scholarly journals Biosynthetic pathway of mitochondrial ATPase subunit 9 in Neurospora crassa.

1983 ◽  
Vol 96 (1) ◽  
pp. 248-255 ◽  
Author(s):  
B Schmidt ◽  
B Hennig ◽  
R Zimmermann ◽  
W Neupert
Keyword(s):  
Molecular Weight ◽  
Mitochondrial Membrane ◽  
Proteolytic Cleavage ◽  
Mitochondrial Atpase ◽  
Inner Mitochondrial Membrane ◽  
Intact Cells ◽  
Atpase Subunit ◽  
Free System ◽  
Reticulocyte Lysates ◽  
Subunit 9

Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.

Two Forms of a Mitochondrial Endonuclease in Neurospora crassa

1975 ◽  
Vol 53 (7) ◽  
pp. 823-825 ◽  
Author(s):  
Charles E. Martin ◽  
Robert P. Wagner
Keyword(s):  
Molecular Weight ◽  
Neurospora Crassa ◽  
Mitochondrial Membrane ◽  
Nuclease Activity ◽  
Inner Mitochondrial Membrane ◽  
Detergent Treatment ◽  
Approximate Molecular Weight ◽  
Membrane Bound

Mitochondrial nuclease activity in Neurospora crassa occurs in membrane-bound and soluble forms in approximately equal proportions. These activities apparently are due to the same enzyme, which has an approximate molecular weight of 120 000. A portion of the insoluble enzyme appears to be associated with the inner mitochondrial membrane and is resistant to solubilization by detergent treatment as well as by physical disruption methods.

Spontaneous mitochondrial depolarizations are independent of SR Ca2+ release

2004 ◽  
Vol 286 (5) ◽  
pp. C1139-C1151 ◽  
Author(s):  
Catherine M. O'Reilly ◽  
Kevin E. Fogarty ◽  
Robert M. Drummond ◽  
Richard A. Tuft ◽  
John V. Walsh
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Mitochondrial Membrane ◽  
High Speed ◽  
Ryanodine Receptors ◽  
Muscle Cells ◽  
Inner Mitochondrial Membrane ◽  
Intact Cells ◽  
Mitochondrial Functions ◽  
Intracellular Ca

The mitochondrial membrane potential (ΔΨm) underlies many mitochondrial functions, including Ca2+ influx into the mitochondria, which allows them to serve as buffers of intracellular Ca2+. Spontaneous depolarizations of ΔΨm, flickers, have been observed in isolated mitochondria and intact cells using the fluorescent cationic lipophile tetramethylrhodamine ethyl ester (TMRE), which distributes across the inner mitochondrial membrane in accordance with the Nernst equation. Flickers in cardiomyocytes have been attributed to uptake of Ca2+ released from the sarcoplasmic reticulum (SR) via ryanodine receptors in focal transients called Ca2+ sparks. We have shown previously that an increase in global Ca2+ in smooth muscle cells causes an increase in mitochondrial Ca2+ and depolarization of ΔΨm. Here we sought to determine whether flickers in smooth muscle cells are caused by uptake of Ca2+ released focally in Ca2+ sparks. High-speed three-dimensional imaging was used to monitor ΔΨm in freshly dissociated myocytes from toad stomach that were simultaneously voltage clamped at 0 mV to ensure the cytosolic TMRE concentration was constant and equal to the low level in the bath (2.5 nM). This approach allows quantitative analysis of flickers as we have previously demonstrated. Depletion of SR Ca2+ not only failed to eliminate flickers but rather increased their magnitude and frequency somewhat. Flickers were not altered in magnitude or frequency by ryanodine or xestospongin C, inhibitors of intracellular Ca2+ release, or by cyclosporin A, an inhibitor of the permeability transition pore. Focal Ca2+ release from the SR does not cause flickers in the cells employed here.

Mitochondrial ion circuits

2010 ◽  
Vol 47 ◽  
pp. 25-35 ◽  
Author(s):  
David G. Nicholls
Keyword(s):  
Electron Transport Chain ◽  
Mitochondrial Membrane ◽  
Primary Energy ◽  
Proton Leak ◽  
Primary Proton ◽  
Inner Mitochondrial Membrane ◽  
Intact Cells ◽  
Transport Chain ◽  
Proton Current ◽  
Circuit Techniques

Proton circuits across the inner mitochondrial membrane link the primary energy generators, namely the complexes of the electron transport chain, to multiple energy utilizing processes, including the ATP synthase, inherent proton leak pathways, metabolite transport and linked circuits of sodium and calcium. These mitochondrial circuits can be monitored in both isolated preparations and intact cells and, for the primary proton circuit techniques, exist to follow both the proton current and proton electrochemical potential components of the circuit in parallel experiments, providing a quantitative means of assessing mitochondrial function and, equally importantly, dysfunction.

atpif1 regulates Mitochondrial Heme Synthesis In Developing Erythroid Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 163-163
Author(s):  
Dhvanit I Shah ◽  
Naoko Takahasi-Makise ◽  
Iman Schultz ◽  
Eric L Pierce ◽  
Liangtao Li ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Positional Cloning ◽  
Conflicts Of Interest ◽  
Microcytic Anemia ◽  
Mitochondrial Atpase ◽  
Cluster Assembly ◽  
Inner Mitochondrial Membrane ◽  
Heme Synthesis

Abstract Abstract 163 Iron plays a key role as a cofactor in many fundamental metabolic processes, which require heme synthesis and Fe/S cluster assembly in the mitochondria. Defects in the transport of iron into the mitochondria would lead to anemias due to a deficiency in heme and hemoglobin synthesis. Here we describe a zebrafish genetic mutant, pinotage (pnttq209), which exhibits a profound hypochromic, microcytic anemia. Erythrocytes from pnt mutants have a defect in hemoglobinization and decreased red cell indices (mean corpuscular volume and hemoglobin content, hematocrit, hemoglobin concentration). Through positional cloning, we showed that the mitochondrial ATPase Inhibitory Factor 1 (atpif1), which regulates the inner mitochondrial membrane potential, is the gene disrupted in pnt. The identity of the pnt gene was verified by: (a) decreased atpif1 steady-state mRNA in pnt mutants, (b) phenocopying the anemia with anti-sense atpif1 morpholinos, (c) functional complementation of the anemia with atpif1 cRNA, and (d) a genetic polymorphism in the 3'UTR co-segregating with the mutant phenotype that destabilizes the atpif1 mRNA. Consistent with the conserved function of atpif1 in higher vertebrates, the silencing of the murine ortholog of atpif1 in Friend mouse erythroleukemia (MEL) cells showed a defect in hemoglobinization by o-dianisidine staining and reduction of 59Fe incorporation into heme in 59Fe-metabolically labeled cells. Moreover, Atpif1 knockdown destabilizes their mitochondrial membrane potential and volume. Therefore, the identification of atpif1 in pnt functionally demonstrates the role of atpif1 in regulating the proton motive gradient across the inner mitochondrial membrane for mitochondrial iron incorporation in heme biosynthesis. These results uncover a novel hematopoiesis-related function of atpif1, which will directly contribute to our understanding and potential treatment of human congenital and acquired anemias. Disclosures: No relevant conflicts of interest to declare.

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