The SNARE motif is essential for the formation of syntaxin clusters in the plasma membrane.

In the plasma membrane, syntaxin 1 and syntaxin 4 clusters define sites at which secretory granules and caveolae fuse, respectively. It is widely believed that lipid phases are mandatory for cluster formation, as cluster integrity depends on cholesterol. Here we report that the native lipid environment is not sufficient for correct syntaxin 1 clustering and that additional cytoplasmic protein-protein interactions, primarily involving the SNARE motif, are required. Apparently no specific cofactors are needed because i), clusters form equally well in nonneuronal cells, and ii), as revealed by nanoscale subdiffraction resolution provided by STED microscopy, the number of clusters directly depends on the syntaxin 1 concentration. For syntaxin 4 clustering the N-terminal domain and the linker region are also dispensable. Moreover, clustering is specific because in both cluster types syntaxins mutually exclude one another at endogenous levels. We suggest that the SNARE motifs of syntaxin 1 and 4 mediate specific syntaxin clustering by homooligomerization, thereby spatially separating sites for different biological activities. Thus, syntaxin clustering represents a mechanism of membrane patterning that is based on protein-protein interactions.

[1]  T. Weimbs,et al.  Syntaxins 3 and 4 are concentrated in separate clusters on the plasma membrane before the establishment of cell polarity. , 2005, Molecular biology of the cell.

[2]  D. Predescu,et al.  Cholesterol-dependent Syntaxin-4 and SNAP-23 Clustering Regulates Caveolar Fusion with the Endothelial Plasma Membrane* , 2005, Journal of Biological Chemistry.

[3]  S. Pantano,et al.  SNARE complexes and neuroexocytosis: how many, how close? , 2005, Trends in biochemical sciences.

[4]  W. Hong SNAREs and traffic. , 2005, Biochimica et biophysica acta.

[5]  Akihiro Kusumi,et al.  Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules. , 2005, Annual review of biophysics and biomolecular structure.

[6]  Thorsten Lang,et al.  A dual function for Munc‐18 in exocytosis of PC12 cells , 2005, The European journal of neuroscience.

[7]  S. Terakawa,et al.  The Activation of Exocytotic Sites by the Formation of Phosphatidylinositol 4,5-Bisphosphate Microdomains at Syntaxin Clusters* , 2005, Journal of Biological Chemistry.

[8]  Volker Westphal,et al.  Nanoscale resolution in the focal plane of an optical microscope. , 2005, Physical review letters.

[9]  Axel T Brunger,et al.  Structure and function of SNARE and SNARE-interacting proteins , 2005, Quarterly Reviews of Biophysics.

[10]  L. Chamberlain,et al.  Lipid Rafts and the Regulation of Exocytosis , 2004, Traffic.

[11]  K. Kumakura,et al.  Site of Docking and Fusion of Insulin Secretory Granules in Live MIN6 β Cells Analyzed by TAT-conjugated Anti-syntaxin 1 Antibody and Total Internal Reflection Fluorescence Microscopy* , 2004, Journal of Biological Chemistry.

[12]  Colin Rickman,et al.  High Affinity Interaction of Syntaxin and SNAP-25 on the Plasma Membrane Is Abolished by Botulinum Toxin E* , 2004, Journal of Biological Chemistry.

[13]  S. Munro Lipid Rafts Elusive or Illusive? , 2003, Cell.

[14]  S. Hell Toward fluorescence nanoscopy , 2003, Nature Biotechnology.

[15]  Marcus Dyba,et al.  Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission. , 2003, Applied optics.

[16]  T. Lang Imaging SNAREs at work in 'unroofed' cells--approaches that may be of general interest for functional studies on membrane proteins. , 2003, Biochemical Society transactions.

[17]  Heike Hering,et al.  Lipid Rafts in the Maintenance of Synapses, Dendritic Spines, and Surface AMPA Receptor Stability , 2003, The Journal of Neuroscience.

[18]  M. Eck,et al.  Homotetrameric Structure of the SNAP-23 N-terminal Coiled-coil Domain* , 2003, The Journal of Biological Chemistry.

[19]  G. Gould,et al.  The Vesicle- and Target-SNARE Proteins That Mediate Glut4 Vesicle Fusion Are Localized in Detergent-insoluble Lipid Rafts Present on Distinct Intracellular Membranes* , 2002, The Journal of Biological Chemistry.

[20]  R. Jahn,et al.  SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs , 2002, The Journal of cell biology.

[21]  R. Tsien,et al.  Partitioning of Lipid-Modified Monomeric GFPs into Membrane Microdomains of Live Cells , 2002, Science.

[22]  D. Bruns,et al.  SNAREs are concentrated in cholesterol‐dependent clusters that define docking and fusion sites for exocytosis , 2001, The EMBO journal.

[23]  G. Gould,et al.  SNARE proteins are highly enriched in lipid rafts in PC12 cells: Implications for the spatial control of exocytosis , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[24]  K. Misura,et al.  Self-association of the H3 Region of Syntaxin 1A , 2001, The Journal of Biological Chemistry.

[25]  R. Jahn,et al.  Homo- and Heterooligomeric SNARE Complexes Studied by Site-directed Spin Labeling* , 2001, The Journal of Biological Chemistry.

[26]  IMPLICATIONS FOR INTERMEDIATES IN SNARE COMPLEX ASSEMBLY , 2001 .

[27]  S. Hell,et al.  Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[28]  T. Südhof,et al.  Selective Interaction of Complexin with the Neuronal SNARE Complex , 2000, The Journal of Biological Chemistry.

[29]  R. Fairman,et al.  Structural analysis of the neuronal SNARE protein syntaxin-1A. , 2000, Biochemistry.

[30]  D. Langosch,et al.  A Conserved Membrane-spanning Amino Acid Motif Drives Homomeric and Supports Heteromeric Assembly of Presynaptic SNARE Proteins* , 2000, The Journal of Biological Chemistry.

[31]  D. Ellis,et al.  A Cell-Free System for Regulated Exocytosis in Pc12 Cells , 2000, The Journal of cell biology.

[32]  T. Südhof,et al.  A conformational switch in syntaxin during exocytosis: role of munc18 , 1999, The EMBO journal.

[33]  P. Hanson,et al.  Membrane fusion: SNAREs line up in new environment , 1998, Nature.

[34]  D. Brown,et al.  Functions of lipid rafts in biological membranes. , 1998, Annual review of cell and developmental biology.

[35]  P. Bucher,et al.  A conserved domain is present in different families of vesicular fusion proteins: a new superfamily. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[36]  S. Hell,et al.  Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. , 1994, Optics letters.

[37]  Mark K. Bennett,et al.  A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion , 1993, Cell.

[38]  R. Scheller,et al.  The syntaxin family of vesicular transport receptors , 1993, Cell.

[39]  K. Akagawa,et al.  Neuron specific expression of a membrane protein, HPC-1: tissue distribution, and cellular and subcellular localization of immunoreactivity and mRNA. , 1993, Brain research. Molecular brain research.

[40]  G J Brakenhoff,et al.  Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy. , 1992, Journal of cell science.

[41]  Deborah A. Brown,et al.  Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface , 1992, Cell.

[42]  C. Barnstable,et al.  A marker of early amacrine cell development in rat retina. , 1985, Brain research.

[43]  H. Thoenen,et al.  Relationship between NGF-mediated volume increase and "priming effect" in fast and slow reacting clones of PC12 pheochromocytoma cells. Role of cAMP. , 1983, Experimental cell research.