The N-terminal peptide of the syntaxin Tlg2p modulates binding of its closed conformation to Vps45p
The N-terminal peptide of the syntaxin Tlg2p modulates binding of its closed conformation to Vps45p
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DOI：
10.1073/pnas.0902976106
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发表时间：
2009-08-25
影响因子：
11.1
通讯作者：
Munson, Mary
中科院分区：
文献类型：
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作者：
Furgason, Melonnie L. M.;MacDonald, Chris;Munson, Mary
Sec 1/Munc 18（SM）蛋白家族通过与单个SNARE蛋白和组装的SNARE复合物相互作用来调节细胞内运输。揭示这种调节的共同机制一直具有挑战性，主要是因为SM蛋白和它们的同源突触融合蛋白型SNARE之间观察到的多种相互作用模式。这些模式包括SM与突触融合蛋白的闭合构象结合、与突触融合蛋白的N-末端肽结合、与组装的SNARE复合物结合和/或与非突触融合蛋白SNARE结合。SM蛋白Vps 45 p，其调节酵母中的内体运输，结合突触融合蛋白Tlg 2 p的保守N-末端肽。我们使用尺寸排阻色谱法和定量荧光凝胶迁移率变动分析，以揭示一个额外的结合位点，不需要Tlg 2 p N-肽。Tlg 2 p突变体和截短的表征表明，该结合位点对应于Tlg 2 p的闭合构象。此外，Tlg 2 p N-肽与闭合构象竞争结合，表明SM-突触融合蛋白相互作用在SNARE组装和膜融合中的基本调节机制。
The Sec1/Munc18 (SM) protein family regulates intracellular trafficking through interactions with individual SNARE proteins and assembled SNARE complexes. Revealing a common mechanism of this regulation has been challenging, largely because of the multiple modes of interaction observed between SM proteins and their cognate syntaxin-type SNAREs. These modes include binding of the SM to a closed conformation of syntaxin, binding to the N-terminal peptide of syntaxin, binding to assembled SNARE complexes, and/or binding to nonsyntaxin SNAREs. The SM protein Vps45p, which regulates endosomal trafficking in yeast, binds the conserved N-terminal peptide of the syntaxin Tlg2p. We used size exclusion chromatography and a quantitative fluorescent gel mobility shift assay to reveal an additional binding site that does not require the Tlg2p N-peptide. Characterization of Tlg2p mutants and truncations indicate that this binding site corresponds to a closed conformation of Tlg2p. Furthermore, the Tlg2p N-peptide competes with the closed conformation for binding, suggesting a fundamental regulatory mechanism for SM-syntaxin interactions in SNARE assembly and membrane fusion.