Mapping the GRIF-1 binding domain of the kinesin, KIF5C, substantiates a role for GRIF-1 as an adaptor protein in the anterograde trafficking of cargoes
Mapping the GRIF-1 binding domain of the kinesin, KIF5C, substantiates a role for GRIF-1 as an adaptor protein in the anterograde trafficking of cargoes
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DOI：
10.1074/jbc.m600522200
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发表时间：
2006-09-15
影响因子：
4.8
通讯作者：
Stephenson, F. Anne
中科院分区：
文献类型：
--
作者：
Smith, Miriam J.;Pozo, Karine;Stephenson, F. Anne
γ-氨基丁酸，A型（GABA（A））受体相互作用因子-1（GRIF-1）和N-乙酰葡糖胺转移酶相互作用蛋白（OIP）106都是新鉴定的卷曲螺旋蛋白家族的成员。它们是驱动蛋白相关蛋白，被认为在细胞器到突触的顺行运输中起衔接子的作用。在这里，我们更详细地研究了原型驱动蛋白重链、KIF 5C、驱动蛋白轻链和GRIF-1之间的相互作用。在哺乳动物细胞中表达后，在酵母双杂交相互作用测定、共免疫沉淀和共定位研究中使用截短构建体绘制KIF 5C的GRIF-1结合位点。使用这些方法，显示GRIF-1和GRIF-1的KIF 5C结合结构域GRIF-1-（124-283）与KIF 5C非运动结构域相关。使用酵母双杂交相互作用和免疫共沉淀的精细研究表明，GRIF-1和GRIF-1-（124-283）与KIF 5C非马达结构域内的货物结合区相关。通过使用荧光标记的GRIF-1和KIF 5C构建体的荧光共振能量转移分析证实GRIF-1-KIF 5C相互作用是直接的。在C端EYFP标记的KIF 5C和ECFP-GRIF-1之间、C端EYFP标记的KIF 5C非马达结构域和ECFP-GRIF-1之间发现了显著的荧光共振能量转移值，但在N端EYFP标记的KIF 5C和EYFP-KIF 5C马达结构域与ECFP-GRIF-1之间均未发现，因此证实了两种蛋白质在KIF 5C C端和GRIF-1 N端区域之间的直接关联。免疫共沉淀和共聚焦成像策略进一步表明GRIF-1可以结合四聚体驱动蛋白轻链/驱动蛋白重链复合物。这些发现支持GRIF-1作为驱动蛋白衔接分子的作用，所述驱动蛋白衔接分子是在脑中顺行递送确定的货物（例如并入含有β 2亚基的GABA（A）受体的线粒体和/或囊泡）所必需的。
gamma-Aminobutyric acid, type A (GABA(A)) receptor interacting factor-1 (GRIF-1) and N-acetylglucosamine transferase interacting protein (OIP) 106 are both members of a newly identified coiled-coil family of proteins. They are kinesin-associated proteins proposed to function as adaptors in the anterograde trafficking of organelles to synapses. Here we have studied in more detail the interaction between the prototypic kinesin heavy chain, KIF5C, kinesin light chain, and GRIF-1. The GRIF-1 binding site of KIF5C was mapped using truncation constructs in yeast two-hybrid interaction assays, co-immunoprecipitations, and co-localization studies following expression in mammalian cells. Using these approaches, it was shown that GRIF-1 and the KIF5C binding domain of GRIF-1, GRIF-1-(124-283), associated with the KIF5C non-motor domain. Refined studies using yeast two-hybrid interactions and co-immunoprecipitations showed that GRIF-1 and GRIF-1-(124-283) associated with the cargo binding region within the KIF5C non-motor domain. Substantiation that the GRIF-1-KIF5C interaction was direct was shown by fluorescence resonance energy transfer analyses using fluorescently tagged GRIF-1 and KIF5C constructs. A significant fluorescence resonance energy transfer value was found between the C-terminal EYFP-tagged KIF5C and ECFP-GRIF-1, the C-terminal EYFP-tagged KIF5C non-motor domain and ECFP-GRIF-1, but not between the N-terminal EYFP-tagged KIF5C nor the EYFP-KIF5C motor domain and ECFP-GRIF-1, thus confirming direct association between the two proteins at the KIF5C C-terminal and GRIF-1 N-terminal regions. Co-immunoprecipitation and confocal imaging strategies further showed that GRIF-1 can bind to the tetrameric kinesin light-chain/kinesin heavy-chain complex. These findings support a role for GRIF-1 as a kinesin adaptor molecule requisite for the anterograde delivery of defined cargoes such as mitochondria and/or vesicles incorporating beta 2 subunit-containing GABA(A) receptors, in the brain.