In vivo and in vitro evidence for a proton leakage through the inner mitochondrial membrane in a mutant of Saccharomyces cerevisiae

1988 ◽  
Vol 174 (2) ◽  
pp. 399-404 ◽  
Author(s):  
Stephen MANON ◽  
Felicien RAKOTOMANANA ◽  
Martine GUERIN
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Membrane ◽  
Inner Mitochondrial Membrane ◽  
Proton Leakage

scholarly journals Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (11) ◽  
pp. 5487-5496 ◽  
Author(s):  
M E Dumont ◽  
T S Cardillo ◽  
M K Hayes ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Mitochondrial Membrane ◽  
Intermembrane Space ◽  
Inner Mitochondrial Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
Apocytochrome C ◽  
Cytochrome C Heme Lyase

Heme is covalently attached to cytochrome c by the enzyme cytochrome c heme lyase. To test whether heme attachment is required for import of cytochrome c into mitochondria in vivo, antibodies to cytochrome c have been used to assay the distributions of apo- and holocytochromes c in the cytoplasm and mitochondria from various strains of the yeast Saccharomyces cerevisiae. Strains lacking heme lyase accumulate apocytochrome c in the cytoplasm. Similar cytoplasmic accumulation is observed for an altered apocytochrome c in which serine residues were substituted for the two cysteine residues that normally serve as sites of heme attachment, even in the presence of normal levels of heme lyase. However, detectable amounts of this altered apocytochrome c are also found inside mitochondria. The level of internalized altered apocytochrome c is decreased in a strain that completely lacks heme lyase and is greatly increased in a strain that overexpresses heme lyase. Antibodies recognizing heme lyase were used to demonstrate that the enzyme is found on the outer surface of the inner mitochondrial membrane and is not enriched at sites of contact between the inner and outer mitochondrial membranes. These results suggest that apocytochrome c is transported across the outer mitochondrial membrane by a freely reversible process, binds to heme lyase in the intermembrane space, and is then trapped inside mitochondria by an irreversible conversion to holocytochrome c accompanied by folding to the native conformation. Altered apocytochrome c lacking the ability to have heme covalently attached accumulates in mitochondria only to the extent that it remains bound to heme lyase.

107 DIFFERENCES IN THE INNER MITOCHONDRIAL MEMBRANE POTENTIAL BETWEEN NON-CULTURED AND CULTURED PORCINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 159
Author(s):  
M. Romek ◽  
B. Gajda ◽  
M. Rolka ◽  
Z. Smorag
Keyword(s):  
Fatty Acids ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Developmental Competence ◽  
Critical Event ◽  
Inner Mitochondrial Membrane ◽  
Oxidation Rates

In comparison to in vivo derived pig embryos, in vitro culture conditions produce embryos with altered metabolic rates of carbohydrates and fatty acids (Romek M et al. 2010 Theriogenology 74, 265–276), which may compromise embryo viability. Because various energy substrates are metabolized via several aerobic pathways leading to generation of the inner mitochondrial membrane potential (ΔΨm), value of ΔΨm is a key indicator of embryo metabolic activity, closely related to oxygen consumption and cellular energy needs. Therefore, the aim of this study was to compare ΔΨm between non-cultured and cultured pig embryos during early development. The non-cultured embryos were obtained from 6-month-old gilts, whereas those derived in vitro were cultured from zygotes to the appropriate stage in North Carolina State University 23 (NCSU-23) medium supplemented with 4 mg mL–1 of bovine serum albumin. The ΔΨm measurements were carried out on both non-cultured and cultured 4 to 8 cell embryos, morulae, blastocysts and late blastocysts. For this, embryos were labelled with 0.5 μM Mito Tracker Orange CMTMRos (MtOR) for 30 min at 39°C and then with 0.5 μM Mito Tracker Deep Red (MtDR) for 30 min at 10°C. Using a LSM 510 Meta Zeiss confocal microscope, we measured the amounts of fluorescence (IMtOR and IMtDR) emitted from embryos and values of ΔΨm were estimated as the IMtOR/IMtDR ratios. The results were analysed by ANOVA and Tukey's test. From the zygote to morula stages, ΔΨm remained unchanged and did not differ between developmentally matched non-cultured and cultured embryos (P < 0.001). The value of ΔΨm increased significantly (P < 0.05) from 0.90 ± 0.26 arbitrary units (a.u.) for morulae to 3.92 ± 0.63 and 2.06 ± 0.38 a.u. for non-cultured and cultured early blastocysts, respectively. Whereas the mean value of ΔΨm was almost 2 times higher in non-cultured than in cultured early blastocysts, the mitochondrial membrane potential was statistically similar (P < 0.05) in the in vivo derived (2.10 ± 0.37 a.u.) compared to cultured (1.87 ± 0.30 a.u.) blastocysts. The lower ΔΨm in cultured early blastocysts may be explained by several-fold higher glucose concentration in NCSU-23 medium than in the oviductal fluid. It was reported that high levels of glucose decreases the Krebs cycle metabolism of pyruvate, glutamine, and glucose, and reduces oxidation rates of fatty acids in cultured pig embryos in comparison with in vivo counterparts. Hence, this impaired metabolism reflected by decreased ΔΨm may be responsible for insufficient energy production and reduced developmental competence of cultured early blastocysts. Therefore, because embryo-cavitation is a critical event in pig development, further effort should be focused on proper blastocyst culture. Research was partially supported by Grant NR 12 0036 06 from NCBiR, Poland.

Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (11) ◽  
pp. 5487-5496
Author(s):  
M E Dumont ◽  
T S Cardillo ◽  
M K Hayes ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Mitochondrial Membrane ◽  
Intermembrane Space ◽  
Inner Mitochondrial Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
Apocytochrome C ◽  
Cytochrome C Heme Lyase

Heme is covalently attached to cytochrome c by the enzyme cytochrome c heme lyase. To test whether heme attachment is required for import of cytochrome c into mitochondria in vivo, antibodies to cytochrome c have been used to assay the distributions of apo- and holocytochromes c in the cytoplasm and mitochondria from various strains of the yeast Saccharomyces cerevisiae. Strains lacking heme lyase accumulate apocytochrome c in the cytoplasm. Similar cytoplasmic accumulation is observed for an altered apocytochrome c in which serine residues were substituted for the two cysteine residues that normally serve as sites of heme attachment, even in the presence of normal levels of heme lyase. However, detectable amounts of this altered apocytochrome c are also found inside mitochondria. The level of internalized altered apocytochrome c is decreased in a strain that completely lacks heme lyase and is greatly increased in a strain that overexpresses heme lyase. Antibodies recognizing heme lyase were used to demonstrate that the enzyme is found on the outer surface of the inner mitochondrial membrane and is not enriched at sites of contact between the inner and outer mitochondrial membranes. These results suggest that apocytochrome c is transported across the outer mitochondrial membrane by a freely reversible process, binds to heme lyase in the intermembrane space, and is then trapped inside mitochondria by an irreversible conversion to holocytochrome c accompanied by folding to the native conformation. Altered apocytochrome c lacking the ability to have heme covalently attached accumulates in mitochondria only to the extent that it remains bound to heme lyase.

scholarly journals Opa1 Prevents Apoptosis and Cisplatin-Induced Ototoxicity in Murine Cochleae

2021 ◽  
Vol 9 ◽  
Author(s):  
Tingting Dong ◽  
Xuejie Zhang ◽  
Yiqing Liu ◽  
Shan Xu ◽  
Haishuang Chang ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Death ◽  
Mitochondrial Membrane ◽  
Vital Role ◽  
Mitochondrial Morphology ◽  
Oxygen Species ◽  
Inner Mitochondrial Membrane ◽  
Cochlear Damage

Optic atrophy1 (OPA1) is crucial for inner mitochondrial membrane (IMM) fusion and essential for maintaining crista structure and mitochondrial morphology. Optic atrophy and hearing impairment are the most prevalent clinical features associated with mutations in the OPA1 gene, but the function of OPA1 in hearing is still unknown. In this study, we examined the ability of Opa1 to protect against cisplatin-induced cochlear cell death in vitro and in vivo. Our results revealed that knockdown of Opa1 affects mitochondrial function in HEI-OC1 and Neuro 2a cells, as evidenced by an elevated reactive oxygen species (ROS) level and reduced mitochondrial membrane potential. The dysfunctional mitochondria release cytochrome c, which triggers apoptosis. Opa1 expression was found to be significantly reduced after cell exposed to cisplatin in HEI-OC1 and Neuro 2a cells. Loss of Opa1 aggravated the apoptosis and mitochondrial dysfunction induced by cisplatin treatment, whereas overexpression of Opa1 alleviated cisplatin-induced cochlear cell death in vitro and in explant. Our results demonstrate that overexpression of Opa1 prevented cisplatin-induced ototoxicity, suggesting that Opa1 may play a vital role in ototoxicity and/or mitochondria-associated cochlear damage.

The ligands of translocator protein: design and biological properties

2020 ◽  
Vol 26 ◽  
Author(s):  
Grigory V. Mokrov ◽  
Olga A. Deeva ◽  
Tatiana A. Gudasheva
Keyword(s):  
Protein Design ◽  
Mitochondrial Membrane ◽  
Benzodiazepine Receptor ◽  
Biological Properties ◽  
Translocator Protein ◽  
Peripheral Benzodiazepine Receptor ◽  
Inner Mitochondrial Membrane ◽  
Limiting Stage

: In 2020, it is already 43 years since Braestrup and Squires discovered 18 kDa translocator protein (TSPO), known until 2006 as "peripheral benzodiazepine receptor". During this time the functions of this receptor which is located on the outer membrane of mitochondria were studied in detail. One of the key functions of TSPO is the transfer of cholesterol from the outer to the inner mitochondrial membrane, which is the limiting stage in the synthesis of neurosteroids. TSPO is also involved in the transport of porphyrins, mitochondrial respiration, the opening of mitochondrial pores, apoptosis and cell proliferation. This review presents current information on the structure of TSPO, the mechanism of its participation in neurosteroidogenesis, as well as endogenous and synthetic TSPO ligands. Particular emphasis is placed on the analysis of approaches to the design of synthetic ligands and their neuropsychotropic activity in vitro and in vivo. The presented review demonstrates the promise of constructing new neuropsychotropic drugs in the series of TSPO ligands.

scholarly journals The mitochondrial Ca2+ uniporter MCU is required for normal glucose-stimulated insulin secretion in vitro and in vivo

2019 ◽  
Author(s):  
Elizabeth Haythorne ◽  
Eleni Georgiadou ◽  
Matthew T. Dickerson ◽  
Livia Lopez-Noriega ◽  
Timothy J. Pullen ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Glucose Tolerance ◽  
Mitochondrial Membrane ◽  
Inner Mitochondrial Membrane ◽  
Β Cells ◽  
Β Cell ◽  
Compensatory Mechanisms ◽  
Glucose Stimulated Insulin Secretion

AbstractMitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic β-cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in β-cells has not previously been examined in vivo. Here, we inactivated the pore-forming subunit MCUa (MCU) selectively in the β-cell in mice using Ins1Cre-mediated recombination. Glucose-stimulated mitochondrial Ca2+ accumulation, ATP production and insulin secretion were strongly (p<0.05 and p<0.01) inhibited in MCU null animals (βMCU-KO) in vitro. Interestingly, cytosolic Ca2+ concentrations increased (p<0.001) whereas mitochondrial membrane depolarisation improved in βMCU-KO animals. Male βMCU-KO mice displayed impaired in vivo insulin secretion at 5 (p<0.001) but not 15 min. post intraperitoneal (IP) injection of glucose while the opposite phenomenon was observed following an oral gavage at 5 min. Unexpectedly, glucose tolerance was improved (p<0.05) in young βMCU-KO (<12 weeks), but not older animals. We conclude that MCU is crucial for mitochondrial Ca2+ uptake in pancreatic β-cells and is required for normal GSIS. The apparent compensatory mechanisms which maintain glucose tolerance in βMCU-KO mice remain to be established.

scholarly journals Review of T-2307, an Investigational Agent That Causes Collapse of Fungal Mitochondrial Membrane Potential

2021 ◽  
Vol 7 (2) ◽  
pp. 130
Author(s):  
Nathan P. Wiederhold
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Candida Species ◽  
Mammalian Cells ◽  
Mitochondrial Membrane ◽  
Fungal Infections ◽  
Published Data ◽  
Candida Auris

Invasive infections caused by Candida that are resistant to clinically available antifungals are of increasing concern. Increasing rates of fluconazole resistance in non-albicans Candida species have been documented in multiple countries on several continents. This situation has been further exacerbated over the last several years by Candida auris, as isolates of this emerging pathogen that are often resistant to multiple antifungals. T-2307 is an aromatic diamidine currently in development for the treatment of invasive fungal infections. This agent has been shown to selectively cause the collapse of the mitochondrial membrane potential in yeasts when compared to mammalian cells. In vitro activity has been demonstrated against Candida species, including C. albicans, C. glabrata, and C. auris strains, which are resistant to azole and echinocandin antifungals. Activity has also been reported against Cryptococcus species, and this has translated into in vivo efficacy in experimental models of invasive candidiasis and cryptococcosis. However, little is known regarding the clinical efficacy and safety of this agent, as published data from studies involving humans are not currently available.

scholarly journals Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion

2007 ◽  
Vol 6 (12) ◽  
pp. 2214-2221 ◽  
Author(s):  
Lois M. Douglas ◽  
Li Li ◽  
Yang Yang ◽  
A. M. Dranginis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biofilm Formation ◽  
In Vitro System ◽  
Glycosylphosphatidylinositol Anchor ◽  
Fungal Adhesion ◽  
Very High ◽  
Molecular Weight Form

ABSTRACT The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

scholarly journals Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875 ◽  
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

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