An immunohistochemical method that distinguishes free from complexed SNAP-25.
Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes composed of target (t-) SNAREs syntaxin and SNAP-25 and vesicle SNARE synaptobrevin play an essential role in neurosecretion. It is hypothesized that a transient intermediate complex between the t-SNAREs is formed during the assembly of the ternary complex. The existence of the t-SNARE binary complexes in vivo, however, has not been demonstrated. By using an affinity absorption scheme with preformed syntaxin-SNAP-25 complexes, we isolated antibodies capable of distinguishing free SNAP-25 from those associated with syntaxin. By semiquantitative immunohistochemistry, we estimated that, in cultured cerebellar neurons, the majority of SNAP-25 existed as complexes. Compared with the cultured neurons, PC12 cells expressed significantly less syntaxin, and we found that SNAP-25 was primarily in free forms. In contrast, a PC12 line that stably expressed a recombinant syntaxin showed a marked increase in SNAP-25 complexes. By using fluorescence resonance energy transfer (FRET) techniques, we observed FRET between cyan fluorescence protein-syntaxin and yellow fluorescence protein-SNAP-25 fusion proteins expressed in COS-7 and PC12 cells, suggesting a physiological interaction between syntaxin and SNAP-25. Our results demonstrate that, unlike what was previously hypothesized, syntaxin and SNAP-25 exist preferably as stable binary complexes in neurons. These findings offer novel insight into the mechanisms underlying the initiation and regulation of SNARE complex assembly.
Xiao
Jingnan
J
Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
Xia
Zongping
Z
Pradhan
Anuradha
A
Zhou
Qiong
Q
Liu
Yuechueng
Y
D000818:Animals; D000831:Animals, Newborn; D000906:Antibodies / Q000276:immunology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D002522:Chlorocebus aethiops; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry / Q000379:methods*; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation / Q000276:immunology; D020932:Nerve Growth Factor / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D050825:Synaptosomal-Associated Protein 25; D013997:Time Factors
D000906:Antibodies; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D049452:Green Fluorescent Proteins; D020932:Nerve Growth Factor