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1.
J Biol Chem ; 289(48): 33617-28, 2014 Nov 28.
Article in English | MEDLINE | ID: mdl-25326390

ABSTRACT

Munc18-1 plays essential dual roles in exocytosis: (i) stabilizing and trafficking the central SNARE protein, syntaxin-1 (i.e. chaperoning function), by its domain-1; and (ii) priming/stimulating exocytosis by its domain-3a. Here, we examine whether or not domain-3a also plays a significant role in the chaperoning of syntaxin-1 and, if so, how these dual functions of domain-3a are regulated. We demonstrate that introduction of quintuple mutations (K332E/K333E/P335A/Q336A/Y337L) in domain-3a of Munc18-1 abolishes its ability to bind syntaxin-1 and fails to rescue the level and trafficking of syntaxin-1 as well as to restore exocytosis in Munc18-1/2 double knockdown cells. By contrast, a quadruple mutant (K332E/K333E/Q336A/Y337L) sparing the Pro-335 residue retains all of these capabilities. A single point mutant of P335A reduces the ability to bind syntaxin-1 and rescue syntaxin-1 levels. Nonetheless, it surprisingly outperforms the wild type in the rescue of exocytosis. However, when additional mutations in the neighboring residues are combined with P335A mutation (K332E/K333E/P335A, P335A/Q336A/Y337L), the ability of the Munc18-1 variants to chaperone syntaxin-1 and to rescue exocytosis is strongly impaired. Our results indicate that residues from Lys-332 to Tyr-337 of domain-3a are intimately tied to the chaperoning function of Munc18-1. We also propose that Pro-335 plays a pivotal role in regulating the balance between the dual functions of domain-3a. The hinged conformation of the α-helix containing Pro-335 promotes the syntaxin-1 chaperoning function, whereas the P335A mutation promotes its priming function by facilitating the α-helix to adopt an extended conformation.


Subject(s)
Exocytosis/physiology , Molecular Chaperones/metabolism , Munc18 Proteins/metabolism , Amino Acid Substitution , Gene Knockdown Techniques , HEK293 Cells , Humans , Molecular Chaperones/genetics , Munc18 Proteins/genetics , Mutation, Missense , Proline/genetics , Proline/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , 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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