A novel assay reveals preferential binding between Rabs, kinesins, and specific endosomal subpopulations.
A novel assay reveals preferential binding between Rabs, kinesins, and specific endosomal subpopulations.
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DOI：
10.1083/jcb.201408056
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发表时间：
2015-02-02
期刊：
影响因子：
--
通讯作者：
Banker G
中科院分区：
文献类型：
--
作者：
Bentley M;Decker H;Luisi J;Banker G
一项基于表达frb标记的候选囊泡结合蛋白的新实验表明，KIF13A和KIF13B优先结合早期核内体，而KIF1A和KIF1Bβ优先结合晚期核内体和溶酶体。识别调节囊泡运输的蛋白质是细胞生物学的一个基本问题。在本文中，我们介绍了一种新的实验，该实验涉及fkbp12 -雷帕霉素结合结构域标记的候选囊泡结合蛋白的表达，该蛋白可以诱导与动力蛋白或动力蛋白连接。囊泡可以用任何方便的方法标记。如果候选蛋白结合标记的囊泡，添加连接药物会导致囊泡定位的可预测的，高度独特的变化。该分析产生稳健且易于解释的结果，为候选蛋白与感兴趣的囊泡群之间的结合提供了直接的实验证据。我们使用这种方法来比较不同内体群体的Kinesin-3家族成员的结合。我们发现KIF13A和KIF13B优先结合早期核内体，KIF1A和KIF1Bβ优先结合晚期核内体和溶酶体。该分析方法对于鉴定与不同囊泡群结合的转运蛋白具有广泛的实用价值。
A novel assay based on expressing FRB-tagged candidate vesicle-binding protein reveals that KIF13A and KIF13B bind preferentially to early endosomes, whereas KIF1A and KIF1Bβ bind preferentially to late endosomes and lysosomes. Identifying the proteins that regulate vesicle trafficking is a fundamental problem in cell biology. In this paper, we introduce a new assay that involves the expression of an FKBP12-rapamycin–binding domain–tagged candidate vesicle-binding protein, which can be inducibly linked to dynein or kinesin. Vesicles can be labeled by any convenient method. If the candidate protein binds the labeled vesicles, addition of the linker drug results in a predictable, highly distinctive change in vesicle localization. This assay generates robust and easily interpretable results that provide direct experimental evidence of binding between a candidate protein and the vesicle population of interest. We used this approach to compare the binding of Kinesin-3 family members with different endosomal populations. We found that KIF13A and KIF13B bind preferentially to early endosomes and that KIF1A and KIF1Bβ bind preferentially to late endosomes and lysosomes. This assay may have broad utility for identifying the trafficking proteins that bind to different vesicle populations.
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DOI：
10.1083/jcb.201104034
发表时间：
2011-07-25
期刊：
The Journal of cell biology
影响因子：
--
作者：
Nakata T;Niwa S;Okada Y;Perez F;Hirokawa N
通讯作者：
Hirokawa N
DOI：
10.1083/jcb.201205070
发表时间：
2012-08-20
期刊：
The Journal of cell biology
影响因子：
--
作者：
Jenkins B;Decker H;Bentley M;Luisi J;Banker G
通讯作者：
Banker G
影响因子：
16.2
作者：
Jacobson, C;Schnapp, B;Banker, GA
通讯作者：
Banker, GA
影响因子：
9.8
作者：
Lewis TL Jr;Mao T;Arnold DB
通讯作者：
Arnold DB
影响因子：
3.4
作者：
Duclos, Sophie;Clavarino, Giovanna;Desjardins, Michel
通讯作者：
Desjardins, Michel