Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 4 de 4
Filter
Add more filters










Database
Language
Publication year range
1.
J Cell Sci ; 128(10): 1946-60, 2015 May 15.
Article in English | MEDLINE | ID: mdl-25795302

ABSTRACT

Understanding how Munc18 proteins govern exocytosis is crucial because mutations of this protein cause severe secretion deficits in neuronal and immune cells. Munc18-2 has indispensable roles in the degranulation of mast cell, partly by binding and chaperoning a subset of syntaxin isoforms. However, the key syntaxin that, crucially, participates in the degranulation ­ whose levels and intracellular localization are regulated by Munc18-2 ­ remains unknown. Here, we demonstrate that double knockdown of Munc18-1 and Munc-2 in mast cells results in greatly reduced degranulation accompanied with strikingly compromised expression levels and localization of syntaxin-3. This phenotype is fully rescued by wild-type Munc18 proteins but not by the K46E, E59K and K46E/E59K mutants of Munc-18 domain 1, each of which exhibits completely abolished binding to 'closed' syntaxin-3. Furthermore, knockdown of syntaxin-3 strongly impairs degranulation. Collectively, our data argue that residues Lys46 and Glu59 of Munc18 proteins are indispensable for mediating the interaction between Munc18 and closed syntaxin-3, which is essential for degranulation by chaperoning syntaxin-3. Our results also indicate that the functional contribution of these residues differs between immune cell degranulation and neuronal secretion.


Subject(s)
Mast Cells/metabolism , Molecular Chaperones/metabolism , Munc18 Proteins/metabolism , Protein Binding/genetics , Qa-SNARE Proteins/metabolism , Animals , Exocytosis , Humans , Rats
2.
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
3.
J Biol Chem ; 288(32): 23050-63, 2013 Aug 09.
Article in English | MEDLINE | ID: mdl-23801330

ABSTRACT

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.


Subject(s)
Calcium-Binding Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neurosecretion/physiology , Syntaxin 1/metabolism , Animals , Calcium-Binding Proteins/genetics , HEK293 Cells , Humans , Mice , Nerve Tissue Proteins/genetics , PC12 Cells , Peptide Mapping , Protein Binding/physiology , Protein Stability , Protein Structure, Tertiary , Rats , SNARE Proteins/genetics , SNARE Proteins/metabolism , Saccharomyces cerevisiae/genetics , Syntaxin 1/genetics , Two-Hybrid System Techniques
4.
Proc Natl Acad Sci U S A ; 110(12): 4610-5, 2013 Mar 19.
Article in English | MEDLINE | ID: mdl-23487749

ABSTRACT

The function of the Munc18-1 protein hydrophobic pocket, which interacts with the syntaxin-1 N-terminal peptide, has been highly controversial in neurosecretion. Recent analysis of patients with familial hemophagocytic lymphohistiocytosis type 5 has identified the E132A mutation in the hydrophobic pocket of Munc18-2, prompting us to examine the role of this region in the context of immune cell secretion. Double knockdown of Munc18-1 and Munc18-2 in RBL-2H3 mast cells eliminates both IgE-dependent and ionomycin-induced degranulation and causes a significant reduction in syntaxin-11 without altering expressions of the other syntaxin isoforms examined. These phenotypes were effectively rescued on reexpression of wild-type Munc18-1 or Munc18-2 but not the mutants (F115E, E132A, and F115E/E132A) in the hydrophobic pocket of Munc18. In addition, these mutants show that they are unable to directly interact with syntaxin-11, as tested through protein interaction experiments. Our results demonstrate the crucial roles of the hydrophobic pocket of Munc18 in mast cell degranulation, which include the regulation of syntaxin-11. We also suggest that the functional importance of this region is significantly different between neuronal and immune cell exocytosis.


Subject(s)
Cell Degranulation , Lymphohistiocytosis, Hemophagocytic/metabolism , Mast Cells/metabolism , Munc18 Proteins/metabolism , Amino Acid Substitution , Animals , Calcium Ionophores/pharmacology , HEK293 Cells , Humans , Hydrophobic and Hydrophilic Interactions , Immunoglobulin E/metabolism , Immunoglobulin E/pharmacology , Ionomycin/pharmacology , Lymphohistiocytosis, Hemophagocytic/genetics , Lymphohistiocytosis, Hemophagocytic/pathology , Mast Cells/pathology , Mice , Munc18 Proteins/genetics , Mutation, Missense , PC12 Cells , Protein Structure, Tertiary , Qa-SNARE Proteins/genetics , Qa-SNARE Proteins/metabolism , Rats
SELECTION OF CITATIONS
SEARCH DETAIL
...