scholarly journals Type II NADH Dehydrogenase Inhibitor 1-Hydroxy-2-Dodecyl- 4(1H)Quinolone Leads to Collapse of Mitochondrial Inner- Membrane Potential and ATP Depletion in Toxoplasma gondii

2009 ◽  
Vol 8 (6) ◽  
pp. 877-887 ◽  
Author(s):  
San San Lin ◽  
Uwe Groß ◽  
Wolfgang Bohne
Keyword(s):  
Membrane Potential ◽  
Toxoplasma Gondii ◽  
Mammalian Cells ◽  
Proton Pumping ◽  
Atp Depletion ◽  
Prolonged Treatment ◽  
Type Ii ◽  
Atpase Inhibitor ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane

ABSTRACT The apicomplexan parasite Toxoplasma gondii expresses type II NADH dehydrogenases (NDH2s) instead of canonical complex I at the inner mitochondrial membrane. These non-proton-pumping enzymes are considered to be promising drug targets due to their absence in mammalian cells. We recently showed by inhibition kinetics that T. gondii NDH2-I is a target of the quinolone-like compound 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ), which inhibits T. gondii replication in the nanomolar range. In this study, the cationic fluorescent probes Mitotracker and DiOC6(3) (3,3′-dihexyloxacarbocyanine iodine) were used to monitor the influence of HDQ on the mitochondrial inner membrane potential (ΔΨm) in T. gondii. Real-time imaging revealed that nanomolar HDQ concentrations led to a ΔΨm collapse within minutes, which is followed by severe ATP depletions of 30% after 1 h and 70% after 24 h. ΔΨm depolarization was attenuated when substrates for other dehydrogenases that can donate electrons to ubiquinone were added to digitonin-permeabilized cells or when infected cultures were treated with the Fo-ATPase inhibitor oligomycin. A prolonged treatment with sublethal concentrations of HDQ induced differentiation into bradyzoites. This dormant stage is likely to be less dependent on the ΔΨm, since ΔΨm-positive parasites were found at a significantly lower frequency in alkaline-pH-induced bradyzoites than in tachyzoites. Together, our studies reveal that oxidative phosphorylation is essential for maintaining the ATP level in the fast-growing tachyzoite stage and that HDQ interferes with this pathway by inhibiting the electron transport chain at the level of ubiquinone reduction.

Abstract 332: Mitochondrial Inner-Membrane Potential Instability Promotes Sarcolemmal Electrical Instability after Ischemia-Reperfusion in Monolayers of Cardiac Myocytes

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Soroosh Solhjoo ◽  
Brian O’Rourke
Keyword(s):  
Membrane Potential ◽  
Cardiac Myocytes ◽  
Mitochondrial Function ◽  
Ischemia Reperfusion ◽  
Neonatal Rat ◽  
Partial Recovery ◽  
Mitochondrial Network ◽  
Electrical Excitability ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane

Mitochondrial uncoupling due to oxidative stress can, through opening of sarcolemmal KATP channels, alter cellular electrical excitability, and it has been proposed that mitochondrial function is a major factor in arrhythmogenesis during ischemia-reperfusion. Here, we examine the effects of ischemia-reperfusion on mitochondrial inner membrane potential (ΔΨm) and corresponding changes in electrical excitability and wave propagation in monolayer cultures of neonatal rat ventricular myocytes. Changes in ΔΨm were observed using TMRM and changes in the sarcolemmal voltage were recorded with a 464-element photodiode array using di-4-ANEPPS. Ischemia was induced by covering the center part of the monolayer (D = 22 mm) with a coverslip (D = 15 mm). Cell contractions ceased after approximately 6 min of ischemia; however, electrical activity continued for 11.3 ± 4.2 min (N = 5). Amplitude and conduction velocity of the action potentials in the ischemic region decreased over the same time period. ΔΨm was lost in two phases: a reversible phase of partial depolarization, after 11.2 ± 1.3 min of ischemia, and a nonreversible phase, which happened after 30 ± 6 min of ischemia, during which the whole mitochondrial network of the myocyte became depolarized and the cells underwent contracture (N = 4). Reperfusion after the long ischemia resulted in only partial recovery and the observance of oscillations of ΔΨm in the mitochondrial network or rapid flickering of individual mitochondrial clusters and was associated with heterogeneous electrical recovery, and formation of wavelets and reentry (4/5 monolayers). In contrast, mitochondria fully recovered and reentry was rare (1/5 monolayers) for reperfusion after the short ischemia (10-12 min). 4’-chlorodiazepam, an inhibitor of inner membrane anion channels, stabilized mitochondrial function after the long ischemia, and prevented wavelets (5/5 monolayers) and reentry (4/5 monolayers). In conclusion, incomplete or unstable recovery of mitochondrial function after ischemia correlates with reentrant arrhythmias in monolayers of cardiac myocytes. Our findings suggest that stabilization of mitochondrial network dynamics is an important strategy for preventing ischemia/reperfusion-related arrhythmias.

External alternative NADH:ubiquinone oxidoreductase redirected to the internal face of the mitochondrial inner membrane rescues complex I deficiency in Yarrowia lipolytica

2001 ◽  
Vol 114 (21) ◽  
pp. 3915-3921 ◽  
Author(s):  
Stefan J. Kerscher ◽  
Andrea Eschemann ◽  
Pamela M. Okun ◽  
Ulrich Brandt
Keyword(s):  
Yarrowia Lipolytica ◽  
Complex I ◽  
Functional Expression ◽  
Proton Pumping ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
The Matrix ◽  
Cytosolic Side ◽  
Respiratory Membrane

Alternative NADH:ubiquinone oxidoreductases are single subunit enzymes capable of transferring electrons from NADH to ubiquinone without contributing to the proton gradient across the respiratory membrane. The obligately aerobic yeast Yarrowia lipolytica has only one such enzyme, encoded by the NDH2 gene and located on the external face of the mitochondrial inner membrane. In sharp contrast to ndh2 deletions, deficiencies in nuclear genes for central subunits of proton pumping NADH:ubiquinone oxidoreductases (complex I) are lethal. We have redirected NDH2 to the internal face of the mitochondrial inner membrane by N-terminally attaching the mitochondrial targeting sequence of NUAM, the largest subunit of complex I. Lethality of complex I mutations was rescued by the internal, but not the external version of alternative NADH:ubiquinone oxidoreductase. Internal NDH2 also permitted growth in the presence of complex I inhibitors such as 2-decyl-4-quinazolinyl amine (DQA). Functional expression of NDH2 on both sides of the mitochondrial inner membrane indicates that alternative NADH:ubiquinone oxidoreductase requires no additional components for catalytic activity. Our findings also demonstrate that shuttle mechanisms for the transfer of redox equivalents from the matrix to the cytosolic side of the mitochondrial inner membrane are insufficient in Y. lipolytica.

scholarly journals Acidocalcisomes in Toxoplasma gondii tachyzoites

1996 ◽  
Vol 313 (2) ◽  
pp. 655-659 ◽  
Author(s):  
Silvia N. J. MORENO ◽  
Li ZHONG
Keyword(s):  
Endoplasmic Reticulum ◽  
Toxoplasma Gondii ◽  
Mammalian Cells ◽  
Specific Inhibitor ◽  
Mitochondrial Atpase ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Extracellular Medium ◽  
Acidic Compartment ◽  
Acidic Organelles

Toxoplasma gondii tachyzoites were loaded with the fluorescent indicator fura 2 to investigate the transport mechanisms involved in maintaining their intracellular Ca2+ homoeostasis. The mitochondrial ATPase inhibitor oligomycin and the endoplasmic-reticulum Ca2+-ATPase inhibitor thapsigargin increased the intracellular Ca2+ concentration ([Ca2+]i), thus indicating the requirement for ATP and the involvement of the endoplasmic reticulum in maintaining intracellular Ca2+ homoeostasis. The effect of thapsigargin was more accentuated in the presence of extracellular Ca2+, clearly showing that, as occurs with other eukaryotic cells, depletion of intracellular Ca2+ pools led to an increase in the uptake of Ca2+ from the extracellular medium. In addition to these results, we found evidence that, in contrast with what occurs in mammalian cells, T. gondii tachyzoites possess a significant amount of Ca2+ stored in an acidic compartment, termed the acidocalcisome, as indicated by: (1) the increase in [Ca2+]i induced by bafilomycin A1 (a specific inhibitor of H+-ATPases), nigericin (a K+/H+ exchanger) or the weak base NH4Cl, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry; and (2) the effect of ionomycin, a Ca2+-releasing ionophore that cannot take Ca2+ out of acidic organelles and that was more effective after alkalinization of these compartments by addition of bafilomycin A1, nigericin or NH4Cl. Considering the relative importance of the ionomycin-releasable and the ionomycin+NH4Cl-releasable Ca2+ pools, it is apparent that T. gondii tachyzoites contain a significant amount of Ca2+ stored in acidocalcisomes.

scholarly journals Inducible proteolytic inactivation of OPA1 mediated by the OMA1 protease in mammalian cells

2009 ◽  
Vol 187 (7) ◽  
pp. 959-966 ◽  
Author(s):  
Brian Head ◽  
Lorena Griparic ◽  
Mandana Amiri ◽  
Shilpa Gandre-Babbe ◽  
Alexander M. van der Bliek
Keyword(s):  
Membrane Potential ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Mitochondrial Network ◽  
Membrane Fusion Protein ◽  
Zinc Metalloprotease ◽  
Mitochondrial Inner Membrane ◽  
Proteolytic Cascade ◽  
And Function ◽  
Interfering Rna

The mammalian mitochondrial inner membrane fusion protein OPA1 is controlled by complex patterns of alternative splicing and proteolysis. A subset of OPA1 isoforms is constitutively cleaved by YME1L. Other isoforms are not cleaved by YME1L, but they are cleaved when mitochondria lose membrane potential or adenosine triphosphate. In this study, we show that this inducible cleavage is mediated by a zinc metalloprotease called OMA1. We find that OMA1 small interfering RNA inhibits inducible cleavage, helps retain fusion competence, and slows the onset of apoptosis, showing that OMA1 controls OPA1 cleavage and function. We also find that OMA1 is normally cleaved from 60 to 40 kD by another as of yet unidentified protease. Loss of membrane potential causes 60-kD protein to accumulate, suggesting that OMA1 is attenuated by proteolytic degradation. We conclude that a proteolytic cascade controls OPA1. Inducible cleavage provides a mechanism for quality control because proteolytic inactivation of OPA1 promotes selective removal of defective mitochondrial fragments by preventing their fusion with the mitochondrial network.

scholarly journals The translocator maintenance protein Tam41 is required for mitochondrial cardiolipin biosynthesis

2008 ◽  
Vol 183 (7) ◽  
pp. 1213-1221 ◽  
Author(s):  
Stephan Kutik ◽  
Michael Rissler ◽  
Xue Li Guan ◽  
Bernard Guiard ◽  
Guanghou Shui ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Respiratory Chain ◽  
Matrix Protein ◽  
Mitochondrial Protein ◽  
Pleiotropic Effects ◽  
Mitochondrial Matrix ◽  
Carrier Proteins ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane

The mitochondrial inner membrane contains different translocator systems for the import of presequence-carrying proteins and carrier proteins. The translocator assembly and maintenance protein 41 (Tam41/mitochondrial matrix protein 37) was identified as a new member of the mitochondrial protein translocator systems by its role in maintaining the integrity and activity of the presequence translocase of the inner membrane (TIM23 complex). Here we demonstrate that the assembly of proteins imported by the carrier translocase, TIM22 complex, is even more strongly affected by the lack of Tam41. Moreover, respiratory chain supercomplexes and the inner membrane potential are impaired by lack of Tam41. The phenotype of Tam41-deficient mitochondria thus resembles that of mitochondria lacking cardiolipin. Indeed, we found that Tam41 is required for the biosynthesis of the dimeric phospholipid cardiolipin. The pleiotropic effects of the translocator maintenance protein on preprotein import and respiratory chain can be attributed to its role in biosynthesis of mitochondrial cardiolipin.

scholarly journals A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 determines the direct and strain-specific activation of Stat3

2009 ◽  
Vol 206 (12) ◽  
pp. 2747-2760 ◽  
Author(s):  
Masahiro Yamamoto ◽  
Daron M. Standley ◽  
Seiji Takashima ◽  
Hiroyuki Saiga ◽  
Megumi Okuyama ◽  
...  
Keyword(s):  
Amino Acid ◽  
Toxoplasma Gondii ◽  
Immune Responses ◽  
Mammalian Cells ◽  
Single Amino Acid ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Enhanced Production ◽  
Stat3 Phosphorylation

Infection by Toxoplasma gondii down-regulates the host innate immune responses, such as proinflammatory cytokine production, in a Stat3-dependent manner. A forward genetic approach recently demonstrated that the type II strain fails to suppress immune responses because of a potential defect in a highly polymorphic parasite-derived kinase, ROP16. We generated ROP16-deficient type I parasites by reverse genetics and found a severe defect in parasite-induced Stat3 activation, culminating in enhanced production of interleukin (IL) 6 and IL-12 p40 in the infected macrophages. Furthermore, overexpression of ROP16 but not ROP18 in mammalian cells resulted in Stat3 phosphorylation and strong activation of Stat3-dependent promoters. In addition, kinase-inactive ROP16 failed to activate Stat3. Comparison of type I and type II ROP16 revealed that a single amino acid substitution in the kinase domain determined the strain difference in terms of Stat3 activation. Moreover, ROP16 bound Stat3 and directly induced phosphorylation of this transcription factor. These results formally establish an essential and direct requirement of ROP16 in parasite-induced Stat3 activation and the significance of a single amino acid replacement in the function of type II ROP16.

scholarly journals Yeast mitochondria lacking the phosphate carrier/p32 are blocked in phosphate transport but can import preproteins after regeneration of a membrane potential.

1996 ◽  
Vol 16 (11) ◽  
pp. 6524-6531 ◽  
Author(s):  
V Zara ◽  
K Dietmeier ◽  
A Palmisano ◽  
A Vozza ◽  
J Rassow ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Outer Membrane ◽  
Protein Import ◽  
Outer Membrane Proteins ◽  
Phosphate Transport ◽  
Inner Membrane ◽  
Saccharomyces Cerevisiae Mitochondria ◽  
Mitochondrial Inner Membrane ◽  
Precursor Proteins ◽  
Phosphate Carrier

Two different functions have been proposed for the phosphate carrier protein/p32 of Saccharomyces cerevisiae mitochondria: transport of phosphate and requirement for import of precursor proteins into mitochondria. We characterized a yeast mutant lacking the gene for the phosphate carrier/p32 and found both a block in the import of phosphate and a strong reduction in the import of preproteins transported to the mitochondrial inner membrane and matrix. Binding of preproteins to the surface of mutant mitochondria and import of outer membrane proteins were not inhibited, indicating that the inhibition of protein import occurred after the recognition step at the outer membrane. The membrane potential across the inner membrane of the mutant mitochondria was strongly reduced. Restoration of the membrane potential restored preprotein import but did not affect the block of phosphate transport of the mutant mitochondria. We conclude that the inhibition of protein import into mitochondria lacking the phosphate carrier/p32 is indirectly caused by a reduction of the mitochondrial membrane potential (delta(gamma)), and we propose a model that the reduction of delta(psi) is due to the defective phosphate import, suggesting that phosphate transport is the primary function of the phosphate carrier/p32.

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