Lipid Raft Association of SNARE Proteins Regulates Exocytosis in PC12 Cells

Abstract

SNAP25 and SNAP23 are plasma membrane SNARE proteins essential for regulated exocytosis in diverse cell types. Several recent studies have shown that these proteins are partly localized in lipid rafts, domains of the plasma membrane enriched in sphingolipids, and cholesterol. Here, we have employed cysteine mutants of SNAP25/SNAP23, which have modified affinities for raft domains, to examine whether raft association of these proteins is important for the regulation of exocytosis. PC12 cells were engineered that express the light chain of botulinum neurotoxin; in these cells all of the SNAP25 was cleaved to a lower molecular weight form, and regulated exocytosis was essentially absent. Exocytosis was rescued by expressing toxin-resistant SNAP25 or wildtype SNAP23, which is naturally toxin-resistant. Remarkably, a mutant SNAP25 protein with an increased affinity for rafts displayed a reduced ability to support exocytosis, whereas SNAP23 mutants with a decreased affinity for rafts displayed an enhancement of exocytosis when compared with wild-type SNAP23. The effects of the mutant proteins on exocytosis were dependent upon the integrity of the plasma membrane and lipid rafts. These results provide the first direct evidence that rafts regulate SNARE function and exocytosis and identify the central cysteine-rich region of SNAP25/23 as an important regulatory domain.Exocytosis, the fusion of intracellular vesicles with the plasma membrane, mediates the secretion of molecules from the cell and the insertion of proteins and lipids into the plasma membrane. This membrane fusion event needs to be tightly regulated when vesicles contain molecules such as neurotransmitters, adrenaline, or insulin. A large number of proteins have been identified that function in exocytosis (1, 2). Among these, SNARE 1 proteins have emerged as potential membrane fusion catalysts (3, 4). Membrane fusion requires the interaction of Q-SNAREs present on the plasma membrane with R-SNAREs residing on the vesicle membrane. In neuronal and neuroendocrine cells, the Q-SNAREs that function in regulated exocytosis are syntaxin 1 and SNAP25, whereas the R-SNARE is VAMP/ synaptobrevin (5).Recently, there has been significant interest in the domain distribution of Q-SNAREs present at the plasma membrane. A number of studies have suggested that Q-SNAREs are partly localized in lipid rafts, lipid microdomains in the plasma membrane enriched in sphingolipids, and cholesterol (6 -14). As rafts have been proposed to function in the regulation of numerous signal transduction (15) and membrane traffic pathways (16), these observations raise the intriguing possibility that rafts may regulate SNARE function and, hence, exocytosis. As yet, the importance of the raft association of SNARE proteins for exocytosis has not been examined.Recent work from our group has reported that raft association of SNAP25 and its ubiquitous homologue, SNAP23, is mediated by the cysteine-rich domains of these proteins (17). We identified mutations within SNAP23 that decreased its raft a...

Cholesterol facilitates the native mechanism of Ca2+-triggered membrane fusion

“…Studies were also carried out with polyene antibiotics, a class of molecules known to bind and effectively sequester sterols in the membrane (Weissmann and Sessa, 1967;Norman et al, 1972a;Norman et al, 1972b;Patterson et al, 1979). Cholesterol oxidase was used to metabolically 'remove' cholesterol from the membranes and effectively disrupt functional microdomains (Xu and London, 2000;Samsonov et al, 2001) The results of these experiments support the role of cholesterol as a pre-fusion organizer (Lang et al, 2001;Salaun et al, 2005), but also indicate that cholesterol functions more directly in the native molecular mechanism of bilayer merger.…”

“…In both model and native membranes cholesterol associates to form discrete, functional microdomains (rafts) that serve as sites for specific protein-lipid interactions (Lucero and Robbins, 2004). Several proteins implicated in the exocytotic process have been shown to associate with cholesterol-rich microdomains (Lang et al, 2001): these domains have been suggested to be the sites of membrane fusion, although a more recent study suggests them to be negative regulators of the exocytotic process (Salaun et al, 2005), consistent with other membrane components functioning downstream in the actual triggered membrane fusion event Peters and Mayer, 1998;Peters et al, 1999;Peters et al, 2001;Coorssen et al, 2003). This is at least potentially consistent with either the protein pore or proximity models for fusion.…”

Brain-Derived Neurotrophic Factor Regulates Cholesterol Metabolism for Synapse Development

“…It was demonstrated that (1) cholesterol itself regulates the availability of synaptic vesicles via a direct interaction with synaptophysin (Thiele et al, 2000), (2) SNAP receptor (SNARE) complex formation is cholesterol-dependent (Lang et al, 2001;Mitter et al, 2003), and (3) the association of SNARE complex with lipid rafts regulates exocytosis (Sala͐n et al, 2005). Presynaptic proteins are increased in cholesterol-rich lipid raft fractions concomitantly with cortical development (data not shown).…”

Differential palmitoylation regulates intracellular patterning of SNAP25

“…5). To determine whether functional differences between SNAP25b and SNAP23 might arise owing to the effects of the different number of cysteine residues on RE and TGN targeting, we examined a SNAP25b mutant with an extra cysteine (F84C) to mimic SNAP23 (Salaun et al, 2005a) (Fig. 5A).…”