Synaptotagmin (syt) 7 is one of three syt isoforms found in all metazoans; it is ubiquitously expressed, yet its function in neurons remains obscure. Here, we resolved Ca2+-dependent and Ca2+-independent synaptic vesicle (SV) replenishment pathways, and found that syt 7 plays a selective and critical role in the Ca2+-dependent pathway. Mutations that disrupt Ca2+-binding to syt 7 abolish this function, suggesting that syt 7 functions as a Ca2+-sensor for replenishment. The Ca2+-binding protein calmodulin (CaM) has also been implicated in SV replenishment, and we found that loss of syt 7 was phenocopied by a CaM antagonist. Moreover, we discovered that syt 7 binds to CaM in a highly specific and Ca2+-dependent manner; this interaction requires intact Ca2+-binding sites within syt 7. Together, these data indicate that a complex of two conserved Ca2+-binding proteins, syt 7 and CaM, serve as a key regulator of SV replenishment in presynaptic nerve terminals.DOI: http://dx.doi.org/10.7554/eLife.01524.001

SUMMARY Synaptotagmin-I (syt) is a Ca2+ sensor that triggers synchronous neurotransmitter release. The first documented biochemical property of syt was its ability to aggregate membranes in response to Ca2+. However, the mechanism and function of syt-mediated membrane aggregation are poorly understood. Here, we demonstrate that syt-mediated vesicle aggregation is driven by trans interactions between syt molecules bound to different membranes. We observed a strong correlation between the ability of Ca2+-syt to aggregate vesicles and to stimulate SNARE-mediated membrane fusion. Moreover, artificial aggregation of membranes - using non-syt proteins - also efficiently promoted fusion of SNARE-bearing liposomes. Finally, using a modified fusion assay, we observed that syt drives the assembly of otherwise non-fusogenic individual t-SNARE proteins into fusion competent heterodimers, in an aggregation-independent manner. Thus, membrane aggregation and t-SNARE assembly appear to be two key aspects of Ca2+-syt-regulated, SNARE-catalyzed fusion reactions.

Mitochondrial homeostasis vitally depends on mitophagy, the programmed degradation of mitochondria. The roster of proteins known to participate in mitophagy remains small. We devised here a multidimensional CRISPR/Cas9 genetic screen, using multiple mitophagy reporter systems and pro-mitophagy triggers, and uncover numerous new components of Parkin-dependent Reprints and permissions information is available at www.nature.com/reprints.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: