrupted mitochondria and may involve enhanced availability of P-450scc to inner membrane cholesterol. When added together, these agents produce more than additive effects on cholesterol metabolism. Preincubation with n-hexane did not increase reactive cholesterol, suggesting that enhanced cholesterol transport occurs only in concert with metabolism of inner membrane cholesterol. Uptake of alkanes into mitochondrial membranes may effect structural changes that facilitate outer to inner membrane cholesterol transfer, but major changes are excluded by the effectiveness of isocitrate as a reductant for P-450scc. In combination, n-hexane and cardiolipin reproduce the effect of the ACTH-sensitive sterol regulatory peptide on mitochondria [R. C. Pedersen and A. C. Brownie (1983) Proc. Natl. Acad. Sci. USA 80, 1882-1886], suggesting that peptide action on adrenal mitochondria may resolve into two analogous components.     

Identification of a GTP-binding protein in the contact sites between inner and outer mitochondrial membranes

Whilst investigating whether GTP hydrolysis may be required for the import of preproteins into mitochondria we have found that a GTP-binding protein is located at the contact sites between mitochondrial inner and outer membranes. When mitochondrial outer membranes purified from rat liver were UV-irradiated in the presence of [alpha-32P]GTP, a 52 kDa protein was radiolabelled, whereas [alpha-32P]ATP did not label this protein. GTP-binding proteins were also labelled in the cytosolic and microsomal fractions, but the 52 kDa protein was concentrated in mitochondrial membranes and was the only protein specifically labelled by GTP in these membranes. Fractionation of mitochondrial membrane vesicles into outer membranes, inner membranes and contact sites between outer and inner membranes showed that the GTP-binding activity was highly enriched in contact sites, the location at which preprotein import is believed to occur. A protein of almost identical size was also found to be labelled in mitochondria from yeast.     

Niemann-Pick Type C2 protein contributes to the transport of endosomal cholesterol to mitochondria without interacting with NPC1

Mitochondrial cholesterol is maintained within a narrow range to regulate steroid and oxysterol synthesis and to ensure mitochondrial function. Mitochondria acquire cholesterol through several pathways from different cellular pools. Here we have characterized mitochondrial import of endosomal cholesterol using Chinese hamster ovary cells expressing a CYP11A1 fusion protein that converts cholesterol to pregnenolone at the mitochondrial inner membrane. RNA interference-mediated depletion of the voltage-dependent anion channel 1 in the mitochondrial outer membrane or of Niemann-Pick Type C2 (NPC2) in the endosome lumen decreased arrival of cholesterol at the mitochondrial inner membrane. Expression of NPC2 mutants unable to transfer cholesterol to NPC1 still restored mitochondrial cholesterol import in NPC2-depleted cells. Transport assays in semi-permeabilized cells showed nonvesicular cholesterol trafficking directly from endosomes to mitochondria that did not require cytosolic transport proteins but that was reduced in the absence of NPC2. Our findings indicate that NPC2 delivers cholesterol to the perimeter membrane of late endosomes, where it becomes available for transport to mitochondria without requiring NPC1.     

In vitro studies on the role of the peripheral-type benzodiazepine receptor in steroidogenesis

In vitro studies using isolated cells, mitochondria and submitochondrial fractions demonstrated that in steroid synthesizing cells, the peripheral-type benzodiazepine receptor (PBR) is an outer mitochondrial membrane protein, preferentially located in the outer/inner membrane contact sites, involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis. Mitochondrial PBR ligand binding characteristics and topography are sensitive to hormone treatment suggesting a role of PBR in the regulation of hormone-mediated steroidogenesis. Targeted disruption of the PBR gene in Leydig cells in vitro resulted in the arrest of cholesterol transport into mitochondria and steroid formation; transfection of the mutant cells with a PBR cDNA rescued steroidogenesis demonstrating an obligatory role for PBR in cholesterol transport. Molecular modeling of PBR suggested that it might function as a channel for cholesterol. This hypothesis was tested in a bacterial system devoid of PBR and cholesterol. Cholesterol uptake and transport by these cells was induced upon PBR expression. Amino acid deletion followed by site-directed mutagenesis studies and expression of mutant PBRs demonstrated the presence in the cytoplasmic carboxy-terminus of the receptor of a cholesterol recognition/interaction amino acid consensus sequence. This amino acid sequence may help for recruiting the cholesterol coming from intracellular sites to the mitochondria.     

Mitochondrial cholesterol import

All animal subcellular membranes require cholesterol, which influences membrane fluidity and permeability, fission and fusion processes, and membrane protein function. The distribution of cholesterol among subcellular membranes is highly heterogeneous and the cholesterol content of each membrane must be carefully regulated. Compared to other subcellular membranes, mitochondrial membranes are cholesterol-poor, particularly the inner mitochondrial membrane (IMM). As a result, steroidogenesis can be controlled through the delivery of cholesterol to the IMM, where it is converted to pregnenolone. The low basal levels of cholesterol also make mitochondria sensitive to changes in cholesterol content, which can have a relatively large impact on the biophysical and functional characteristics of mitochondrial membranes. Increased mitochondrial cholesterol levels have been observed in diverse pathological conditions including cancer, steatohepatitis, Alzheimer disease and Niemann-Pick Type C1-deficiency, and are associated with increased oxidative stress, impaired oxidative phosphorylation, and changes in the susceptibility to apoptosis, among other alterations in mitochondrial function. Mitochondria are not included in the vesicular trafficking network; therefore, cholesterol transport to mitochondria is mostly achieved through the activity of lipid transfer proteins at membrane contact sites or by cytosolic, diffusible lipid transfer proteins. Here we will give an overview of the main mechanisms involved in mitochondrial cholesterol import, focusing on the steroidogenic acute regulatory protein StAR/STARD1 and other members of the StAR-related lipid transfer (START) domain protein family, and we will discuss how changes in mitochondrial cholesterol levels can arise and affect mitochondrial function. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.     

C1orf163/RESA1 Is a Novel Mitochondrial Intermembrane Space Protein Connected to Respiratory Chain Assembly

Oxidative phosphorylation (OXPHOS) in mitochondria takes place at the inner membrane, which folds into numerous cristae. The stability of cristae depends, among other things, on the mitochondrial intermembrane space bridging complex. Its components include inner mitochondrial membrane protein mitofilin and outer membrane protein Sam50. We identified a conserved, uncharacterized protein, C1orf163 [SEL1 repeat containing 1 protein (SELRC1)], as one of the proteins significantly reduced after the knockdown of Sam50 and mitofilin. We show that C1orf163 is a mitochondrial soluble intermembrane space protein. Sam50 depletion affects moderately the import and assembly of C1orf163 into two protein complexes of approximately 60 kDa and 150 kDa. We observe that the knockdown of C1orf163 leads to reduction of levels of proteins belonging to the OXPHOS complexes. The activity of complexes I and IV is reduced in C1orf163-depleted cells, and we observe the strongest defects in the assembly of complex IV. Therefore, we propose C1orf163 to be a novel factor important for the assembly of respiratory chain complexes in human mitochondria and suggest to name it RESA1 (for RESpiratory chain Assembly 1).     

Adenine nucleotide translocase activity and sensitivity to inhibitors in hepatomas. Comparison of the ADP/ATP carrier in mitochondria and in a purified reconstituted liposome system

Adenine nucleotide uptake was found to be lower in mitochondria from hepatoma 7777, 7800, and 9618A than in the host livers. Moreover, in the fast-growing hepatoma 7777 the sensitivity of the adenine nucleotide translocase to inhibition by carboxyatractylate and bongkrekic acid was considerably decreased. Purification of the ADP/ATP carrier from hepatoma 7777 mitochondria and its reconstitution into an artificial liposome system reversed the abnormal kinetics in that the adenine nucleotide uptake and response to inhibitors were identical in proteoliposome preparations from host liver and tumor mitochondria. Analysis of the lipids of the hepatoma inner mitochondrial membrane indicated considerable differences from normal in the levels of phospholipids and cholesterol. Most striking was the increase in cholesterol and sphingomyelin of the hepatoma 7777 inner membrane. An artificial liposome system containing cholesterol in addition to the standard phospholipids could produce alterations in kinetics of the purified ADP/ATP carrier from heart mitochondria similar to those seen in the hepatoma 7777. In general, these results support the suggestion that alterations in the lipid environment of the inner mitochondrial membrane rather than intrinsic changes in the carrier protein itself produce the aberrant observations of adenine nucleotide translocase activity in hepatoma mitochondria.     

An internal segment (residues 58-119) of the hepatitis B virus X protein is sufficient to activate MAP kinase pathways in mouse liver

StAR, a protein synthesized in the cytoplasm and subsequently imported into mitochondria, regulates the rate-determining step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The active form of StAR is the 37 kDa pre-protein, which has a short half-life. To determine whether proteasomes participate in the turnover of StAR, we incubated primary cultures of preovulatory rat granulosa cells and immortalized human granulosa cells in the presence of MG132, a specific inhibitor to proteasome catalysis. This treatment caused accumulation of StAR in unstimulated cells. Moreover, incubation of the cells with MG132 in the presence of forskolin (FK), luteinizing hormone/chorionic gonadotropin or follicular stimulating hormone augmented the accumulation of both the 37 kDa cytoplasmic protein and the 30 kDa mature mitochondrial protein, compared to cells incubated with FK or the gonadotropic hormones alone. Concomitantly, progesterone production was enhanced. In contrast no elevation in the 37 kDa StAR intracellular levels or progesterone production was observed following incubation of the cells with the cysteine protease inhibitor E-64. The increase of the 37 kDa StAR protein was evident after 15 min and 30 min of incubation with MG132 (143% and 187% of control values, respectively) with no significant elevation of the 30 kDa protein. Accumulation of the intermediate mitochondrial 32 kDa protein was evident after 1-2 h and the accumulation of the 30 kDa protein was evident only after 4 h of incubation with MG132. In contrast, no elevation in adrenodoxin, a component of the cytochrome P450scc enzyme system, was found. These data suggest that StAR protein is either directly or indirectly degraded by the proteasome which may explain, in part, its short half-life. Moreover, it seems that the cytosolic 37 kDa protein, which is responsible for the steroidogenic activity of StAR, is the primary proteasomal substrate and that the inhibition of its degradation by MG132 causes the up-regulation of progesterone production.     

Sterol carrier protein2. Identification of adrenal sterol carrier protein2 and site of action for mitochondrial cholesterol utilization

Addition of homogeneous rat liver sterol carrier protein2 (SCP2) or an adrenal cytosolic fraction enhanced pregnenolone production by adrenal mitochondria. Pretreatment of SCP2 or adrenal cytosol with anti-SCP2 IgG abolished the stimulatory effect of both preparations on mitochondrial pregnenolone output. Incubation of mitochondria with aminoglutethimide, which blocks interaction of cholesterol with inner membrane cytochrome P-450scc, resulted in decreased pregnenolone production and a decreased level of mitoplast cholesterol. Addition of SCP2 to the incubation media caused an almost 2-fold increase in cholesterol associated with the mitoplast, but did not enhance mitochondrial pregnenolone production. Studies with reconstituted cytochrome P-450scc in phospholipid vesicles also suggested that SCP2 did not affect interaction of cholesterol with the hemoprotein. Treatment of rats with cycloheximide alone or with adrenocorticotropic hormone resulted in a dramatic increase in mitochondrial cholesterol. However, these mitochondria did not exhibit increased levels of pregnenolone output under control incubation conditions. When SCP2 was included in the mitochondrial incubation media, pregnenolone production was significantly increased over that observed with adrenal mitochondria from untreated or adrenocorticotropic hormone-treated rats. The results imply that SCP2 enhances mitochondrial pregnenolone production by improving transfer of mitochondrial cholesterol to cytochrome P-450scc on the inner membrane, but does not directly influence the interaction of substrate with the hemoprotein.     

Evolutionary Origin of the Mitochondrial Cholesterol Transport Machinery Reveals a Universal Mechanism of Steroid Hormone Biosynthesis in Animals

Steroidogenesis begins with the transport of cholesterol from intracellular stores into mitochondria via a series of protein-protein interactions involving cytosolic and mitochondrial proteins located at both the outer and inner mitochondrial membranes. In adrenal glands and gonads, this process is accelerated by hormones, leading to the production of high levels of steroids that control tissue development and function. A hormone-induced multiprotein complex, the transduceosome, was recently identified, and is composed of cytosolic and outer mitochondrial membrane proteins that control the rate of cholesterol entry into the outer mitochondrial membrane. More recent studies unveiled the steroidogenic metabolon, a bioactive, multimeric protein complex that spans the outer-inner mitochondrial membranes and is responsible for hormone-induced import, segregation, targeting, and metabolism of cholesterol by cytochrome P450 family 11 subfamily A polypeptide 1 (CYP11A1) in the inner mitochondrial membrane. The availability of genome information allowed us to systematically explore the evolutionary origin of the proteins involved in the mitochondrial cholesterol transport machinery (transduceosome, steroidogenic metabolon, and signaling proteins), trace the original archetype, and predict their biological functions by molecular phylogenetic and functional divergence analyses, protein homology modeling and molecular docking. Although most members of these complexes have a history of gene duplication and functional divergence during evolution, phylogenomic analysis revealed that all vertebrates have the same functional complex members, suggesting a common mechanism in the first step of steroidogenesis. An archetype of the complex was found in invertebrates. The data presented herein suggest that the cholesterol transport machinery is responsible for steroidogenesis among all vertebrates and is evolutionarily conserved throughout the entire animal kingdom.     

Immunofluorescent localization of the StAR protein in mitochondria of mouse Leydig cells in vitro

The Steroidogenic Acute Regulatory (StAR) protein is assumed to enhance the rate-limiting step of the steroid biosynthesis. Now, it is the most likely candidate, responsible for acutely regulating transfer of cholesterol from the outer to the inner mitochondrial membrane. In this study, the immunoreactive StAR protein was observed in the mitochondria of mouse cultured Leydig cells stimulated by hCG andtesticular macrophage-conditioned medium. Immunocytochemistry was performed using a polyclonal rabbit antibody against the StAR protein. For selective staining of mitochondria in Leydig cells, the Mito Tracker dye was used. Computerized, superimposed images from double-fluorescence staining showed a remarkable degree of similarity in the distribution of the StAR protein and mitochondria, indicating mitochondrial localization of StAR.     

Peripheral-type benzodiazepine receptor (PBR) aggregation and absence of steroidogenic acute regulatory protein (StAR)/PBR association in the mitochondrial membrane as determined by bioluminescence resonance energy transfer (BRET)

The steroidogenic acute regulatory protein (StAR) is responsible for acute control of cholesterol transport across the mitochondrial membrane, however the mechanism of StAR-associated cholesterol transport is unknown and may involve the peripheral-type benzodiazepine receptor (PBR)/endozepine system. Several molecules of PBR may associate to form a channel through which cholesterol passes to the inner mitochondrial membrane, and endozepine is the natural ligand for PBR. Bioluminescence resonance energy transfer (BRET) was used to test StAR/PBR/endozepine interactions, PBR aggregation, and the effect of second messengers on interactions. There was no evidence of StAR/PBR, StAR/endozepine, or PBR/endozepine interactions. The StAR and PBR fusion proteins were trafficking to the mitochondria as expected, but the endozepine fusion protein was not localized to the mitochondria indicating that it was not biologically active. Data were obtained indicating that PBR forms aggregates in the mitochondrial membrane. Energy transfer between PBR fusion proteins was dose and time dependent, but there was no effect induced by PK11195 ligand binding or pharmacologic activation of PKA or PKC second messenger pathways. It appears that PBR aggregates in the mitochondrial membrane, however there was no evidence that PBR aggregation is regulated in the acute control of steroidogenesis, or that PBR and StAR interact.     

Intramitochondrial fatty acylation of a cytoplasmic imported protein in animal cells

Mitochondrial digitonin particles from mouse liver (and also from other tissues) incorporate [3H]myristic acid into a 52-kilodalton (kDa) protein in an energy-dependent manner. The 52-kDa N-myristylated protein is located inside the mitochondrial inner membrane since it is protected against proteolytic degradation in intact mitoplasts. Disruption of mitochondrial inner membrane by sonication results in severalfold higher labeling of the 52-kDa protein, further confirming that the enzyme system for protein fatty acylation as well as the 52-kDa target protein are compartmentalized inside the mitochondrial inner membrane matrix. The results of in vitro labeling of submitochondrial fractions suggest that both the 52-kDa target protein and the enzyme system for fatty acylation are in the matrix fraction, although the N-myristylated protein is found loosely associated with the inner membrane. Finally, immunoprecipitation of cytoplasmic free polysome translation products and in vitro transport of proteins into isolated mitochondria show that the 52-kDa protein is of cytoplasmic translation origin. These results demonstrate that the intramitochondrial N-myristylation of the 52-kDa protein is not translationally linked.     

A yeast mitochondrial ATPase inhibitor interacts with three proteins that are easy to dissociate from the mitochondrial inner membrane

A mitochondrial ATPase inhibitor is a 7.4 kDa protein that regulates the catalytic activity of ATP synthase (F(1)F(o)-ATPase). In the present study, we examined the binding sites of the inhibitor on the mitochondrial membrane using chemical cross-linkers, disuccinimidyl suberate (DSS) and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Most of the inhibitors were recovered from the inner membrane fraction of mitochondria, indicating that the inhibitor binds to the membrane. Seven different cross-linked products that reacted with the antibody against the inhibitor were detected. The apparent molecular masses of the products were 61, 58, 47, 41, 28, 27, and 26 kDa. The 61 and 58 kDa products were attributed to the inhibitor+alpha and inhibitor+beta adducts on immunoblotting. The proteins cross-linked to the inhibitor in the 28, 27, and 26 kDa products were distinguished from subunit 4 (23 kDa), oligomycin sensitivity conferring protein (21 kDa), and subunit d (20 kDa) of F(1)F(o)-ATPase by analysis of the cross-linked products of mutant mitochondria in which the three proteins were replaced by hemagglutinin-tagged versions. The 28, 27, and 26 kDa products could be gradually dissociated from the mitochondrial membrane by increasing the salt concentration. These results shows that the endogenous inhibitor binds not only to the catalytic part of the enzyme, but also to the 19-21 kDa proteins that loosely associate with the mitochondrial inner membrane.     

Both the p33 and p55 Subunits of the Helicobacter pylori VacA Toxin Are Targeted to Mammalian Mitochondria

Helicobacter pylori infection causes peptic ulcers and gastric cancer. A major toxin secreted by H. pylori is the bipartite vacuolating cytotoxin A, VacA. The toxin is believed to enter host cells as two subunits: the p55 subunit (55 kDa) and the p33 subunit (33 kDa). At the biochemical level, it has been shown that VacA forms through the assembly of large multimeric pores composed of both the p33 subunit and the p55 subunit in biological membranes. One of the major target organelles of VacA is the mitochondria. Since only the p33 subunit has been reported to be translocated into mitochondria and the p55 subunit is not imported, it has been contentious as to whether VacA assembles into pores in a mitochondrial membrane. Here we show the p55 protein is imported into the mitochondria along with the p33 protein subunit. The p33 subunit integrally associates with the mitochondrial inner membrane, and both the p33 subunit and the p55 subunit are exposed to the mitochondrial intermembrane space. Their colocalization suggests that they could reassemble and form a pore in the inner mitochondrial membrane.