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1.
J Biol Chem ; 291(40): 21257-21270, 2016 Sep 30.
Article in English | MEDLINE | ID: mdl-27528604

ABSTRACT

Neurotransmitters and peptide hormones are secreted by regulated vesicle exocytosis. CAPS (also known as CADPS) is a 145-kDa cytosolic and peripheral membrane protein required for vesicle docking and priming steps that precede Ca2+-triggered vesicle exocytosis. CAPS binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and SNARE proteins and is proposed to promote SNARE protein complex assembly for vesicle docking and priming. We characterized purified soluble CAPS as mainly monomer in equilibrium with small amounts of dimer. However, the active form of CAPS bound to PC12 cell membranes or to liposomes containing PI(4,5)P2 and Q-SNARE proteins was mainly dimer. CAPS dimer formation required its C2 domain based on mutation or deletion studies. Moreover, C2 domain mutations or deletions resulted in a loss of CAPS function in regulated vesicle exocytosis, indicating that dimerization is essential for CAPS function. Comparison of the CAPS C2 domain to a structurally defined Munc13-1 C2A domain dimer revealed conserved residues involved in CAPS dimerization. We conclude that CAPS functions as a C2 domain-mediated dimer in regulated vesicle exocytosis. The unique tandem C2-PH domain of CAPS may serve as a PI(4,5)P2-triggered switch for dimerization. CAPS dimerization may be coupled to oligomeric SNARE complex assembly for vesicle docking and priming.


Subject(s)
Calcium-Binding Proteins/metabolism , Exocytosis/physiology , Protein Multimerization/physiology , Secretory Vesicles/metabolism , Animals , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , PC12 Cells , Phosphatidylinositol 4,5-Diphosphate/chemistry , Phosphatidylinositol 4,5-Diphosphate/genetics , Phosphatidylinositol 4,5-Diphosphate/metabolism , Protein Domains , Q-SNARE Proteins/chemistry , Q-SNARE Proteins/genetics , Q-SNARE Proteins/metabolism , Rats , Secretory Vesicles/chemistry , Secretory Vesicles/genetics
2.
Elife ; 3: e01879, 2014 Jan 01.
Article in English | MEDLINE | ID: mdl-24596153

ABSTRACT

Like other intracellular fusion events, the homotypic fusion of yeast vacuoles requires a Rab GTPase, a large Rab effector complex, SNARE proteins which can form a 4-helical bundle, and the SNARE disassembly chaperones Sec17p and Sec18p. In addition to these proteins, specific vacuole lipids are required for efficient fusion in vivo and with the purified organelle. Reconstitution of vacuole fusion with all purified components reveals that high SNARE levels can mask the requirement for a complex mixture of vacuole lipids. At lower, more physiological SNARE levels, neutral lipids with small headgroups that tend to form non-bilayer structures (phosphatidylethanolamine, diacylglycerol, and ergosterol) are essential. Membranes without these three lipids can dock and complete trans-SNARE pairing but cannot rearrange their lipids for fusion. DOI: http://dx.doi.org/10.7554/eLife.01879.001.


Subject(s)
Membrane Fusion , Membrane Lipids/metabolism , Membranes, Artificial , Q-SNARE Proteins/metabolism , R-SNARE Proteins/metabolism , Vacuoles/metabolism , Adenosine Triphosphatases/metabolism , Binding Sites , Membrane Lipids/chemistry , Molecular Structure , Protein Binding , Protein Conformation , Proteolipids/metabolism , Q-SNARE Proteins/chemistry , R-SNARE Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , Time Factors , Vacuoles/chemistry , Vesicular Transport Proteins/metabolism
3.
Biochemistry ; 51(17): 3606-13, 2012 May 01.
Article in English | MEDLINE | ID: mdl-22463803

ABSTRACT

Myocilin is a widely expressed protein with no known function; however, mutations in myocilin appear to manifest uniquely as ocular hypertension and the blinding disease of glaucoma. Using the protein homology/analogy recognition engine (Phyre), we find that the olfactomedin domain of myocilin is similar in sequence motif and structure to a six-blade, kelch repeat motif based on the known crystal structures of such proteins. Additionally, using sequence analysis, we identify a coiled-coil segment of myocilin with homology to human Q-SNARE proteins (inset). Using COS-7 cells expressing full-length human myocilin and a version lacking the C-terminal olfactomedin domain, we identified a membrane-associated protein complex containing myocilin by hydrodynamic analysis. The myocilin construct that included the coiled-coil but lacked the olfactomedin domain formed complexes similar to the full-length protein, indicating that the coiled-coil domain of myocilin is sufficient for myocilin binding to the large detergent-resistant complex. In human retina and retinal pigment epithelium, which express myocilin, we detected the protein in a large, sodium dodecyl sulfate-resistant, membrane-associated complex. We characterized myocilin in human tissues as either a 15 S complex with an M(r) of 405000-440000 yielding a slightly elongated globular shape similar to that of known SNARE complexes or a 6.4 S dimer with an M(r) of 108000. By identifying the Q-SNARE homology within the second coil of myocilin and documenting its participation in a SNARE-like complex, we provide evidence of a SNARE domain-containing protein associated with a human disease.


Subject(s)
Cytoskeletal Proteins/chemistry , Eye Proteins/chemistry , Glycoproteins/chemistry , Membrane Proteins/chemistry , Q-SNARE Proteins/chemistry , Structural Homology, Protein , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , Cytoskeletal Proteins/biosynthesis , Eye Proteins/biosynthesis , Glycoproteins/biosynthesis , Humans , Membrane Proteins/metabolism , Molecular Sequence Data , Multiprotein Complexes/chemistry , Protein Structure, Tertiary/physiology , Q-SNARE Proteins/physiology
4.
Proc Natl Acad Sci U S A ; 108(42): 17325-30, 2011 Oct 18.
Article in English | MEDLINE | ID: mdl-21987819

ABSTRACT

Intracellular membrane fusion requires R-SNAREs and Q-SNAREs to assemble into a four-helical parallel coiled-coil, with their hydrophobic anchors spanning the two apposed membranes. Based on the fusion properties of chemically defined SNARE- proteoliposomes, it has been proposed that the assembly of this helical bundle transduces force through the entire bilayer via the transmembrane SNARE anchor domains to drive fusion. However, an R-SNARE, Nyv1p, with a genetically engineered lipid anchor that spans half of the bilayer suffices for the fusion of isolated vacuoles, although this organelle has other R-SNAREs. To demonstrate unequivocally the fusion activity of lipid-anchored Nyv1p, we reconstituted proteoliposomes with purified lipid-anchored Nyv1p as the only protein. When these proteoliposomes were incubated with those bearing cognate Q-SNAREs, there was trans-SNARE complex assembly but, in accord with prior studies of the neuronal SNAREs, little lipid mixing. However, the addition of physiological fusion accessory proteins (HOPS, Sec17p, and Sec18p) allows lipid-anchored Nyv1p to support fusion, suggesting that trans-SNARE complex function is not limited to force transduction across the bilayers through the transmembrane domains.


Subject(s)
Membrane Fusion/physiology , SNARE Proteins/physiology , Lipid-Linked Proteins/chemistry , Lipid-Linked Proteins/physiology , Membrane Proteins/chemistry , Membrane Proteins/physiology , Protein Multimerization , Proteolipids/chemistry , Proteolipids/physiology , Q-SNARE Proteins/chemistry , Q-SNARE Proteins/physiology , R-SNARE Proteins/chemistry , R-SNARE Proteins/physiology , Recombinant Proteins/chemistry , SNARE Proteins/chemistry , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/physiology , Vacuoles/chemistry , Vacuoles/physiology
5.
J Biol Chem ; 285(50): 39359-65, 2010 Dec 10.
Article in English | MEDLINE | ID: mdl-20937838

ABSTRACT

Phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are essential for rapid SNARE-dependent fusion of yeast vacuoles and other organelles. These phosphoinositides also regulate the fusion of reconstituted proteoliposomes. The reconstituted reaction allows separate analysis of phosphoinositide-responsive subreactions: fusion with SNAREs alone, with the addition of the HOPS tethering factor, and with the further addition of the SNARE complex disassembly chaperones Sec17p and Sec18p. Using assays of membrane tethering, trans-SNARE pairing, and lipid mixing, we found that PI(3)P and PI(4,5)P(2) have distinct functions that are asymmetric with respect to R-SNARE (Nyv1p) and the 3Q-SNAREs (Vam3p, Vti1p, and Vam7p). Fusion reactions with the Q-SNAREs and R-SNARE on separate membranes showed that PI(3)P has two distinct functions. PI(3)P on Q-SNARE proteoliposomes promoted Vam7p binding and association with the other two Q-SNAREs. PI(3)P on R-SNARE proteoliposomes was recognized by the PX domain of Vam7p on Q-SNARE proteoliposomes to promote tethering, although this function could be supplanted by the tethering activity of HOPS. PI(4,5)P(2) stimulated fusion when it was on R-SNARE proteoliposomes, apposed to Q-SNARE proteoliposomes bearing PI(3)P. These functions are essential for the phosphoinositide-dependent synergy between HOPS and Sec17p/Sec18p in promoting rapid fusion.


Subject(s)
Fungi/metabolism , Gene Expression Regulation, Fungal , Phosphatidylinositol 4,5-Diphosphate/chemistry , Phosphatidylinositol Phosphates/chemistry , Q-SNARE Proteins/chemistry , Biological Transport , Cell Membrane/metabolism , Fungal Proteins , Lipids/chemistry , Liposomes/metabolism , Membrane Fusion/physiology , Models, Biological , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry
6.
Biopolymers ; 93(6): 560-70, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20108313

ABSTRACT

Since neurotransmitter releasing into the synaptic space delivers electrical signals from presynaptic neural cell to the postsynaptic cell, neurotransmitter secretion must be much orchestrated. Crowded intracellular vesicles involving neurotransmitters present a question of the how secretory vesicles fuse onto the plasma membrane in a fast synchronized fashion. Complexin is one of the most experimentally studied proteins that regulate assembly of fusogenic four-helix SNARE complex to synchronized neurotransmitter secretion. We used MD simulation to investigate the interaction of complexin with the neural SNARE complex in detail. Our results show that the SNARE complex interacts with the complexin central helix by forming salt bridges and hydrogen bonds. Complexin also can interact with the Q-SNARE complex instead of synaptobrevin to decrease the Q-SNARE flexibility. The complexin alpha-accessory helix and the C-terminal region of synaptobrevin can interact with the same region of syntaxin. Although the alpha-accessory helix aids the tight binding of the central helix to the SNARE complex, its proximity with synaptobrevin causes the destabilization of syntaxin and Sn1 helices. This study suggests that the alpha-accessory helix of complexin can be an inhibiting factor for membrane fusion by competing with synaptobrevin for binding to the Q-SNARE complex.


Subject(s)
Q-SNARE Proteins/chemistry , Adaptor Proteins, Vesicular Transport/chemistry , Amino Acid Sequence , Animals , Calcium/chemistry , Computer Simulation , Drosophila melanogaster , Humans , Mice , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Neurotransmitter Agents/chemistry , Protein Binding , Protein Structure, Secondary , Qa-SNARE Proteins/chemistry , R-SNARE Proteins/chemistry , Rats , Sequence Homology, Amino Acid
7.
J Biol Chem ; 281(7): 4495-506, 2006 Feb 17.
Article in English | MEDLINE | ID: mdl-16354670

ABSTRACT

The cellular endomembrane system requires the proper kinetic balance of synthesis and degradation of its individual components, which is maintained in part by a specific membrane fusion apparatus. In this study, we describe the molecular properties of D12, which was identified from a mouse expression library. This C-terminal anchored membrane protein has sequence similarity to both a yeast soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE), Use1p/Slt1p, and a recently identified human syntaxin 18-binding protein, p31. D12 formed a tight complex with syntaxin 18 as well as Sec22b and bound to alpha-SNAP, indicating that D12 is a SNARE protein. Although the majority of D12 is located in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments at steady state, overexpression or knockdown of D12 had no obvious effects on membrane trafficking in the early secretory pathway. However, suppression of D12 expression caused rapid appearance of lipofuscin granules, accompanied by apoptotic cell death without the apparent activation of the unfolded protein response. The typical cause of lipofuscin formation is the impaired degradation of mitochondria by lysosomal degradative enzymes, and, consistent with this, we found that proper post-Golgi maturation of cathepsin D was impaired in D12-deficient cells. This unexpected observation was supported by evidence that D12 associates with VAMP7, a SNARE in the endosomal-lysosomal pathway. Hence, we suggest that D12 participates in the degradative function of lysosomes.


Subject(s)
Endosomes/physiology , Lysosomes/physiology , Q-SNARE Proteins/physiology , Amino Acid Sequence , Animals , Apoptosis , Lipofuscin/biosynthesis , Mice , Molecular Sequence Data , NIH 3T3 Cells , Q-SNARE Proteins/chemistry , R-SNARE Proteins/physiology , RNA, Small Interfering/pharmacology
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