Publications by authors named "Francesco M Lasorsa"

15 Publications

  • Page 1 of 1

KRAS-regulated glutamine metabolism requires UCP2-mediated aspartate transport to support pancreatic cancer growth.

Nat Metab 2020 12 23;2(12):1373-1381. Epub 2020 Nov 23.

Department of Bioscience, Biotechnology and Biopharmaceutics, University of Bari, Bari, Italy.

The oncogenic KRAS mutation has a critical role in the initiation of human pancreatic ductal adenocarcinoma (PDAC) since it rewires glutamine metabolism to increase reduced nicotinamide adenine dinucleotide phosphate (NADPH) production, balancing cellular redox homeostasis with macromolecular synthesis. Mitochondrial glutamine-derived aspartate must be transported into the cytosol to generate metabolic precursors for NADPH production. The mitochondrial transporter responsible for this aspartate efflux has remained elusive. Here, we show that mitochondrial uncoupling protein 2 (UCP2) catalyses this transport and promotes tumour growth. UCP2-silenced KRAS cell lines display decreased glutaminolysis, lower NADPH/NADP and glutathione/glutathione disulfide ratios and higher reactive oxygen species levels compared to wild-type counterparts. UCP2 silencing reduces glutaminolysis also in KRAS PDAC cells but does not affect their redox homeostasis or proliferation rates. In vitro and in vivo, UCP2 silencing strongly suppresses KRAS PDAC cell growth. Collectively, these results demonstrate that UCP2 plays a vital role in PDAC, since its aspartate transport activity connects the mitochondrial and cytosolic reactions necessary for KRAS rewired glutamine metabolism, and thus it should be considered a key metabolic target for the treatment of this refractory tumour.
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http://dx.doi.org/10.1038/s42255-020-00315-1DOI Listing
December 2020

Statins Reduce Intratumor Cholesterol Affecting Adrenocortical Cancer Growth.

Mol Cancer Ther 2020 09 16;19(9):1909-1921. Epub 2020 Jun 16.

Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende, Cosenza, Italy.

Mitotane causes hypercholesterolemia in patients with adrenocortical carcinoma (ACC). We suppose that cholesterol increases within the tumor and can be used to activate proliferative pathways. In this study, we used statins to decrease intratumor cholesterol and investigated the effects on ACC growth related to estrogen receptor α (ERα) action at the nuclear and mitochondrial levels. We first used microarray to investigate mitotane effect on genes involved in cholesterol homeostasis and evaluated their relationship with patients' survival in ACC TCGA. We then blocked cholesterol synthesis with simvastatin and determined the effects on H295R cell proliferation, estradiol production, and ERα activity and in xenograft tumors. We found that mitotane increases intratumor cholesterol content and expression of genes involved in cholesterol homeostasis, among them , whose expression affects patients' survival. Treatment of H295R cells with simvastatin to block cholesterol synthesis decreased cellular cholesterol content, and this affected cell viability. Simvastatin reduced estradiol production and decreased nuclear and mitochondrial ERα function. A mitochondrial target of ERα, the respiratory complex IV (COXIV), was reduced after simvastatin treatment, which profoundly affected mitochondrial respiration activating apoptosis. Additionally, simvastatin reduced tumor volume and weight of grafted H295R cells, intratumor cholesterol content, Ki-67 and ERα, COXIV expression and activity and increase terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. Collectively, these data demonstrate that a reduction in intratumor cholesterol content prevents estradiol production and inhibits mitochondrial respiratory chain-inducing apoptosis in ACC cells. Inhibition of mitochondrial respiration by simvastatin represents a novel strategy to counteract ACC growth.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-1063DOI Listing
September 2020

A Mutation in the Mitochondrial Aspartate/Glutamate Carrier Leads to a More Oxidizing Intramitochondrial Environment and an Inflammatory Myopathy in Dutch Shepherd Dogs.

J Neuromuscul Dis 2019 ;6(4):485-501

Department of Pathology, School of Medicine, University of California San Diego, La Jolla, CA, USA.

Background: Inflammatory myopathies are characterized by infiltration of inflammatory cells into muscle. Typically, immune-mediated disorders such as polymyositis, dermatomyositis and inclusion body myositis are diagnosed.

Objective: A small family of dogs with early onset muscle weakness and inflammatory muscle biopsies were investigated for an underlying genetic cause.

Methods: Following the histopathological diagnosis of inflammatory myopathy, mutational analysis including whole genome sequencing, functional transport studies of the mutated and wild-type proteins, and metabolomic analysis were performed.

Results: Whole genome resequencing identified a pathological variant in the SLC25A12 gene, resulting in a leucine to proline substitution at amino acid 349 in the mitochondrial aspartate-glutamate transporter known as the neuron and muscle specific aspartate glutamate carrier 1 (AGC1). Functionally reconstituting recombinant wild-type and mutant AGC1 into liposomes demonstrated a dramatic decrease in AGC1 transport activity and inability to transfer reducing equivalents from the cytosol into mitochondria. Targeted, broad-spectrum metabolomic analysis from affected and control muscles demonstrated a proinflammatory milieu and strong support for oxidative stress.

Conclusions: This study provides the first description of a metabolic mechanism in which ablated mitochondrial glutamate transport markedly reduced the import of reducing equivalents into mitochondria and produced a highly oxidizing and proinflammatory muscle environment and an inflammatory myopathy.
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http://dx.doi.org/10.3233/JND-190421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918910PMC
April 2020

SLC25A10 biallelic mutations in intractable epileptic encephalopathy with complex I deficiency.

Hum Mol Genet 2018 02;27(3):499-504

Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari, 70125 Bari, Italy.

Mitochondrial diseases are a plethora of inherited neuromuscular disorders sharing defects in mitochondrial respiration, but largely different from one another for genetic basis and pathogenic mechanism. Whole exome sequencing was performed in a familiar trio (trio-WES) with a child affected by severe epileptic encephalopathy associated with respiratory complex I deficiency and mitochondrial DNA depletion in skeletal muscle. By trio-WES we identified biallelic mutations in SLC25A10, a nuclear gene encoding a member of the mitochondrial carrier family. Genetic and functional analyses conducted on patient fibroblasts showed that SLC25A10 mutations are associated with reduction in RNA quantity and aberrant RNA splicing, and to absence of SLC25A10 protein and its transporting function. The yeast SLC25A10 ortholog knockout strain showed defects in mitochondrial respiration and mitochondrial DNA content, similarly to what observed in the patient skeletal muscle, and growth susceptibility to oxidative stress. Albeit patient fibroblasts were depleted in the main antioxidant molecules NADPH and glutathione, transport assays demonstrated that SLC25A10 is unable to transport glutathione. Here, we report the first recessive mutations of SLC25A10 associated to an inherited severe mitochondrial neurodegenerative disorder. We propose that SLC25A10 loss-of-function causes pathological disarrangements in respiratory-demanding conditions and oxidative stress vulnerability.
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http://dx.doi.org/10.1093/hmg/ddx419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886107PMC
February 2018

Inhibition of the Mitochondrial Glutamate Carrier SLC25A22 in Astrocytes Leads to Intracellular Glutamate Accumulation.

Front Cell Neurosci 2017 31;11:149. Epub 2017 May 31.

INMED, INSERM, Aix-Marseille UniversitéMarseille, France.

The solute carrier family 25 (SLC25) drives the import of a large diversity of metabolites into mitochondria, a key cellular structure involved in many metabolic functions. Mutations of the mitochondrial glutamate carrier (also named ) have been identified in early epileptic encephalopathy (EEE) and migrating partial seizures in infancy (MPSI) but the pathophysiological mechanism of GC1 deficiency is still unknown, hampered by the absence of an model. This carrier is mainly expressed in astrocytes and is the principal gate for glutamate entry into mitochondria. A sufficient supply of energy is essential for the proper function of the brain and mitochondria have a pivotal role in maintaining energy homeostasis. In this work, we wanted to study the consequences of GC1 absence in an model in order to understand if glutamate catabolism and/or mitochondrial function could be affected. First, short hairpin RNA (shRNA) designed to specifically silence were validated in rat C6 glioma cells. Silencing in C6 resulted in a reduction of the mRNA combined with a decrease of the mitochondrial glutamate carrier activity. Then, primary astrocyte cultures were prepared and transfected with shRNA-GC1 or mismatch-RNA (mmRNA) constructs using the Neon® Transfection System in order to target a high number of primary astrocytes, more than 64%. Silencing in primary astrocytes resulted in a reduced nicotinamide adenine dinucleotide (Phosphate) (NAD(P)H) formation upon glutamate stimulation. We also observed that the mitochondrial respiratory chain (MRC) was functional after glucose stimulation but not activated by glutamate, resulting in a lower level of cellular adenosine triphosphate (ATP) in silenced astrocytes compared to control cells. Moreover, GC1 inactivation resulted in an intracellular glutamate accumulation. Our results show that mitochondrial glutamate transport via GC1 is important in sustaining glutamate homeostasis in astrocytes. The mitochondrial respiratory chain is functional in absence of GC1Lack of glutamate oxidation results in a lower global ATP levelLack of mitochondrial glutamate transport results in intracellular glutamate accumulation.
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http://dx.doi.org/10.3389/fncel.2017.00149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449474PMC
May 2017

AGC1 Deficiency Causes Infantile Epilepsy, Abnormal Myelination, and Reduced N-Acetylaspartate.

JIMD Rep 2014 11;14:77-85. Epub 2014 Feb 11.

Division of Human Genetics, Department of Pediatrics, The Children's Hospital of Philadelphia and The Perelman School of Medicine, Philadelphia, PA, 19104, USA,

Background: Whole exome sequencing (WES) offers a powerful diagnostic tool to rapidly and efficiently sequence all coding genes in individuals presenting for consideration of phenotypically and genetically heterogeneous disorders such as suspected mitochondrial disease. Here, we report results of WES and functional validation in a consanguineous Indian kindred where two siblings presented with profound developmental delay, congenital hypotonia, refractory epilepsy, abnormal myelination, fluctuating basal ganglia changes, cerebral atrophy, and reduced N-acetylaspartate (NAA).

Methods: Whole blood DNA from one affected and one unaffected sibling was captured by Agilent SureSelect Human All Exon kit and sequenced on the Illumina HiSeq2000. Mutations were validated by Sanger sequencing in all family members. Protein from wild-type and mutant fibroblasts was isolated to assess mutation effects on protein expression and enzyme activity.

Results: A novel SLC25A12 homozygous missense mutation, c.1058G>A; p.Arg353Gln, segregated with disease in this kindred. SLC25A12 encodes the neuronal aspartate-glutamate carrier 1 (AGC1) protein, an essential component of the neuronal malate/aspartate shuttle that transfers NADH and H(+) reducing equivalents from the cytosol to mitochondria. AGC1 activity enables neuronal export of aspartate, the glial substrate necessary for proper neuronal myelination. Recombinant mutant p.Arg353Gln AGC1 activity was reduced to 15% of wild type. One prior reported SLC25A12 mutation caused complete loss of AGC1 activity in a child with epilepsy, hypotonia, hypomyelination, and reduced brain NAA.

Conclusions: These data strongly suggest that SLC25A12 disease impairs neuronal AGC1 activity. SLC25A12 sequencing should be considered in children with infantile epilepsy, congenital hypotonia, global delay, abnormal myelination, and reduced brain NAA.
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http://dx.doi.org/10.1007/8904_2013_287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4213337PMC
October 2014

UCP2 transports C4 metabolites out of mitochondria, regulating glucose and glutamine oxidation.

Proc Natl Acad Sci U S A 2014 Jan 6;111(3):960-5. Epub 2014 Jan 6.

Department of Biosciences, Biotechnologies, and Biopharmaceutics and Center of Excellence in Comparative Genomics, University of Bari, 70125 Bari, Italy.

Uncoupling protein 2 (UCP2) is involved in various physiological and pathological processes such as insulin secretion, stem cell differentiation, cancer, and aging. However, its biochemical and physiological function is still under debate. Here we show that UCP2 is a metabolite transporter that regulates substrate oxidation in mitochondria. To shed light on its biochemical role, we first studied the effects of its silencing on the mitochondrial oxidation of glucose and glutamine. Compared with wild-type, UCP2-silenced human hepatocellular carcinoma (HepG2) cells, grown in the presence of glucose, showed a higher inner mitochondrial membrane potential and ATP:ADP ratio associated with a lower lactate release. Opposite results were obtained in the presence of glutamine instead of glucose. UCP2 reconstituted in lipid vesicles catalyzed the exchange of malate, oxaloacetate, and aspartate for phosphate plus a proton from opposite sides of the membrane. The higher levels of citric acid cycle intermediates found in the mitochondria of siUCP2-HepG2 cells compared with those found in wild-type cells in addition to the transport data indicate that, by exporting C4 compounds out of mitochondria, UCP2 limits the oxidation of acetyl-CoA-producing substrates such as glucose and enhances glutaminolysis, preventing the mitochondrial accumulation of C4 metabolites derived from glutamine. Our work reveals a unique regulatory mechanism in cell bioenergetics and provokes a substantial reconsideration of the physiological and pathological functions ascribed to UCP2 based on its purported uncoupling properties.
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http://dx.doi.org/10.1073/pnas.1317400111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903233PMC
January 2014

Perturbed mitochondrial Ca2+ signals as causes or consequences of mitophagy induction.

Autophagy 2013 Nov 21;9(11):1677-86. Epub 2013 May 21.

Department of Morphology, Surgery and Experimental Medicine; Section of General Pathology; Interdisciplinary Center for the Study of Inflammation (ICSI); Laboratory for Technologies of Advanced Therapies (LTTA); University of Ferrara; Ferrara, Italy.

Mitophagy is an essential process that maintains mitochondrial quality and number, thus limiting cellular degeneration. Along with apoptosis, mitophagy participates in cellular fate decisions by eliminating damaged mitochondria. A variety of mitochondrial parameters, such as structure, membrane potential, and reactive oxygen species, are key determinants in triggering the mitophagic machinery. These parameters are also important regulators of the mitochondrial capacity for calcium (Ca (2+)) uptake. Rapid Ca (2+) accumulation in the mitochondrial matrix allows for prompt stimulation of the organelle. This process requires a close morphofunctional coupling between mitochondria and the main intracellular Ca (2+) stores. In mitophagy, the role of Ca (2+) remains obscure. What role does mitochondrial Ca (2+) play in metabolic sensing or in mitochondrial remodeling? Is endoplasmic reticulum (ER)-Ca (2+) crosstalk involved? These are some of the questions that we address in this review.
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http://dx.doi.org/10.4161/auto.24795DOI Listing
November 2013

Rapamycin reduces oxidative stress in frataxin-deficient yeast cells.

Mitochondrion 2012 Jan 18;12(1):156-61. Epub 2011 Jul 18.

Laboratory of Biochemistry and Molecular Biology, Department of Pharmaco-Biology, University of Bari, Via E. Orabona 4, 70125 Bari, Italy.

Friedreich ataxia (FRDA) is a common form of ataxia caused by decreased expression of the mitochondrial protein frataxin. Oxidative damage of mitochondria is thought to play a key role in the pathogenesis of the disease. Therefore, a possible therapeutic strategy should be directed to an antioxidant protection against mitochondrial damage. Indeed, treatment of FRDA patients with the antioxidant idebenone has been shown to improve neurological functions. The yeast frataxin knock-out model of the disease shows mitochondrial iron accumulation, iron-sulfur cluster defects and high sensitivity to oxidative stress. By flow cytometry analysis we studied reactive oxygen species (ROS) production of yeast frataxin mutant cells treated with two antioxidants, N-acetyl-L-cysteine and a mitochondrially-targeted analog of vitamin E, confirming that mitochondria are the main site of ROS production in this model. Furthermore we found a significant reduction of ROS production and a decrease in the mitochondrial mass in mutant cells treated with rapamycin, an inhibitor of TOR kinases, most likely due to autophagy of damaged mitochondria.
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http://dx.doi.org/10.1016/j.mito.2011.07.001DOI Listing
January 2012

AGC1 deficiency associated with global cerebral hypomyelination.

N Engl J Med 2009 Jul;361(5):489-95

Center for Inherited Metabolic Diseases, Karolinska University Hospital, Stockholm, Sweden.

The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), specific to neurons and muscle, supplies aspartate to the cytosol and, as a component of the malate-aspartate shuttle, enables mitochondrial oxidation of cytosolic NADH, thought to be important in providing energy for neurons in the central nervous system. We describe AGC1 deficiency, a novel syndrome characterized by arrested psychomotor development, hypotonia, and seizures in a child with a homozygous missense mutation in the solute carrier family 25, member 12, gene SLC25A12, which encodes the AGC1 protein. Functional analysis of the mutant AGC1 protein showed abolished activity. The child had global hypomyelination in the cerebral hemispheres, suggesting that impaired efflux of aspartate from neuronal mitochondria prevents normal myelin formation.
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http://dx.doi.org/10.1056/NEJMoa0900591DOI Listing
July 2009

Mitochondrial glutamate carrier GC1 as a newly identified player in the control of glucose-stimulated insulin secretion.

J Biol Chem 2009 Sep 7;284(37):25004-14. Epub 2009 Jul 7.

Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, 1211 Geneva 4, Switzerland.

The SLC25 carrier family mediates solute transport across the inner mitochondrial membrane, a process that is still poorly characterized regarding both the mechanisms and proteins implicated. This study investigated mitochondrial glutamate carrier GC1 in insulin-secreting beta-cells. GC1 was cloned from insulin-secreting cells, and sequence analysis revealed hydropathy profile of a six-transmembrane protein, characteristic of mitochondrial solute carriers. GC1 was found to be expressed at the mRNA and protein levels in INS-1E beta-cells and pancreatic rat islets. Immunohistochemistry showed that GC1 was present in mitochondria, and ultrastructural analysis by electron microscopy revealed inner mitochondrial membrane localization of the transporter. Silencing of GC1 in INS-1E beta-cells, mediated by adenoviral delivery of short hairpin RNA, reduced mitochondrial glutamate transport by 48% (p < 0.001). Insulin secretion at basal 2.5 mM glucose and stimulated either by intermediate 7.5 mM glucose or non-nutrient 30 mM KCl was not modified by GC1 silencing. Conversely, insulin secretion stimulated with optimal 15 mM glucose was reduced by 23% (p < 0.005) in GC1 knocked down cells compared with controls. Adjunct of cell-permeant glutamate (5 mM dimethyl glutamate) fully restored the secretory response at 15 mM glucose (p < 0.005). Kinetics of insulin secretion were investigated in perifused isolated rat islets. GC1 silencing in islets inhibited the secretory response induced by 16.7 mM glucose, both during first (-25%, p < 0.05) and second (-33%, p < 0.05) phases. This study demonstrates that insulin-secreting cells depend on GC1 for maximal glucose response, thereby assigning a physiological function to this newly identified mitochondrial glutamate carrier.
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http://dx.doi.org/10.1074/jbc.M109.015495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757205PMC
September 2009

The insulin-like growth factor-I-mTOR signaling pathway induces the mitochondrial pyrimidine nucleotide carrier to promote cell growth.

Mol Biol Cell 2007 Sep 27;18(9):3545-55. Epub 2007 Jun 27.

Cell Biology Laboratory, Department of Biochemistry, BioSciences Institute, National University of Ireland, Cork, Ireland.

The insulin/insulin-like growth factor (IGF) signaling pathway to mTOR is essential for the survival and growth of normal cells and also contributes to the genesis and progression of cancer. This signaling pathway is linked with regulation of mitochondrial function, but how is incompletely understood. Here we show that IGF-I and insulin induce rapid transcription of the mitochondrial pyrimidine nucleotide carrier PNC1, which shares significant identity with the essential yeast mitochondrial carrier Rim2p. PNC1 expression is dependent on PI-3 kinase and mTOR activity and is higher in transformed fibroblasts, cancer cell lines, and primary prostate cancers than in normal tissues. Overexpression of PNC1 enhances cell size, whereas suppression of PNC1 expression causes reduced cell size and retarded cell cycle progression and proliferation. Cells with reduced PNC1 expression have reduced mitochondrial UTP levels, but while mitochondrial membrane potential and cellular ATP are not altered, cellular ROS levels are increased. Overall the data indicate that PNC1 is a target of the IGF-I/mTOR pathway that is essential for mitochondrial activity in regulating cell growth and proliferation.
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http://dx.doi.org/10.1091/mbc.e06-12-1109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1951771PMC
September 2007

Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins.

Biochim Biophys Acta 2006 Sep-Oct;1757(9-10):1249-62. Epub 2006 May 23.

Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Via E. Orabona 4, 70125 Bari, Italy.

The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane alpha-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established.
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http://dx.doi.org/10.1016/j.bbabio.2006.05.023DOI Listing
December 2006

The yeast peroxisomal adenine nucleotide transporter: characterization of two transport modes and involvement in DeltapH formation across peroxisomal membranes.

Biochem J 2004 Aug;381(Pt 3):581-5

Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, 70125 Bari, Italy.

The yeast peroxisomal adenine nucleotide carrier, Ant1p, was shown to catalyse unidirectional transport in addition to exchange of substrates. In both transport modes, proton movement occurs. Nucleotide hetero-exchange is H+-compensated and electroneutral. Furthermore, microscopic fluorescence imaging of a pH-sensitive green fluorescent protein targeted to peroxisomes shows that Ant1p is involved in the formation of a DeltapH across the peroxisomal membrane, acidic inside.
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http://dx.doi.org/10.1042/BJ20040856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1133865PMC
August 2004