Designing the lipid raft marker protein for synaptic vesicles

Lipid rafts are cholesterol-enriched microdomains and implicated in many essential physiological ac-tivities such as the neurotransmitter release.Many studies have been carried out on the function of rafts inthe plasma membranes,whereas little is known about the information of such microdomains in subcellularcompartments especially synaptic vesicles(SVs).In the well-studied plasma membranes,several proteinshave been recognized as raft markers,which are used to label or trace rafts.But the raft marker proteinon SVs has not been identified yet.Although some SV proteins,including VAMP and CPE,have beenfound in raft fractions,they cannot be used as markers due to their low abundance in rafts.In this work,we designed several chimera proteins and tested their characteristics for using as SV raft makers.First,we detected whether they located in SVs,and then the chimeras exhibiting the better localization in SVswere further examined for their enrichment in raft using detergent treatment and gradient density floatationanalysis.Our results indicate that one of the chimeric proteins is primarily located in SVs and distributedin raft microdomains,which strongly suggests that it could be served as a raft marker for SVs.

Two forms of the membrane-bound state of the first C2 domain (C2A) of synaptotagminⅠand calcium-triggered membrane insertion

The synaptic vesicle protein synaptotagminⅠ (sytⅠ) is a vesicle transmembrane protein present in synap-tic vesicles, which has been proposed as the Ca2+ sensor that regulates secretion. The C2A domain is the membrane proximal part of its cytoplasmic domain. The interaction between C2A and lipid bilayer has been considered to be essential for triggering neurotransmitter release. In the pre-sent work, the measurements of membrane surface tension and surface concentration showed that the C2A domain of sytⅠexhibited two membrane-bound states: the surface adsorption state and the membrane insertion state. The sur-face absorption state formed in a Ca2+-independent manner with lower affinity, while the membrane insertion state formed with high affinity was only found in the presence of Ca2+. Both the Ca2+-independent and Ca2+-dependent sytⅠ- membrane interactions required anionic phospholipids, such as phosphatidylserine (PS). When expressed into rat pheo-chromocytoma (PC12) cells and human embryonic kidney (HEK-293) cells, as demonstrated by immunofluorescence staining and subcellular fractionation, most of the C2A was found at the plasma membrane, even when the cells were depleted of Ca2+ by incubation with EGTA. These results suggested a new molecular mechanism of sytⅠas a Ca2+ sensor in membrane fusion. Ca2+-independent surface ad-sorption might attach sytⅠto the release site during the docking or priming step. When intracellular Ca2+ increased, sytⅠtriggered the neurotransmitter release following the Ca2+-dependent penetration into the target membrane.