Abstract
Membrane fusion for exocytosis is mediated by SNAREs, forming trans-ternary complexes to bridge vesicle and target membranes. There is an array of accessory proteins that directly interact with and regulate SNARE proteins. PRIP (phospholipase C-related but catalytically inactive protein) is likely one of these proteins; PRIP, consisting of multiple functional modules including pleckstrin homology and C2 domains, inhibited exocytosis, probably via the binding to membrane phosphoinositides through the pleckstrin homology domain. However, the roles of the C2 domain have not yet been investigated. In this study, we found that the C2 domain of PRIP directly interacts with syntaxin 1 and SNAP-25 but not with VAMP2. The C2 domain promoted PRIP to co-localize with syntaxin 1 and SNAP-25 in PC12 cells. The binding profile of the C2 domain to SNAP-25 was comparable with that of synaptotagmin I, and PRIP inhibited synaptotagmin I in binding to SNAP-25 and syntaxin 1. It was also shown that the C2 domain was required for PRIP to suppress SDS-resistant ternary SNARE complex formation and inhibit high K(+)-induced noradrenalin release from PC12 cells. These results suggest that PRIP inhibits regulated exocytosis through the interaction of its C2 domain with syntaxin 1 and SNAP-25, potentially competing with other SNARE-binding, C2 domain-containing accessory proteins such as synaptotagmin I and by directly inhibiting trans-SNARE complex formation.
Highlights
PRIP inhibits exocytosis, but the underlying mechanism is unknown
These results suggest that PRIP inhibits regulated exocytosis through the interaction of its C2 domain with syntaxin 1 and synaptosome-associated protein of 25 kDa (SNAP-25), potentially competing with other SNAREbinding, C2 domain-containing accessory proteins such as syn
Strong lipid binding as a positive control was observed as follows: PtdIns[4,5]P2 binding by the pleckstrin homology (PH) domain of phospholipase C (PLC)-␦1 and phosphatidylserine binding by the C2A domain of synaptotagmin I (Syt-C2A), which was abolished in the absence of Ca2ϩ (Fig. 2)
Summary
Results: PRIP interacts with syntaxin-1 and SNAP-25 through its C2 domain and inhibits SNARE complex formation. PRIP (phospholipase C-related but catalytically inactive protein) is likely one of these proteins; PRIP, consisting of multiple functional modules including pleckstrin homology and C2 domains, inhibited exocytosis, probably via the binding to membrane phosphoinositides through the pleckstrin homology domain. It was shown that the C2 domain was required for PRIP to suppress SDS-resistant ternary SNARE complex formation and inhibit high K؉-induced noradrenalin release from PC12 cells. These results suggest that PRIP inhibits regulated exocytosis through the interaction of its C2 domain with syntaxin 1 and SNAP-25, potentially competing with other SNAREbinding, C2 domain-containing accessory proteins such as syn-. We propose that PRIP is a new member of C2 domain-containing proteins that regulate membrane traffic by its negative regulation of exocytosis through a combination of PtdIns[4,5]P2 binding by its PH domain and t-SNARE binding by its C2 domain
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