IRREGULAR TRICHOME BRANCH 2 (ITB2) encodes a putative aminophospholipid translocase that regulates trichome branch elongation in Arabidopsis

The P4 ATPase family in Arabidopsis consists of 12 members that encode putative aminophospholipid translocases (ALA1–12). Until recently, no mutations in these genes have been shown to cause a visible phenotype, although reduced expression of ALA1 in transgenic plants expressing an antisense construct has been shown to result in reduced plant size when plants were grown under cold conditions. During a genetic screen for mutations that affect trichome shape, we isolated several alleles of the irregular trichome branch 2 ( itb2 ) mutation. Subsequent positional cloning of this locus showed that ITB2 encoded ALA3 . Phenotypic and genetic analyses of multiple itb2 alleles, including the T-DNA insertion alleles, showed that the loss of ITB2 / ALA3 function leads to aberrant trichome expansion, reduced primary root growth and longer root hairs. We also found that itb2 / ala3 mutant pollen does not grow as well as wild-type pollen, leading to severe segregation distortion. Our results suggest that aminophospholipid translocases play an important role in the polar growth of plant cells, which is consistent with the proposed role of ALA3 in membrane trafficking. Furthermore, itb2 / ala3 mutants provide a convenient visible phenotype for further genetic analysis of the ALA family in Arabidopsis.     

The mitochondrial TOM complex modulates bax-induced apoptosis in Drosophila

Bax is a pro-apoptotic member of the Bcl-2 family proteins involved in the release of apoptogenic factors from mitochondria to the cytosol. Recently, it has been shown both in mammals and yeast that Bax insertion in the mitochondrial outer membrane involves at least two distinct mechanisms, one of which uses the TOM complex. Here, we show that in Drosophila, heterozygous loss of function mutations of Tom22 or Tom70, two receptors of the TOM complex, attenuates bax-induced phenotypes in vivo. These results argue that the TOM complex may be used as a mitochondrial Bax receptor in Drosophila.     

Switch from inhibition to activation of the mitochondrial permeability transition during hematoporphyrin-mediated photooxidative stress.: Unmasking pore-regulating external thiols

We have studied the mitochondrial permeability transition pore (PTP) under oxidizing conditions with mitochondria-bound hematoporphyrin, which generates reactive oxygen species (mainly singlet oxygen, ¹O₂) upon UV/visible light-irradiation and promotes the photooxidative modification of vicinal targets. We have characterized the PTP-modulating properties of two major critical sites endowed with different degrees of photosensitivity: (i) the most photovulnerable site comprises critical histidines, whose photomodification by vicinal hematoporphyrin causes a drop in reactivity of matrix-exposed (internal), PTP-regulating cysteines thus stabilizing the pore in a closed conformation; (ii) the most photoresistant site coincides with the binding domains of (external) cysteines sensitive to membrane-impermeant reagents, which are easily unmasked when oxidation of internal cysteines is prevented. Photooxidation of external cysteines promoted by vicinal hematoporphyrin reactivates the PTP after the block caused by histidine photodegradation. Thus, hematoporphyrin-mediated photooxidative stress can either inhibit or activate the mitochondrial permeability transition depending on the site of hematoporphyrin localization and on the nature of the substrate; and selective photomodification of different hematoporphyrin-containing pore domains can be achieved by fine regulation of the sensitizer/light doses. These findings shed new light on PTP modulation by oxidative stress.     

The flavonoid quercetin induces changes in mitochondrial permeability by inhibiting adenine nucleotide translocase

This study shows the effects of the flavonoid quercetin on diverse mitochondrial functions, among them membrane permeability. Our findings indicate that the addition of 50 μM quercetin did not produce reactive oxygen derived species; however, it inhibited the oxidative stress induced after the addition of Fe²/H₂O₂ by about 38%. At this concentration, quercetin also promoted a fast calcium release, inhibited oxidative phosphorylation, stimulated oxygen consumption, and decreased membrane potential. In addition 50 μM quercetin inhibited the adenine nucleotide translocase (ANT) by 46%. These effects induced the opening of the permeability transition pore and release of cytochrome c, by its interaction with a component of the non-specific pore complex, fixed to the carrier in the conformation c, as carboxyatractyloside does. Quercetin-induced permeability transition pore opening was inhibited by 0.5 μM cyclosporin A, but, interestingly, the release of cytochrome c was not inhibited by the immunosuppressor, as quercetin was found to disrupt the outer membrane.     

GD3-7-aldehyde is an apoptosis inducer and interacts with adenine nucleotide translocase

We prepared GD3-7-aldehyde (GD3-7) and determined its apoptotic potential. GD3-7 proved to be more efficient to induce pro-apoptotic mitochondrial alterations than GD3 when tested on mouse liver mitochondria. GD3-7-induced mitochondrial swelling and depolarization was blocked by cyclosporin A (CsA) supporting a critical role of the permeability transition pore complex (PTPC) during GD3-7-mediated apoptosis. In contrast to GD3, GD3-7 was able to induce channel formation in proteoliposomes containing adenine nucleotide translocase (ANT). This suggests that ANT is the molecular target of GD3-7. Using a specific antiserum, GD3-7 was detected in the lipid extract of the myeloid tumor cell line HL-60 after apoptosis induction, but not in living cells. Therefore, GD3-7 might be a novel mediator of PTPC-dependent apoptosis in cancer cells.     

Two Modular Forms of the Mitochondrial Sorting and Assembly Machinery Are Involved in Biogenesis of α-Helical Outer Membrane Proteins

The mitochondrial outer membrane contains two translocase machineries for precursor proteins—the translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The TOM complex functions as the main mitochondrial entry gate for nuclear-encoded proteins, whereas the SAM complex was identified according to its function in the biogenesis of β-barrel proteins of the outer membrane. The SAM complex is required for the assembly of precursors of the TOM complex, including not only the β-barrel protein Tom40 but also a subset of α-helical subunits. While the interaction of β-barrel proteins with the SAM complex has been studied in detail, little is known about the interaction between the SAM complex and α-helical precursor proteins. We report that the SAM is not static but that the SAM core complex can associate with different partner proteins to form two large SAM complexes with different functions in the biogenesis of α-helical Tom proteins. We found that a subcomplex of TOM, Tom5-Tom40, associates with the SAM core complex to form a new large SAM complex. This SAM-Tom5/Tom40 complex binds the α-helical precursor of Tom6 after the precursor has been inserted into the outer membrane in an Mim1 (mitochondrial import protein 1)-dependent manner. The second large SAM complex, SAM-Mdm10 (mitochondrial distribution and morphology protein), binds the α-helical precursor of Tom22 and promotes its membrane integration. We suggest that the modular composition of the SAM complex provides a flexible platform to integrate the sorting pathways of different precursor proteins and to promote their assembly into oligomeric complexes.     

Adenine nucleotide translocase mediates the K(ATP)-channel-openers-induced proton and potassium flux to the mitochondrial matrix

K_ATP channel openers have been shown to protect ischemic-reperfused myocardium by mimicking ischemic preconditioning, although their mechanisms of action have not been fully clarified. In this study we investigated the influence of the adenine nucleotide translocase (ANT) inhibitors–carboxyatractyloside (CAT) and bongkrekic acid (BA)–on the diazoxide- and pinacidil-induced uncoupling of isolated rat heart mitochondria respiring on pyruvate and malate (6 + 6 mM). We found that both CAT (1.3 M) and BA (20 M) markedly reduced the uncoupling of mitochondrial oxidative phosphorylation induced by the K_ATP channel openers. Thus, the uncoupling effect of diazoxide and pinacidil is evident only when ANT is not fixed by inhibitors in neither the C- nor the M-conformation. Moreover, the uncoupling effect of diazoxide and pinacidil was diminished in the presence of ADP or ATP, indicating a competition of K_ATP channel openers with adenine nucleotides. CAT also abolished K⁺-dependent mitochondrial respiratory changes. Thus ANT could also be involved in the regulation of K_ATP-channel-openers-induced K⁺ flux through the inner mitochondrial membrane.     

Carnitine-acylcarnitine translocase deficiency, clinical, biochemical and genetic aspects

The carnitine–acylcarnitine translocase (CACT) is one of the components of the carnitine cycle. The carnitine cycle is necessary to shuttle long-chain fatty acids from the cytosol into the intramitochondrial space where mitochondrial β-oxidation of fatty acids takes place. The oxidation of fatty acids yields acetyl-coenzyme A (CoA) units, which may either be degraded to CO₂ and H₂O in the citric acid cycle to produce ATP or converted into ketone bodies which occurs in liver and kidneys.

Metabolic consequences of a defective CACT are hypoketotic hypoglycaemia under fasting conditions, hyperammonemia, elevated creatine kinase and transaminases, dicarboxylic aciduria, very low free carnitine and an abnormal acylcarnitine profile with marked elevation of the long-chain acylcarnitines.

Clinical signs and symptoms in CACT deficient patients, are a combination of energy depletion and endogenous toxicity. The predominantly affected organs are brain, heart and skeletal muscle, and liver, leading to neurological abnormalities, cardiomyopathy and arrythmias, skeletal muscle damage and liver dysfunction. Most patients become symptomatic in the neonatal period with a rapidly progressive deterioration and a high mortality rate. However, presentations at a later age with a milder phenotype have also been reported.

The therapeutic approach is the same as in other long-chain fatty acid disorders and includes intravenous glucose (± insulin) administration to maximally inhibit lipolysis and subsequent fatty acid oxidation during the acute deterioration, along with other measures such as ammonia detoxification, depending on the clinical features. Long-term strategy consists of avoidance of fasting with frequent meals and a special diet with restriction of long-chain fatty acids. Due to the extremely low free carnitine concentrations, carnitine supplementation is often needed.

Acylcarnitine profiling in plasma is the assay of choice for the diagnosis at a metabolite level. However, since the acylcarnitine profile observed in CACT-deficient patients is identical to that in CPT2-deficient patients, definitive identification of CACT-deficiency in a certain patient requires determination of the activity of CACT. Subsequently, mutational analysis of the CACT gene can be performed. So far, 9 different mutations have been identified in the CACT gene.