A Post-Docking Role of Synaptotagmin 1-C2B Domain Bottom Residues R398/399 in Mouse Chromaffin Cells

Abstract

Synaptotagmin-1 (Syt1) is the principal Ca 2ϩ sensor for vesicle fusion and is also essential for vesicle docking in chromaffin cells. Docking depends on interactions of the Syt1-C2B domain with the t-SNARE SNAP25/Syntaxin1 complex and/or plasma membrane phospholipids. Here, we investigated the role of the positively charged "bottom" region of the C2B domain, proposed to help crosslink membranes, in vesicle docking and secretion in mouse chromaffin cells and in cell-free assays. We expressed a double mutation shown previously to interfere with lipid mixing between proteoliposomes and with synaptic transmission, Syt1-R398/399Q (RQ), in syt1 null mutant cells. Ultrastructural morphometry revealed that Syt1-RQ fully restored the docking defect observed previously in syt1 null mutant cells, similar to wild type Syt1 (Syt1-wt). Small unilamellar lipid vesicles (SUVs) that contained the v-SNARE Synaptobrevin2 and Syt1-R398/399Q also docked to t-SNARE-containing giant vesicles (GUVs), similar to Syt1-wt. However, unlike Syt1-wt, Syt1-RQinduced docking was strictly PI(4,5)P 2 -dependent. Unlike docking, neither synchronized secretion in chromaffin cells nor Ca 2ϩ -triggered SUV-GUV fusion was restored by the Syt1 mutants. Finally, overexpressing the RQ-mutant in wild type cells produced no effect on either docking or secretion. We conclude that the positively charged bottom region in the C2B domain-and, by inference, Syt1-mediated membrane crosslinking-is required for triggering fusion, but not for docking. Secretory vesicles dock by multiple, PI(4,5)P 2 -dependent and PI(4,5)P 2 -independent mechanisms. The R398/399 mutations selectively disrupt the latter and hereby help to discriminate protein regions involved in different aspects of Syt1 function in docking and fusion.

C2‐domain containing calcium sensors in neuroendocrine secretion

“…The functions of syt‐1 in vesicle docking and fusion triggering are distinct, as shown by mutations (R398,399Q) that selectively impair the later (Kedar et al . ). However, a recent study only found minor impairments of docking in central synapses of syt‐1 knockout mice (Imig et al .…”

“…It also plays fundamental roles in the docking of secretory vesicles to the plasma membrane in neuroendocrine cells (de Wit et al 2009;Mohrmann et al 2013), a function already suggested over a decade earlier (Perin 1994). The functions of syt-1 in vesicle docking and fusion triggering are distinct, as shown by mutations (R398,399Q) that selectively impair the later (Kedar et al 2015). However, a recent study only found minor impairments of docking in central synapses of syt-1 knockout mice (Imig et al 2014), and indeed, the mechanism of docking appears to differ fundamentally between neuroendocrine cells and synapses (Man et al 2015).…”

Mechanism of neurotransmitter release coming into focus

“…In this model, the Cpx1 accessory helix becomes unstructured due to steric clashes as binding of the C 2 B domains to the two membranes and C‐terminal zippering of the SNARE complex bring the membranes together . This model also accounts for the strong impairment of release caused by mutations in R398‐R399 together or individually, and for functional data suggesting that these residues are important for triggering fusion . Note in this context that both arginines participate in the primary interface defined by X‐ray crystallography, but R398 does not appear to make strong contacts and mutation of R398‐R399 did not significantly affect the co‐immunoprecipitation of Syt1 with the SNAREs, suggesting that R398‐R399 interact with another key target [e.g., the membranes; Fig.…”

Complexin suppresses spontaneous exocytosis by capturing the membrane-proximal regions of VAMP2 and SNAP25

“…syntaxin1A 1-288 sequence: MKDRTQELRTAKDSDDDDDVTVTVDRDRFMDEFFEQVEEIRGFIDKIAENVEEVKRK HSAILASPNPDEKTKEELEELMSDIKKTANKVRSKLKSIEQSIEQEEGLNRSSADLRIR KTQHSTLSRKFVEVMSEYNATQSDYRERCKGRIQRQLEITGRTTTSEELEDMLESGNP AIFASGIIMDSSISKQALSEIETRHSEIIKLENSIRELHDMFMDMAMLVESQGEMIDRIE YNVEHAVDYVERAVSDIKKAVKYQSKARRKKIMIIICCVILGIIIASTIGGIFG His 6 -SNAP25B 1-206 sequence: MRGSHHHHHHGSMAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDA GIRTLVMLDEQGEQLERIEEGMDQINKDMKEAEKNLTDLGKFCGLCVCPCNKLKSSD AYKKAWGNNQDGVVASQPARVVDEREQMAISGGFIRRVTNDARENEMDENLEQVS GIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSG t-SNARE complex with C-terminally truncated SNAP25 (1-200/1-194): pTW34 (Parlati et al, 1999) The amino acid positions and the hydrophobic layers (highlighted in red) of the SNAP25 SNARE motif are numbered and indicated above the sequences. VAMP2 (1-116): A DNA construct encoding GST-tagged mouse VAMP2 (pSK28) was described previously (Kedar et al, 2015). The GST-tag was removed by thrombin cleavage resulting in a short N-terminal extension highlighted in red.…”

“…t-SNARE expression and purification was performed as described previously (Parlati et al, 1999). VAMP2 was expressed and purified as described previously (Kedar et al, 2015). Synaptotagmin1 containing a C-terminal twin-strep-tag was expressed and purified as described previously for the His 6 -tagged protein (pLM6) (Malsam et al, 2012) with the following modifications: imidazole was omitted from all buffers and 50 mM biotin was used for protein elution from Strep-Tactin XT superflow high capacity resin.…”