Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo.

摘要: Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor essential to neurotransmitter release, in isolation forms closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of H3 domain cognate SNAREs. Munc18-1, member Sec1/Munc18 family, binds syntaxin1A, stabilizing this conformation. We used fluorescence resonance energy transfer (FRET) characterize Munc18-1/syntaxin1A intact cells. Enhanced cyan fluorescent protein-Munc18-1 and citrine variant enhanced yellow protein-syntaxin1A, or mutants these proteins, were expressed as donor acceptor pairs human embryonic kidney HEK293-S3 adrenal chromaffin Apparent FRET efficiency was measured using two independent approaches complementary results that unambiguously verified provided spatial map efficiency. In addition, protein-syntaxin1A colocalized Golgi marker exhibited at early expression times, whereas strong plasma membrane colocalization, similar values, apparent later times. Trafficking syntaxin1A dependent on presence Munc18-1. Both syntaxin1A(L165A/E166A), constitutively open mutant, syntaxin1A(I233A), point demonstrated reduced approximately 70% from control. contrast, mutant syntaxin1A(I209A) had no effect. By phosphomimetic we also established Ser-313, Munc18-1 kinase C phosphorylation site, Thr-574, cyclin-dependent 5 regulate conclude imaging living cells may allow correlated regulation interactions Ca(2+)-regulated secretory events.