Conserved Prefusion Protein Assembly in Regulated Exocytosis

Abstract

The regulated release of hormones and neurotransmitters is a fundamental process throughout the animal kingdom. The short time scale for the calcium triggering of vesicle fusion in regulated secretion suggests that the calcium sensor synaptotagmin and the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) membrane fusion machinery are well ordered before the calcium signal. To gain insight into the organization of the prefusion protein assembly in regulated exocytosis, we undertook a structural/functional study of the vesicular synaptotagmin1 and the plasma membrane SNARE proteins, which copurify from the brain in the absence of calcium. Based on an evolutionary analysis, mutagenesis screens, and a computational protein docking approach, we now provide the first testable description of the supramolecular prefusion assembly. Perturbing the determined synaptotagmin/SNARE-interacting interface in several models of regulated exocytosis altered the secretion of hormones and neurotransmitters. These mutations also disrupted the constitutive synaptotagmin/SNARE link in full agreement with our model. We conclude that the interaction of synaptotagmin with preassembled plasma membrane SNARE proteins, before the action of calcium, can provide a precisely organized "tethering" scaffold that underlies regulated secretion throughout evolution. INTRODUCTIONIn regulated secretion, fusion of docked secretory vesicles with the plasma membrane is triggered by the rapid elevation of the intracellular calcium concentration (Katz and Miledi, 1967). The membrane fusion itself is brought about by the action of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, whereas the vesicular protein synaptotagmin (SYT) acts as the calcium sensor (Sollner et al., 1993;Sudhof and Scheller, 2001;Bonifacino and Glick, 2004). In neuroendocrine cells, the principal SNAREs are syntaxin1 and synaptosome-associated protein of 25 kDa (SNAP-25) on the plasma membrane (so-called target SNAREs or t-SNAREs), and vesicular synaptobrevin, also known as VAMP. Interaction between synaptobrevin and the two t-SNAREs leads to the formation of the ternary SNARE complex, a twisted parallel fourhelical bundle that probably drives membrane fusion (Sutton et al., 1998). With all the major players involved in vesicle fusion identified, it is becoming possible to tackle a central problem of this cellular process-how does calcium trigger vesicle fusion? Arguably, to understand the action of calcium it is essential to know 1) the extent of the assembly of the SNARE fusion proteins and 2) their organization in relation to the calcium sensor, SYT, before calcium-triggered events.It would be reasonable to assume that the SNAREs are at least partially preassembled, and the plasma membrane syntaxin and SNAP-25 are the first obvious candidates for being in such a ready state (An and Almers, 2004;Rickman et al., 2004b). Importantly, synaptobrevin binds syntaxin and SNAP-25 with high-affinity only when the two...

Primed Vesicles Can Be Distinguished from Docked Vesicles by Analyzing Their Mobility

“…The effect of TeNt-LC was small because the interaction of synaptotagmin with syntaxin probably masked some of the priming defect (Rickman et al, 2006). However, the effect was significant.…”

A Quaternary SNARE–Synaptotagmin–Ca2+–Phospholipid Complex in Neurotransmitter Release

“…refs. 40,42,54 ), yet there were contradictory data on these interactions and it was unclear how they might be coupled with Ca 2+ -phospholipid binding to synaptotagmin 1. The results described here now shed light on these questions, showing that synaptotagmin 1 forms an intimate quaternary complex with the SNAREs, Ca 2+ and phospholipids, and suggesting that membrane-anchoring of SNARE complexes increases the specificity of their interactions with synaptotagmin 1.…”

The Diversity of Calcium Sensor Proteins in the Regulation of Neuronal Function

“…Ca 2þ entry through VGCCs leading to Ca 2þ elevation in local microdomains close to the mouth of the Ca 2þ channel is able to trigger very rapid (within less than 1 ms) fusion of synaptic vesicles. Synaptotagmin can bind to both syntaxin and SNAP-25, and fast neurotransmitter release requires synaptotagmin (Geppert et al 1994) probably prebound to assembled or partially assembled SNARE complexes (Schiavo et al 1997;Rickman et al 2006) so that Ca 2þ -induced interaction with phospholipids can occur rapidly (Xue et al 2008). It is still under debate how important synaptotagmin is in vesicle docking (de Wit et al 2009) and how it acts at the plasma membrane in fusion itself (Tang et al 2006;Hui et al 2009).…”