Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo.
Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced approximately 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca(2+)-regulated secretory events.
Department of Molecular and Integrative Physiology, The Medical School, University of Michigan, 7808 Medical Sciences II Building, Ann Arbor, MI 48109, USA.
Jiang
J
Liu
Stephen A
SA
Ernst
Svetlana E
SE
Gladycheva
Yue Ying F
YY
Lee
Stephen I
SI
Lentz
Chi S
CS
Ho
Quanwen
Q
Li
Edward L
EL
Stuenkel
D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019439:Chromaffin Cells / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000737:chemistry; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D051938:Munc18 Proteins; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D050827:Syntaxin 1; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000378:metabolism
D000954:Antigens, Surface; D001426:Bacterial Proteins; D015220:Calcium Channels; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; C497937:STXBP1 protein, human; C497938:Stxbp1 protein, mouse; C497939:Stxbp1 protein, rat; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D002118:Calcium