Conserved function of the lysine-based KXD/E motif in Golgi retention for endomembrane proteins among different organisms.
Conserved function of the lysine-based KXD/E motif in Golgi retention for endomembrane proteins among different organisms.
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DOI：
10.1091/mbc.e15-06-0361
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发表时间：
2015-11-15
影响因子：
3.3
通讯作者：
Jiang L
中科院分区：
文献类型：
--
作者：
Woo CH;Gao C;Yu P;Tu L;Meng Z;Banfield DK;Yao X;Jiang L
内膜蛋白（EMPs） c端胞质尾中与copi相互作用的KXD/E基序是EMPs的重要高尔基体保留机制。在真核生物中，KXD/E在COPI相互作用和高尔基保留中是一个保守的基序。我们最近在拟南芥细胞膜蛋白12 （AtEMP12）的c端胞质尾（CT）中发现了一个新的与copi相互作用的KXD/E基序，作为AtEMP12的关键高尔基保留机制。这种KXD/E基序在植物、酵母和人类中发现的所有EMPs的ct中都是保守的，并且也存在于数百种其他膜蛋白中。在这里，通过克隆来自植物、酵母和哺乳动物的选择性EMP异构体，我们研究了EMP在不同表达系统中的定位，因为关于EMP定位的报道是相互矛盾的。我们发现，在植物、酵母和哺乳动物细胞中，n端和c端gfp标记的EMP融合体分别定位于高尔基区室和后高尔基区室。体外拉下实验进一步证实了KXD/E基序与酵母中COPI涂层的相互作用。在酵母和植物中，COPI的功能丧失会导致EMPs或含有KXD/E基序的蛋白错误定位到液泡中。超微结构研究进一步表明，RNA干扰（RNAi）敲低coatomer在转基因拟南芥植物中的表达会导致高尔基体发生严重的形态变化。综上所述，我们的研究结果表明，n端GFP融合反映了EMPs的真实定位，KXD/E是真核生物中COPI相互作用和高尔基体保留的保守基序。
The COPI-interacting KXD/E motif in the C-terminal cytosolic tail of endomembrane proteins (EMPs) is a crucial Golgi retention mechanism for EMPs. KXD/E is a conserved motif in COPI interaction and Golgi retention in eukaryotes. We recently identified a new COPI-interacting KXD/E motif in the C-terminal cytosolic tail (CT) of Arabidopsis endomembrane protein 12 (AtEMP12) as being a crucial Golgi retention mechanism for AtEMP12. This KXD/E motif is conserved in CTs of all EMPs found in plants, yeast, and humans and is also present in hundreds of other membrane proteins. Here, by cloning selective EMP isoforms from plants, yeast, and mammals, we study the localizations of EMPs in different expression systems, since there are contradictory reports on the localizations of EMPs. We show that the N-terminal and C-terminal GFP-tagged EMP fusions are localized to Golgi and post-Golgi compartments, respectively, in plant, yeast, and mammalian cells. In vitro pull-down assay further proves the interaction of the KXD/E motif with COPI coatomer in yeast. COPI loss of function in yeast and plants causes mislocalization of EMPs or KXD/E motif–containing proteins to vacuole. Ultrastructural studies further show that RNA interference (RNAi) knockdown of coatomer expression in transgenic Arabidopsis plants causes severe morphological changes in the Golgi. Taken together, our results demonstrate that N-terminal GFP fusions reflect the real localization of EMPs, and KXD/E is a conserved motif in COPI interaction and Golgi retention in eukaryotes.