Ca2+ Regulates the Drosophila Stoned-A and Stoned-B Proteins Interaction with the C2B Domain of Synaptotagmin-1

The dicistronic Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled-coil domain of the Stoned-A and the µ-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. Here we report that these interactions are increased in the presence of Ca2+. The Ca2+-dependent interaction between the µ-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca2+ binding loop region that modulate the Ca2+-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca2+-binding loop region of C2B domain. The results indicate that Ca2+-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca2+-bound Synaptotagmin-1 associated synaptic vesicles

Synaptotagmins I and IV promote transmitter release independently of Ca2+ binding in the C2A domain

At nerve terminals, a focal and transient increase in intracellular Ca2+ triggers the fusion of neurotransmitter-filled vesicles with the plasma membrane. The most extensively studied candidate for the Ca2+-sensing trigger is synaptotagmin I, whose Ca2+-dependent interactions with acidic phospholipids and syntaxin1 have largely been ascribed to its C2A domain, although the C2B domain also binds Ca2+ (refs 7, 8). Genetic tests of synaptotagmin I have been equivocal as to whether it is the Ca2+ -sensing trigger of fusion. Synaptotagmin IV, a related isoform that does not bind Ca2+ in the C2A domain, might be an inhibitor of release. We mutated an essential aspartate of the Ca2+-binding site of the synaptotagmin I C2A domain and expressed it in Drosophila lacking synaptotagmin I. Here we show that, despite the disruption of the binding site, the Ca2+-dependent properties of transmission were not altered. Similarly, we found that synaptotagmin IV could substitute for synaptotagmin I. We conclude that the C2A domain of synaptotagmin is not required for Ca2+-dependent synaptic transmission, and that synaptotagmin IV promotes rather than inhibits transmission

Coordinated regulation of sulfur and phospholipid metabolism reflects the importance of methylation in the growth of yeast

The sulfur assimilation and phospholipid biosynthesis pathways interact metabolically and transcriptionally. Genetic analysis, genome-wide sequencing, and expression microarrays show that regulators of these pathways, Met4p and Opi1p, control cellular methylation capacity that can limit the growth rate