Structural Basis for the Golgi Association by the Pleckstrin Homology Domain of the Ceramide Trafficking Protein (CERT)
Structural Basis for the Golgi Association by the Pleckstrin Homology Domain of the Ceramide Trafficking Protein (CERT)
复制标题
DOI：
10.1074/jbc.m112.367730
复制
发表时间：
2012-09-28
影响因子：
4.8
通讯作者：
Shimada, Ichio
中科院分区：
文献类型：
--
作者：
Sugiki, Toshihiko;Takeuchi, Koh;Shimada, Ichio
神经酰胺从内质网转运到高尔基体在鞘脂生物合成中是至关重要的，这一过程依赖于神经酰胺运输蛋白（CERT），其中包含pleckstrin同源性（PH）和star相关的脂质转运结构域。CERT PH结构域特异性识别高尔基膜中特有的磷酸肌醇4-单磷酸（PtdIns(4)P），是CERT将神经酰胺从内质网到高尔基体运输所不可或缺的。在本研究中，我们利用溶液核磁共振技术确定了CERT PH结构域的三维结构。该结构显示在典型PtdIns(4) P识别位点附近存在特征性的基本凹槽。利用核磁共振和其他生物物理技术进行的广泛相互作用研究表明，基本凹槽协调CERT PH结构域，以有效识别和定位高尔基体中的PtdIns(4)P。这一观点也得到了活细胞中CERT突变体高尔基错误定位的支持。不同的结合模式反映了PH结构域的功能，因为基本槽仅在与PtdIns(4) p依赖性脂质转运活性相关的PH结构域中保守，而在具有信号转导活性的PH结构域中则不保守。
Ceramide transport from the endoplasmic reticulum to the Golgi apparatus is crucial in sphingolipid biosynthesis, and the process relies on the ceramide trafficking protein (CERT), which contains pleckstrin homology (PH) and StAR-related lipid transfer domains. The CERT PH domain specifically recognizes phosphatidylinositol 4-monophosphate (PtdIns(4)P), a characteristic phosphoinositide in the Golgi membrane, and is indispensable for the endoplasmic reticulum-to-Golgi transport of ceramide by CERT. In this study, we determined the three-dimensional structure of the CERT PH domain by using solution NMR techniques. The structure revealed the presence of a characteristic basic groove near the canonical PtdIns(4) P recognition site. An extensive interaction study using NMRand other biophysical techniques revealed that the basic groove coordinates the CERT PH domain for efficient PtdIns(4)P recognition and localization in the Golgi apparatus. The notion was also supported by Golgi mislocalization of the CERT mutants in living cells. The distinctive binding modes reflect the functions of PH domains, as the basic groove is conserved only in thePHdomains involved with the PtdIns(4)P-dependent lipid transport activity but not in those with the signal transduction activity.