SNARE proteins and 'membrane rafts'

Abstract

The original 'lipid raft' hypothesis proposed that lipid-platforms/rafts form in the exoplasmic plasmalemmal leaflet by tight clustering of sphingolipids and cholesterol. Their physical state, presumably similar to liquid-ordered phases in model membranes, would confer detergent resistance to rafts and enriched proteins therein. Based on this concept, detergent resistant membranes (DRMs) from solubilized cells were considered to reflect pre-existing 'lipid rafts' in live cells. To date, more than 200 proteins were found in DRMs including also members of the SNARE superfamily, which are small membrane proteins involved in intracellular fusion steps. Their raft association indicates that they are not uniformly distributed, and, indeed, microscopic studies revealed that SNAREs concentrate in submicrometre-sized, cholesterol-dependent clusters at which vesicles fuse. However, the idea that SNARE clusters are 'lipid rafts' was challenged, as they do not colocalize with raft markers, and SNAREs are excluded from liquid-ordered phases in model membranes. Independent from this disagreement, in recent years the solubilization criterion has been criticized for several reasons, calling for a more exact definition of rafts. At a recent consensus on a revised raft model, the term 'lipid rafts' was replaced by 'membrane rafts' that were defined as 'small (10-200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes'. As a result, after dismissing the terms 'detergent resistant' and 'liquid-ordered', it now appears that SNARE clusters are bona fide 'membrane rafts'.

Figure 1. Structure and localization of SNAREs involved in exocytosis

SNAP-25 and SNAP-23 possess two SNARE motifs connected by a linker region which is attached to the membrane via 4 (SNAP-25) or 5 (SNAP-23) palmitoyl anchors. All other exocytotic SNAREs are associated with the membrane via a TMR that is C-terminally attached to their SNARE motif, and only syntaxins have in addition an independently folded N-terminal domain connected to the SNARE motif via a linker region. According to the SNARE hypothesis, membrane fusion is mediated by complex formation between the SNARE motifs of the SNAREs localized on the opposed membranes destined to fuse.

Figure 2. Nanoscale resolution STED microscopic analysis of SNAP-25 organization

Confocal and STED micrograph of a plasma membrane sheet immunostained for SNAP-25. Encircled regions show linearly deconvolved data. Conventional resolution provided by confocal microscopy (A) is not sufficient to resolve individual SNAP-25 clusters present at high density. STED microscopic resolution (B) reveals that individual clusters are smaller than 60 nm average size (published with permission from Willig et al. 2006; Copyright Institute of Physics Publishing).