oligomycins and Starvation

Increasing evidence reveals that a subset of proteins participates in both the autophagy and apoptosis pathways, and this intersection is important in normal physiological contexts and in pathological settings. In this paper, we show that the Drosophila effector caspase, Drosophila caspase 1 (Dcp-1), localizes within mitochondria and regulates mitochondrial morphology and autophagic flux. Loss of Dcp-1 led to mitochondrial elongation, increased levels of the mitochondrial adenine nucleotide translocase stress-sensitive B (SesB), increased adenosine triphosphate (ATP), and a reduction in autophagic flux. Moreover, we find that SesB suppresses autophagic flux during midoogenesis, identifying a novel negative regulator of autophagy. Reduced SesB activity or depletion of ATP by oligomycin A could rescue the autophagic defect in Dcp-1 loss-of-function flies, demonstrating that Dcp-1 promotes autophagy by negatively regulating SesB and ATP levels. Furthermore, we find that pro-Dcp-1 interacts with SesB in a nonproteolytic manner to regulate its stability. These data reveal a new mitochondrial-associated molecular link between nonapoptotic caspase function and autophagy regulation in vivo.

Topics: Adenosine Triphosphate; Animals; Autophagy; Caspases; Caspases, Effector; Cells, Cultured; Drosophila melanogaster; Drosophila Proteins; Female; Gene Expression Regulation, Developmental; Mitochondria; Mitochondrial ADP, ATP Translocases; Oligomycins; Oogenesis; Ovary; Starvation

1. The conversion of inactive (phosphorylated) pyruvate dehydrogenase complex into active (dephosphorylated) complex by pyruvate dehydrogenase phosphate phosphatase is inhibited in heart mitochondria prepared from alloxan-diabetic or 48h-starved rats, in mitochondria prepared from acetate-perfused rat hearts and in mitochondria prepared from normal rat hearts incubated with respiratory substrates for 6 min (as compared with 1 min). 2. This conclusion is based on experiments with isolated intact mitochondria in which the pyruvate dehydrogenase kinase reaction was inhibited by pyruvate or ATP depletion (by using oligomycin and carbonyl cyanide m-chlorophenylhydrazone), and in experiments in which the rate of conversion of inactive complex into active complex by the phosphatase was measured in extracts of mitochondria. The inhibition of the phosphatase reaction was seen with constant concentrations of Ca2+ and Mg2+ (activators of the phosphatase). The phosphatase reaction in these mitochondrial extracts was not inhibited when an excess of exogenous pig heart pyruvate dehydrogenase phosphate was used as substrate. It is concluded that this inhibition is due to some factor(s) associated with the substrate (pyruvate dehydrogenase phosphate complex) and not to inhibition of the phosphatase as such. 3. This conclusion was verified by isolating pyruvate dehydrogenase phosphate complex, free of phosphatase, from hearts of control and diabetic rats an from heart mitochondria incubed for 1min (control) or 6min with respiratory substrates. The rates of re-activation of the inactive complexes were then measured with preparations of ox heart or rat heart phosphatase. The rates were lower (relative to controls) with inactive complex from hearts of diabetic rats or from heart mitochondria incubated for 6min with respiratory substrates. 4. The incorporation of 32Pi into inactive complex took 6min to complete in rat heart mitocondria. The extent of incorporation was consistent with three or four sites of phosphorylation in rat heart pyruvate dehydrogenase complex. 5. It is suggested that phosphorylation of sites additional to an inactivating site may inhibit the conversion of inactive complex into active complex by the phosphatase in heart mitochondria from alloxan-diabetic or 48h-starved rats or in mitochondria incubated for 6min with respiratory substrates.

Topics: Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Diabetes Mellitus, Experimental; Enzyme Activation; In Vitro Techniques; Mitochondria, Heart; Oligomycins; Phosphorylation; Pyruvate Dehydrogenase (Lipoamide)-Phosphatase; Pyruvate Dehydrogenase Complex; Pyruvates; Rats; Starvation

Topics: Adenosine Triphosphate; Adipose Tissue; Animals; Azides; Cricetinae; Cyanides; Depression, Chemical; Glycolysis; Iodoacetates; Kinetics; Models, Biological; Norepinephrine; Oligomycins; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen Consumption; Rotenone; Starvation; Stimulation, Chemical