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/geneticsABSTRACT
The Ca(2+)-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro-scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2-dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly.
Subject(s)
Calcium-Binding Proteins/physiology , Exocytosis , Membrane Fusion/physiology , Neuroendocrine Cells/metabolism , SNARE Proteins/metabolism , Secretory Vesicles/metabolism , Vesicle-Associated Membrane Protein 2/metabolism , Animals , Biological Transport , Calcium/metabolism , Calcium/physiology , Calcium-Binding Proteins/genetics , Cell Membrane/metabolism , HEK293 Cells , Humans , Microscopy, Fluorescence , Neuroendocrine Cells/physiology , PC12 Cells , RNA Interference , RNA, Small Interfering/genetics , RatsABSTRACT
Message-specific translational control is required for gametogenesis. In yeast, the RNA-binding protein Rim4 mediates translational repression of numerous mRNAs, including the B-type cyclin CLB3, which is essential for establishing the meiotic chromosome segregation pattern. Here, we show that Rim4 forms amyloid-like aggregates and that it is the amyloid-like form of Rim4 that is the active, translationally repressive form of the protein. Our data further show that Rim4 aggregation is a developmentally regulated process. Starvation induces the conversion of monomeric Rim4 into amyloid-like aggregates, thereby activating the protein to bring about repression of translation. At the onset of meiosis II, Rim4 aggregates are abruptly degraded allowing translation to commence. Although amyloids are best known for their role in the etiology of diseases such as Alzheimer's, Parkinson's, and diabetes by forming toxic protein aggregates, our findings show that cells can utilize amyloid-like protein aggregates to function as central regulators of gametogenesis.
Subject(s)
Gametogenesis , Protein Aggregates , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Amyloidogenic Proteins/chemistry , Amyloidogenic Proteins/metabolism , Animals , Cyclin B/genetics , Gene Expression Regulation , Male , Meiosis , Mice , Mice, Inbred C57BL , Protein Aggregates/drug effects , Protein Biosynthesis , RNA-Binding Proteins/chemistry , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Sodium Dodecyl Sulfate/pharmacologyABSTRACT
Nuclear pore complexes (NPCs) are indispensable for cell function and are at the center of several human diseases. NPCs provide access to the nucleus and regulate the transport of proteins and RNA across the nuclear envelope. They are aqueous channels generated from a complex network of evolutionarily conserved proteins known as nucleporins. In this Cell Science at a Glance article and the accompanying poster, we discuss how transport between the nucleoplasm and the cytoplasm is regulated, what we currently know about the structure of individual nucleoporins and the assembled NPC, and how the cell regulates assembly and disassembly of such a massive structure. Our aim is to provide a general overview on what we currently know about the nuclear pore and point out directions of research this area is heading to.
Subject(s)
Active Transport, Cell Nucleus/physiology , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore/metabolism , RNA/metabolism , Cell Line, Tumor , Cytoplasm/metabolism , HeLa Cells , HumansABSTRACT
The nuclear pore complex (NPC) is the principal gateway for transport into and out of the nucleus. Selectivity is achieved through the hydrogel-like core of the NPC. The structural integrity of the NPC depends on ~15 architectural proteins, which are organized in distinct subcomplexes to form the >40-MDa ring-like structure. Here we present the 4.1-Å crystal structure of a heterotetrameric core element ('hub') of the Y complex, the essential NPC building block, from Myceliophthora thermophila. Using the hub structure together with known Y-complex fragments, we built the entire ~0.5-MDa Y complex. Our data reveal that the conserved core of the Y complex has six rather than seven members. Evolutionarily distant Y-complex assemblies share a conserved core that is very similar in shape and dimension, thus suggesting that there are closely related architectural codes for constructing the NPC in all eukaryotes.
Subject(s)
Nuclear Pore Complex Proteins/analysis , Nuclear Pore/ultrastructure , Sordariales/metabolism , Amino Acid Sequence , Cell Nucleus/metabolism , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Docking Simulation , Protein Structure, Tertiary , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sequence AlignmentABSTRACT
Phosphoinositides provide compartment-specific signals for membrane trafficking. Plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) is required for Ca(2+)-triggered vesicle exocytosis, but whether vesicles fuse into PIP2-rich membrane domains in live cells and whether PIP2 is metabolized during Ca(2+)-triggered fusion were unknown. Ca(2+)-dependent activator protein in secretion 1 (CAPS-1; CADPS/UNC31) and ubMunc13-2 (UNC13B) are PIP2-binding proteins required for Ca(2+)-triggered vesicle exocytosis in neuroendocrine PC12 cells. These proteins are likely effectors for PIP2, but their localization during exocytosis had not been determined. Using total internal reflection fluorescence microscopy in live cells, we identify PIP2-rich membrane domains at sites of vesicle fusion. CAPS is found to reside on vesicles but depends on plasma membrane PIP2 for its activity. Munc13 is cytoplasmic, but Ca(2+)-dependent translocation to PIP2-rich plasma membrane domains is required for its activity. The results reveal that vesicle fusion into PIP2-rich membrane domains is facilitated by sequential PIP2-dependent activation of CAPS and PIP2-dependent recruitment of Munc13. PIP2 hydrolysis only occurs under strong Ca(2+) influx conditions sufficient to activate phospholipase Cη2 (PLCη2). Such conditions reduce CAPS activity and enhance Munc13 activity, establishing PLCη2 as a Ca(2+)-dependent modulator of exocytosis. These studies provide a direct view of the spatial distribution of PIP2 linked to vesicle exocytosis via regulation of lipid-dependent protein effectors CAPS and Munc13.
Subject(s)
Calcium-Binding Proteins/metabolism , Calcium/metabolism , Nerve Tissue Proteins/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphoinositide Phospholipase C/metabolism , Transport Vesicles/metabolism , Amino Acid Sequence , Animals , Biological Transport , Calcium-Binding Proteins/genetics , Cell Membrane/metabolism , Cytoplasm/metabolism , Exocytosis , Gene Expression Regulation , Isoenzymes/genetics , Isoenzymes/metabolism , Microscopy, Video , Models, Molecular , Molecular Sequence Data , Nerve Tissue Proteins/genetics , PC12 Cells , Phosphoinositide Phospholipase C/genetics , Rats , Sequence Alignment , Signal TransductionABSTRACT
Neuropeptide and peptide hormone secretion from neural and endocrine cells occurs by Ca(2+)-triggered dense-core vesicle exocytosis. The membrane fusion machinery consisting of vesicle and plasma membrane SNARE proteins needs to be assembled for Ca(2+)-triggered vesicle exocytosis. The related Munc13 and CAPS/UNC31 proteins that prime vesicle exocytosis are proposed to promote SNARE complex assembly. CAPS binds SNARE proteins and stimulates SNARE complex formation on liposomes, but the relevance of SNARE binding to CAPS function in cells had not been determined. Here we identify a core SNARE-binding domain in CAPS as corresponding to Munc13 homology domain-1 (MHD1). CAPS lacking a single helix in MHD1 was unable to bind SNARE proteins or to support the Ca(2+)-triggered exocytosis of either docked or newly arrived dense-core vesicles. The results show that MHD1 is a SNARE-binding domain and that SNARE protein binding is essential for CAPS function in dense-core vesicle exocytosis.
