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1.
Mol Biol Cell ; 27(4): 669-85, 2016 02 15.
Article in English | MEDLINE | ID: mdl-26700321

ABSTRACT

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.


Subject(s)
Caenorhabditis elegans Proteins/chemistry , Exocytosis , Munc18 Proteins/metabolism , Neurons/physiology , Syntaxin 1/chemistry , Amino Acid Sequence , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cell Membrane/metabolism , Gene Knockdown Techniques , Molecular Sequence Data , Neurons/metabolism , PC12 Cells , Peptides/chemistry , Peptides/metabolism , Point Mutation , Protein Binding , Protein Structure, Tertiary , Protein Transport , Rats , Syntaxin 1/genetics , Syntaxin 1/metabolism
2.
J Cell Sci ; 126(Pt 11): 2361-71, 2013 Jun 01.
Article in English | MEDLINE | ID: mdl-23525015

ABSTRACT

Munc18-1 is believed to prime or stimulate SNARE-mediated membrane fusion/exocytosis through binding to the SNARE complex, in addition to chaperoning its cognate syntaxins. Nevertheless, a Munc18-1 mutant that selectively loses the priming function while retaining the syntaxin chaperoning activity has not been identified. As a consequence, the mechanism that mediates Munc18-1-dependent priming remains unclear. In the course of analyzing the functional outcomes of a variety of point mutations in domain 3a of Munc18-1, we discovered insertion mutants (K332E/K333E with insertions of 5 or 39 residues). These mutants completely lose their ability to rescue secretion whereas they effectively restore syntaxin-1 expression at the plasma membrane as well as dense-core vesicle docking in Munc18-1 and Munc18-2 double-knockdown PC12 cells. The mutants can bind syntaxin-1A in a stoichiometric manner. However, binding to the SNARE complex is impaired compared with the wild type or the hydrophobic pocket mutant (F115E). Our results suggest that the domain 3a of Munc18-1 plays a crucial role in priming of exocytosis, which is independent of its syntaxin-1 chaperoning activity and is downstream of dense-core vesicle docking. We also suggest that the priming mechanism of Munc18-1 involves its domain-3a-dependent interaction with the SNARE complex.


Subject(s)
Exocytosis/physiology , Membrane Fusion/physiology , Munc18 Proteins/metabolism , SNARE Proteins/metabolism , Animals , Munc18 Proteins/genetics , PC12 Cells , Point Mutation , Protein Structure, Tertiary , Qa-SNARE Proteins/genetics , Qa-SNARE Proteins/metabolism , Rats , SNARE Proteins/genetics
3.
Mol Biol Cell ; 22(18): 3394-409, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21795392

ABSTRACT

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.


Subject(s)
Neurotransmitter Agents/metabolism , Norepinephrine/metabolism , Protein Subunits/metabolism , Secretory Vesicles/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , Alkaline Phosphatase/metabolism , Animals , Dopamine/metabolism , Endosomes/metabolism , Gene Knockdown Techniques , Hydrogen-Ion Concentration , Membrane Fusion , Neurons/metabolism , Neuropeptide Y/metabolism , PC12 Cells , Protein Isoforms/metabolism , Protein Subunits/genetics , Protein Transport , Rats , Recombinant Fusion Proteins/metabolism , Secretory Vesicles/chemistry , Synaptotagmins/metabolism , Up-Regulation , Vacuolar Proton-Translocating ATPases/genetics
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