Conformational states of syntaxin-1 govern the necessity of N-peptide binding in exocytosis of PC12 cells and Caenorhabditis elegans.

Syntaxin-1 is the central SNARE protein for neuronal exocytosis. It interacts with Munc18-1 through its cytoplasmic domains, including the N-terminal peptide (N-peptide). Here we examine the role of the N-peptide binding in two conformational states ("closed" vs. "open") of syntaxin-1 using PC12 cells and Caenorhabditis elegans. We show that expression of "closed" syntaxin-1A carrying N-terminal single point mutations (D3R, L8A) that perturb interaction with the hydrophobic pocket of Munc18-1 rescues impaired secretion in syntaxin-1-depleted PC12 cells and the lethality and lethargy of unc-64 (C. elegans orthologue of syntaxin-1)-null mutants. Conversely, expression of the "open" syntaxin-1A harboring the same mutations fails to rescue the impairments. Biochemically, the L8A mutation alone slightly weakens the binding between "closed" syntaxin-1A and Munc18-1, whereas the same mutation in the "open" syntaxin-1A disrupts it. Our results reveal a striking interplay between the syntaxin-1 N-peptide and the conformational state of the protein. We propose that the N-peptide plays a critical role in intracellular trafficking of syntaxin-1, which is dependent on the conformational state of this protein. Surprisingly, however, the N-peptide binding mode seems dispensable for SNARE-mediated exocytosis per se, as long as the protein is trafficked to the plasma membrane.

Calcium-dependent activator protein for secretion 1 (CAPS1) binds to syntaxin-1 in a distinct mode from Munc13-1.

Calcium-dependent activator protein for secretion 1 (CAPS1) is a multidomain protein containing a Munc13 homology domain 1 (MHD1). Although CAPS1 and Munc13-1 play crucial roles in the priming stage of secretion, their functions are non-redundant. Similar to Munc13-1, CAPS1 binds to syntaxin-1, a key t-SNARE protein in neurosecretion. However, whether CAPS1 interacts with syntaxin-1 in a similar mode to Munc13-1 remains unclear. Here, using yeast two-hybrid assays followed by biochemical binding experiments, we show that the region in CAPS1 consisting of the C-terminal half of the MHD1 with the corresponding C-terminal region can bind to syntaxin-1. Importantly, the binding mode of CAPS1 to syntaxin-1 is distinct from that of Munc13-1; CAPS1 binds to the full-length of cytoplasmic syntaxin-1 with preference to its "open" conformation, whereas Munc13-1 binds to the first 80 N-terminal residues of syntaxin-1. Unexpectedly, the majority of the MHD1 of CAPS1 is dispensable, whereas the C-terminal 69 residues are crucial for the binding to syntaxin-1. Functionally, a C-terminal truncation of 69 or 134 residues in CAPS1 abolishes its ability to reconstitute secretion in permeabilized PC12 cells. Our results reveal a novel mode of binding between CAPS1 and syntaxin-1, which play a crucial role in neurosecretion. We suggest that the distinct binding modes between CAPS1 and Munc13-1 can account for their non-redundant functions in neurosecretion. We also propose that the preferential binding of CAPS1 to open syntaxin-1 can contribute to the stabilization of the open state of syntaxin-1 during its transition from "closed" state to the SNARE complex formation.

RalA GTPase tethers insulin granules to L- and R-type calcium channels through binding α2 δ-1 subunit.

RalA GTPase has been implicated in the regulated delivery of exocytotic vesicles to the plasma membrane (PM) in mammalian cells. We had reported that RalA regulates biphasic insulin secretion, which we have now determined to be contributed by RalA direct interaction with voltage-gated calcium (Cav ) channels. RalA knockdown (KD) in INS-1 cells and primary rat ß-cells resulted in a reduction in Ca(2+) currents arising specifically from L-(Cav 1.2 and Cav 1.3) and R-type (Cav 2.3) Ca(2+) channels. Restoration of RalA expression in RalA KD cells rescued these defects in Ca(2+) currents. RalA co-immunoprecipitated with the Cav α2 δ-1 auxiliary subunit known to bind the three Cav s. Moreover, the functional molecular interactions between Cav α2 δ-1 and RalA on the PM shown by total internal reflection fluorescent microscopy/FRET analysis could be induced by glucose stimulation. KD of RalA inhibited trafficking of α2 δ-1 to insulin granules without affecting the localization of the other Cav subunits. Furthermore, we confirmed that RalA and α2 δ-1 functionally interact since RalA KD-induced inhibition of Cav currents could not be recovered by RalA when α2 δ-1 was simultaneously knocked down. These data provide a mechanism for RalA function in insulin secretion, whereby RalA binds α2 δ-1 on insulin granules to tether these granules to PM Ca(2+) channels. This acts as a chaperoning step prior to and in preparation for sequential assembly of exocyst and excitosome complexes that mediate biphasic insulin secretion.

Vacuolar H(+)-ATPase subunits Voa1 and Voa2 cooperatively regulate secretory vesicle acidification, transmitter uptake, and storage.

The Vo sector of the vacuolar H(+)-ATPase is a multisubunit complex that forms a proteolipid pore. Among the four isoforms (a1-a4) of subunit Voa, the isoform(s) critical for secretory vesicle acidification have yet to be identified. An independent function of Voa1 in exocytosis has been suggested. Here we investigate the function of Voa isoforms in secretory vesicle acidification and exocytosis by using neurosecretory PC12 cells. Fluorescence-tagged and endogenous Voa1 are primarily localized on secretory vesicles, whereas fluorescence-tagged Voa2 and Voa3 are enriched on the Golgi and early endosomes, respectively. To elucidate the functional roles of Voa1 and Voa2, we engineered PC12 cells in which Voa1, Voa2, or both are stably down-regulated. Our results reveal significant reductions in the acidification and transmitter uptake/storage of dense-core vesicles by knockdown of Voa1 and more dramatically of Voa1/Voa2 but not of Voa2. Overexpressing knockdown-resistant Voa1 suppresses the acidification defect caused by the Voa1/Voa2 knockdown. Unexpectedly, Ca(2+)-dependent peptide secretion is largely unaffected in Voa1 or Voa1/Voa2 knockdown cells. Our data demonstrate that Voa1 and Voa2 cooperatively regulate the acidification and transmitter uptake/storage of dense-core vesicles, whereas they might not be as critical for exocytosis as recently proposed.