Brox, a novel farnesylated Bro1 domain-containing protein that associates with charged multivesicular body protein 4 (CHMP4)
Brox, a novel farnesylated Bro1 domain-containing protein that associates with charged multivesicular body protein 4 (CHMP4)
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DOI：
10.1111/j.1742-4658.2007.06230.x
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发表时间：
2008-02-01
期刊：
影响因子：
5.4
通讯作者：
Maki, Masatoshi
中科院分区：
文献类型：
--
作者：
Ichioka, Fumitaka;Kobayashi, Ryota;Maki, Masatoshi
人类Brox是一种新发现的46 kDa蛋白，具有Bro1结构域样序列和C端类异戊二烯脂类硫酯连锁位点（CAAX基序）（C代表半胱氨酸，a代表一般脂肪氨基酸，X代表任何氨基酸）。哺乳动物Alix及其酵母同源物Bro1通过其Bro1结构域与携带电荷的多泡体蛋白4 （CHMP4）相关，CHMP4是运输III所需的内体分选复合体的组成部分，并在泛素化货物的分选中发挥作用。基于Brox与chmp4相互作用的能力，我们研究了Brox是否具有真正的Bro1结构域。Strep- tacn结合序列（Strep）标记的野生型Brox（Strep-Brox(WT)）和Strep标记的法酰化缺陷突变体（Cys -> Ser突变；Strep-Brox(C408S)）都能拉低HEK293细胞中共表达的flag标记的CHMP4b。用法尼基转移酶抑制剂FTI-277处理细胞，导致Strep-Brox（WT）的电泳迁移率发生变化，并且这种迁移率与Strep-Brox（C408S）的迁移率一致。该抑制剂还引起内源性Brox的迁移，通过western blotting检测到Brox的多克隆抗体，表明Brox在体内有法尼化作用。荧光显微镜分析显示，Strep-Brox（WT）在核周区域积聚，并引起FLAG-CHMP4b的点状模式，该点状模式在HEK293细胞中组成性表达。另一方面，Strep-Brox（C408S）在包括细胞核在内的整个细胞中表现出弥漫性模式，并且不会引起FLAG-CHMP4b的积累。单体绿色荧光蛋白(mGFP)-融合Brox（WT）的荧光信号部分与高尔基标记和HeLa细胞中AAA型atp酶SKD1/Vps4B显性阴性突变体过表达诱导的异常内体的荧光信号融合，但这种共定位对mGFP-Brox（C408S）的效率较低。这些结果表明，在体内，Brox的法尼化在其亚细胞分布和与chmp4的有效相互作用中具有生理意义。
Human Brox is a newly identified 46 kDa protein that has a Bro1 domain-like sequence and a C-terminal thioester-linkage site of isoprenoid lipid (CAAX motif) (C standing for cysteine, A for generally aliphatic amino acid, and X for any amino acid). Mammalian Alix and its yeast ortholog, Bro1, are known to associate with charged multivesicular body protein 4 (CHMP4), a component of endosomal sorting complex required for transport III, via their Bro1 domains and to play roles in sorting of ubiquitinated cargoes. We investigated whether Brox has an authentic Bro1 domain on the basis of its capacity for interacting with CHMP4s. Both Strep Tactin binding sequence (Strep)-tagged wild-type Brox (Strep-Brox(WT)) and Strep-tagged farnesylation-defective mutant (Cys -> Ser mutation; Strep-Brox(C408S)) pulled down FLAG-tagged CHMP4b that was coexpressed in HEK293 cells. Treatment of cells with a farnesyltransferase inhibitor, FTI-277, caused an electrophoretic mobility shift of Strep-Brox(WT), and the mobility coincided with that of Strep-Brox(C408S). The inhibitor also caused a mobility shift of endogenous Brox detected by western blotting using polyclonal antibodies to Brox, suggesting farnesylation of Brox in vivo. Fluorescence microscopic analyses revealed that Strep-Brox(WT) exhibited accumulation in the perinuclear area and caused a punctate pattern of FLAG-CHMP4b that was constitutively expressed in HEK293 cells. On the other hand, Strep-Brox(C408S) showed a diffuse pattern throughout the cell, including the nucleus, and did not cause accumulation of FLAG-CHMP4b. Fluorescent signals of monomeric green fluorescent protein (mGFP)-fused Brox(WT) merged partly with those of Golgi markers and with those of abnormal endosomes induced by overexpression of a dominant negative mutant of AAA type ATPase SKD1/Vps4B in HeLa cells, but such colocalization was less efficient for mGFP-Brox(C408S). These results suggest a physiological significance of farnesylation of Brox in its subcellular distribution and efficient interaction with CHMP4s in vivo.