SNAREs, 14-3-3 proteins and cholesterol dependent membrane domains

Lipid rafts are suggested to be sphingolipid and cholesterol rich domains that segregate out from the bulk plasma membrane by forming a more ordered lipid phase. Lipid raft-like domains have been described as cell signalling platforms and have been implicated in the regulation of an array of signal transduction events. This study investigates the association of two classes of protein, 14-3-3 proteins and plasma membrane SNAREs, with lipid raft-like domains. The 14-3-3 family of proteins are important regulators of numerous cell signalling pathways and are essential for cell survival; recently there has been some interest in the roles of these soluble proteins at the membrane, though this area remains poorly characterised. 14-3-3 has also been linked to CJD progression, which is directed by the lipid raft associated prion protein. SNAREs are essential mediators of exocytosis, a process that is also reported to depend on cholesterol, implying lipid raft involvement. SNAREs have also been isolated in detergent resistant membranes (DRMs) that are believed to represent clustered lipid rafts.To examine the association of 14-3-3 and SNAREs with lipid raft-like domains in N2a and PC 12 cells two approaches were taken. Initially, detergent resistant membranes were isolated and analysed for protein association. The second approach involved quantitative analysis of the colocalisation of 14-3-3 and SNAREs with membrane domains in intact cells by confocal microscopy, using the lipid raft marker cholera toxin B subunit (CTXB). Discrepancies between results from these two methods add to evidence implying that DRMs do not necessarily represent pre¬ existing membrane domains. Cholesterol depletion, which affects the integrity of lipid raft-like domains, caused a rearrangement CTXB labelled clusters in N2a and PC 12 cells. The colocalisation of 14-3-3 with CTXB was unaffected by cholesterol depletion, a result which does not support the localisation of 14-3-3 to lipid raft-like domains. Interestingly however, the membrane distribution of the lipid raft marker Thy-1, a GPI-anchored protein, was also unaltered when cholesterol was depleted. In contrast to previous reports, disruption of SNAP-25 or syntaxinla (SNARE) clusters was not observed following cholesterol depletion. However, in N2a cells, the colocalisation of SNAP-25 with CTXB was reduced, though this was not the case in PC 12 cells. Taken together these results suggest that cholesterol depletion may affect various raft-associated proteins and cell types in different ways. The findings from N2a cells indicate a role for lipid raft-like domains in controlling the spatial distribution of SNAP-25 on the plasma membrane. The membrane distribution of syntaxinla appears to be differently regulated from that of SNAP-25, which may have implications for the regulation of exocytosis