Munc18-dependent and -independent clustering of syntaxin in the plasma membrane of cultured endocrine cells

Syntaxin helps in catalyzing membrane fusion during exocytosis. It also forms clusters in the plasma membrane, where both its transmembrane and SNARE domains are thought to homo-oligomerize. To study syntaxin clustering in live PC12 cells, we labeled granules with neuropeptide-Y-mCherry and syntaxin clusters with syntaxin-1a green fluorescent protein (GFP). Abundant clusters appeared under total internal reflection (TIRF) illumination, and some of them associated with granules (“on-granule clusters”). Syntaxin-1a-GFP or its mutants were expressed at low levels and competed with an excess of endogenous syntaxin for inclusion into clusters. On-granule inclusion was diminished by mutations known to inhibit binding to Munc18-1 in vitro. Knock-down of Munc18-1 revealed Munc18-dependent and -independent on-granule clustering. Clustering was inhibited by mutations expected to break salt bridges between syntaxin’s Hb and SNARE domains and was rescued by additional mutations expected to restore them. Most likely, syntaxin is in a closed conformation when it clusters on granules, and its SNARE and Hb domains approach to within atomic distances. Pairwise replacements of Munc18-contacting residues with alanines had only modest effects, except that the pair R114A/I115A essentially abolished on-granule clustering. In summary, an on-granule cluster arises from the specific interaction between a granule and a dense cluster of syntaxin-Munc18-1 complexes. Off-granule clusters, by contrast, were resistant to even the strongest mutations we tried and required neither Munc18-1 nor the presence of a SNARE domain. They may well form through the nonstoichiometric interactions with membrane lipids that others have observed in cell-free systems.

The mesoscale organization of syntaxin 1A and SNAP25 is determined by SNARE-SNARE-interactions

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 suggests 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 this mechanisms preserves active syntaxin 1A-SNAP25 complexes at the plasma membrane.

Syntaxin 1A Gene Is Negatively Regulated in a Cell/Tissue Specific Manner by YY1 Transcription Factor, Which Binds to the −183 to −137 Promoter Region Together with Gene Silencing Factors Including Histone Deacetylase

The HPC-1/syntaxin 1A (Stx1a) gene, which is involved in synaptic transmission and neurodevelopmental disorders, is a TATA-less gene with several transcription start sites. It is activated by the binding of Sp1 and acetylated histone H3 to the −204 to +2 core promoter region (CPR) in neuronal cell/tissue. Furthermore, it is depressed by the association of class 1 histone deacetylases (HDACs) to Stx1a–CPR in non-neuronal cell/tissue. To further clarify the factors characterizing Stx1a gene silencing in non-neuronal cell/tissue not expressing Stx1a, we attempted to identify the promoter region forming DNA–protein complex only in non-neuronal cells. Electrophoresis mobility shift assays (EMSA) demonstrated that the −183 to −137 OL2 promoter region forms DNA–protein complex only in non-neuronal fetal rat skin keratinocyte (FRSK) cells which do not express Stx1a. Furthermore, the Yin-Yang 1 (YY1) transcription factor binds to the −183 to −137 promoter region of Stx1a in FRSK cells, as shown by competitive EMSA and supershift assay. Chromatin immunoprecipitation assay revealed that YY1 in vivo associates to Stx1a–CPR in cell/tissue not expressing Stx1a and that trichostatin A treatment in FRSK cells decreases the high-level association of YY1 to Stx1a-CPR in default. Reporter assay indicated that YY1 negatively regulates Stx1a transcription. Finally, mass spectrometry analysis showed that gene silencing factors, including HDAC1, associate onto the −183 to −137 promoter region together with YY1. The current study is the first to report that Stx1a transcription is negatively regulated in a cell/tissue-specific manner by YY1 transcription factor, which binds to the −183 to −137 promoter region together with gene silencing factors, including HDAC.