Abstract
SNARE proteins have been described as the effectors of fusion events in the secretory pathway more than two decades ago. The strong interactions between SNARE domains are clearly important in membrane fusion, but it is unclear whether they are involved in any other cellular processes. Here, we analyzed two classical SNARE proteins, syntaxin 1A and SNAP25. Although they are supposed to be engaged in tight complexes, we surprisingly find them largely segregated in the plasma membrane. Syntaxin 1A only occupies a small fraction of the plasma membrane area. Yet, we find it is able to redistribute the far more abundant SNAP25 on the mesoscale by gathering crowds of SNAP25 molecules onto syntaxin clusters in a SNARE-domain-dependent manner. Our data suggest that SNARE domain interactions are not only involved in driving membrane fusion on the nanoscale, but also play an important role in controlling the general organization of proteins on the mesoscale. Further, we propose these mechanisms preserve active syntaxin 1A-SNAP25 complexes at the plasma membrane.
Highlights
Molecular crowding is a basic feature of biological cells
28 Syntaxin 1A only occupies a small fraction of the plasma membrane area
Two proteins organized in such multi-protein assemblies are the classical SNAREs syntaxin 1A and SNAP25
Summary
Molecular crowding is a basic feature of biological cells. In some cellular regions, the volume occupied by macromolecules even exceeds that taken by solvating water molecules. Electrophoretic protein migration into a single, maximally crowded patch shows that half of the membrane volume is occupied by proteins (Sowers and Hackenbrock, 1981) These protein-rich regions consist of closely associated membrane domains/protein clusters and are referred to as multi-protein assemblies. Different from the SNARE-domain of syntaxin 1A, the SNARE-domains of SNAP25 are not helical in the isolated protein, yet they become helical in SNARE-complexes (Fasshauer et al, 1997) Taken together, both proteins, even though very different in structure, form crowds of similar copy number. We study the lateral distribution of syntaxin 1A and SNAP25 in the plasma membrane We find that both proteins, forming tight complexes in vitro (Brunger et al, 2019; Jahn and Fasshauer, 2012), are highly segregated in entities, and that these entities organize via SNARE-SNARE interactions. Because syntaxin 1A is able to determine the distribution of an excess of SNAP25, we propose the syntaxin 1A/SNAP25 entities largely preserve their internal architecture and interact only at their peripheries
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