Munc 18a binding to syntaxin 1A and 1B isoforms defines its localization at the plasma membrane and blocks SNARE assembly in a three-hybrid system assay.
Syntaxin 1 and synaptobrevin/VAMP play an essential role in synaptic vesicle exocytosis. Two isoforms for each of these proteins, syntaxins 1A and 1B and synaptobrevin/VAMPs 1 and 2, have been found in nerve endings. Morphological and biochemical studies have revealed a characteristic colocalization and selective interactions patterns of syntaxin 1 and synaptobrevin/VAMP isoforms in nervous and endocrine systems. Moreover, studies in vitro with recombinant proteins have shown characteristic interaction patterns for each syntaxin 1-synaptobrevin/VAMP pair. The cytosolic protein Munc-18a modulates neurotransmission by inhibiting the binding of synaptobrevin/VAMP and SNAP-25 to syntaxin 1A. In the present study, several binding assays were used to demonstrate that Munc-18a significantly binds both isoforms of syntaxin 1 (syntaxins 1A and 1B). Moreover, the coexpression of Munc-18a and syntaxin 1A or syntaxin 1B in 29.3 T cells revealed syntaxin 1-dependent localization of Munc-18a in the plasma membrane. By using the three-hybrid system, we showed the inhibitory role of Munc-18a in the formation of syntaxin 1-synaptobrevin/VAMP complexes regardless of the isoforms. Since Munc-18a can bind both isoforms of syntaxin 1, the present data suggest that this protein is a general modulator of the formation of different SNARE complexes in the nerve endings.
Pérez-Brangulí
Francesc
F
Departament de Biologia Cel.lular i Anatomia Patològica, Campus de Bellvitge, Universitat de Barcelona, C/Feixa Llarga s/n, E-08907 L'Hospitalet de Llobregat, Spain.
Muhaisen
Ashraf
A
Blasi
Juan
J
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D020033:Protein Isoforms / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D050600:SNARE Proteins; D013570:Synaptic Membranes / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology*; D050827:Syntaxin 1; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011506:Proteins; D050600:SNARE Proteins; C494212:STX1A protein, human; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins