This study provides novel mechanistic insights into how Syt-1 and Syt-7 C2B domains regulate the later stages of exocytosis. We show that small structural differences within this region exert significant effects on fusion pore properties. We propose that these effects may arise from differences in C2B domain affinity for the plasma membrane.

Chromaffin cells release transmitters from populations of granules to which synaptotagmin-1 and synaptotagmin-7 are selectively sorted. Rao et al. characterize the functional properties of these granules and show that synaptotagmin-7 confers fast kinetics and high efficacy to the exocytotic event.

Edited by George M. Carman Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial bioenergetics, recent evidence in the yeast model indicates that CL is also essential for intermediary metabolism. To gain insight into the function of CL in energy metabolism in mammalian cells, here we analyzed the metabolic flux of [U-13 C]glucose in a mouse C2C12 myoblast cell line, TAZ-KO, which is CL-deficient because of CRISPR/Cas9-mediated knockout of the CL-remodeling enzyme tafazzin (TAZ). TAZ-KO cells exhibited decreased flux of [U-13 C]glucose to [ 13 C]acetyl-CoA and M2 and M4 isotopomers of tricarboxylic acid (TCA) cycle intermediates. The activity of pyruvate carboxylase, the predominant enzyme for anaplerotic replenishing of the TCA cycle, was elevated in TAZ-KO cells, which also exhibited increased sensitivity to the pyruvate carboxylase inhibitor phenylacetate. We attributed a decreased carbon flux from glucose to acetyl-CoA in the TAZ-KO cells to a ϳ50% decrease in pyruvate dehydrogenase (PDH) activity, which was observed in both TAZ-KO cells and cardiac tissue from TAZ-KO mice. Protein-lipid overlay experiments revealed that PDH binds to CL, and supplementing digitonin-solubilized TAZ-KO mitochondria with CL restored PDH activity to WT levels. Mitochondria from TAZ-KO cells exhibited an increase in phosphorylated PDH, levels of which were reduced in the presence of supplemented CL. These findings indicate that CL is required for optimal PDH activation, generation of acetyl-CoA, and TCA cycle function, findings that link the key mitochondrial lipid CL to TCA cycle function and energy metabolism. The phospholipid cardiolipin (CL) 8 is the signature lipid of mitochondrial membranes. It interacts with a wide variety of mitochondrial proteins (1) and has multiple roles in mitochondrial biogenesis as well as other cellular functions (2-4). Loss of CL leads to deficiencies in respiratory function, mitochondrial membrane potential, and ATP synthesis (1, 5-7). CL that is synthesized de novo has predominantly saturated fatty acids (8). However, the lipid is then remodeled via phospholipase-mediated deacylation to form monolysocardiolipin (MLCL), which is subsequently acylated by the enzyme tafazzin to synthesize CL with predominantly unsaturated fatty acids (9, 10). Abnormal CL composition is associated with several human disorders, including diabetes, Alzheimer's disease, and Parkinson's disease (11-13). However, Barth syndrome (BTHS) is the only disorder identified to date that is caused by altered CL metabolism (14, 15). BTHS is a severe X-linked disorder characterized by dilated cardiomyopathy, skeletal myopathy, neutropenia, exercise intolerance, lactic acidosis, and sudden death from arrhythmia (16-18). The underlying cause of BTHS is mutations in the tafazzin (TAZ) gene, which codes for the transacylase that remodels CL (10, 19). As a result, BTHS patients exhibit an aberrant CL profile characterized by decreased tot...

Previous studies have shown that the cardiolipin (CL)-deficient yeast mutant, crd1Δ, has decreased levels of acetyl-CoA and decreased activities of the TCA cycle enzymes aconitase and succinate dehydrogenase. These biochemical phenotypes are expected to lead to defective TCA cycle function. In this study, we report that signaling and anaplerotic metabolic pathways that supplement defects in the TCA cycle are essential in crd1Δ mutant cells. The crd1Δ mutant is synthetically lethal with mutants in the TCA cycle, retrograde (RTG) pathway, glyoxylate cycle, §

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