Pancreatic plasma membranes: promiscuous partners in membrane fusion.
Ontology highlight
ABSTRACT:
SUBMITTER: Lee EG
PROVIDER: S-EPMC1137901 | biostudies-other | 1994 Mar
REPOSITORIES: biostudies-other
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Project description:SNAP-25 (25 kDa synaptosome-associated protein) is found in cells that release neurotransmitters and hormones, and plays a central role in the fusion of secretory vesicles with the plasma membrane. SNAP-25 has been shown to interact specifically with syntaxin 1, a 35 kDa membrane protein, to mediate the fusion process. Here, we investigated whether other known syntaxin isoforms found at the plasma membrane can serve as binding partners for SNAP-25 in vivo. In our analysis, we employed rat phaeochromocytoma PC12 cells that are often used as a model of neuronal functions. We now show that these cells contain large amounts of SNAP-25, which interacts not only with syntaxin 1, but also with ubiquitous syntaxins 2, 3 and 4. The plasma membrane syntaxins appear to occupy complementary domains at the plasma membrane. In defined reactions, the ubiquitous plasma membrane syntaxin isoforms, when in binary complexes with SNAP-25, readily bound vesicular synaptobrevin to form SDS-resistant SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complexes implicated in membrane fusion. However, vesicular synaptotagmin and cytosolic complexin, both implicated in the fusion process, exhibited differential ability to interact with the SNARE complexes formed by syntaxins 1-4, suggesting that the plasma membrane syntaxins may mediate vesicle fusion events with different properties.
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Project description:TRPM7, of the transient receptor potential (TRP) family, is both an ion channel and a kinase. Previously, we showed that TRPM7 is located in the membranes of acetylcholine (ACh)-secreting synaptic vesicles of sympathetic neurons, forms a molecular complex with proteins of the vesicular fusion machinery, and is critical for stimulated neurotransmitter release. Here, we targeted pHluorin to small synaptic-like vesicles (SSLV) in PC12 cells and demonstrate that it can serve as a single-vesicle plasma membrane fusion reporter. In PC12 cells, as in sympathetic neurons, TRPM7 is located in ACh-secreting SSLVs. TRPM7 knockdown by siRNA, or abolishing channel activity by expression of a dominant negative TRPM7 pore mutant, decreased the frequency of spontaneous and voltage-stimulated SSLV fusion events without affecting large dense core vesicle secretion. We conclude that the conductance of TRPM7 across the vesicle membrane is important in SSLV fusion.
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