Autoinhibition of Munc18-1 modulates synaptobrevin binding and helps to enable Munc13-dependent regulation of membrane fusion
Ewa Sitarska,
Junjie Xu,
Seungmee Park,
Xiaoxia Liu,
Bradley Quade,
Karolina Stepien,
Kyoko Sugita,
Chad A Brautigam,
Shuzo Sugita,
Josep Rizo
Affiliations
Ewa Sitarska
Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States; Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States; Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States
Junjie Xu
Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States; Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States; Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States
Seungmee Park
Department of Physiology, University of Toronto, Toronto, Canada
Xiaoxia Liu
Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States; Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States; Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States
Bradley Quade
Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States; Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States; Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States
Karolina Stepien
Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States; Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States; Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States
Kyoko Sugita
Department of Physiology, University of Toronto, Toronto, Canada
Chad A Brautigam
Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States; Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, United States
Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States; Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States; Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States
Munc18-1 orchestrates SNARE complex assembly together with Munc13-1 to mediate neurotransmitter release. Munc18-1 binds to synaptobrevin, but the relevance of this interaction and its relation to Munc13 function are unclear. NMR experiments now show that Munc18-1 binds specifically and non-specifically to synaptobrevin. Specific binding is inhibited by a L348R mutation in Munc18-1 and enhanced by a D326K mutation designed to disrupt the ‘furled conformation’ of a Munc18-1 loop. Correspondingly, the activity of Munc18-1 in reconstitution assays that require Munc18-1 and Munc13-1 for membrane fusion is stimulated by the D326K mutation and inhibited by the L348R mutation. Moreover, the D326K mutation allows Munc13-1-independent fusion and leads to a gain-of-function in rescue experiments in Caenorhabditis elegans unc-18 nulls. Together with previous studies, our data support a model whereby Munc18-1 acts as a template for SNARE complex assembly, and autoinhibition of synaptobrevin binding contributes to enabling regulation of neurotransmitter release by Munc13-1.
