The Hydrophobic Cysteine-rich Domain of SNAP25 Couples with Downstream Residues to Mediate Membrane Interactions and Recognition by DHHC Palmitoyl Transferases
The Hydrophobic Cysteine-rich Domain of SNAP25 Couples with Downstream Residues to Mediate Membrane Interactions and Recognition by DHHC Palmitoyl Transferases
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DOI：
10.1091/mbc.e08-09-0944
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发表时间：
2009-03-15
影响因子：
3.3
通讯作者：
Chamberlain, Luke H.
中科院分区：
文献类型：
--
作者：
Greaves, Jennifer;Prescott, Gerald R.;Chamberlain, Luke H.
SNAP25是以可溶性蛋白质的形式合成的，但必须与质膜结合才能发挥胞吐作用；然而，这一膜靶向途径尚不清楚。SNAP25含有一个棕榈酰化的半胱氨酸富含结构域和四个半胱氨酸，我们证明了特定的DHHC棕榈酰转移酶的共表达足以促进SNAP25在HEK293细胞中的膜结合。SiRNA介导的SNAP25在PC12细胞中的膜结合并不影响SNAP25的膜相互作用，而半胱氨酸到丙氨酸的特异性突变扰乱了膜结合，这种结合可以通过亮氨酸取代来恢复。这些结果表明，半胱氨酸的疏水性在SNAP25的起始膜相互作用中发挥了作用，实际上，富含半胱氨酸结构域的其他疏水残基对膜结合也是重要的。除了富含半胱氨酸的结构域，Pro-117对SNAP25膜结合也是必不可少的，在HEK293细胞中的实验表明，该残基的突变抑制了与DHHC17共表达而诱导的膜结合，但不抑制DHHC3或DHHC7的共表达。这些结果表明，SNAP25通过其疏水的半胱氨酸富含结构域与膜自主相互作用，只需要充分表达伴侣DHHC蛋白就能稳定地与膜结合。Pro-117在SNAP25棕榈酰化中的作用是对底物蛋白中调节DHHC特异性的元件的最早描述之一。
SNAP25 is synthesized as a soluble protein but must associate with the plasma membrane to function in exocytosis; however, this membrane-targeting pathway is poorly defined. SNAP25 contains a palmitoylated cysteine-rich domain with four cysteines, and we show that coexpression of specific DHHC palmitoyl transferases is sufficient to promote SNAP25 membrane association in HEK293 cells. siRNA-mediated knockdown of its SNARE partner, syntaxin 1A, does not affect membrane interaction of SNAP25 in PC12 cells, whereas specific cysteine-to-alanine mutations perturb membrane binding, which is restored by leucine substitutions. These results suggest a role for cysteine hydrophobicity in initial membrane interactions of SNAP25, and indeed other hydrophobic residues in the cysteine-rich domain are also important for membrane binding. In addition to the cysteine-rich domain, proline-117 is also essential for SNAP25 membrane binding, and experiments in HEK293 cells revealed that mutation of this residue inhibits membrane binding induced by coexpression with DHHC17, but not DHHC3 or DHHC7. These results suggest a model whereby SNAP25 interacts autonomously with membranes via its hydrophobic cysteine-rich domain, requiring only sufficient expression of partner DHHC proteins for stable membrane binding. The role of proline-117 in SNAP25 palmitoylation is one of the first descriptions of elements within substrate proteins that modulate DHHC specificity.