Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo, Japan.
J Biol Chem. 2012 Sep 28;287(40):33706-18. doi: 10.1074/jbc.M112.367730. Epub 2012 Aug 6.
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 NMR and 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 the PH domains involved with the PtdIns(4)P-dependent lipid transport activity but not in those with the signal transduction activity.
内质网到高尔基体的神经酰胺转运对于鞘脂生物合成至关重要，该过程依赖于神经酰胺转运蛋白（CERT），其包含pleckstrin 同源（PH）和 StAR 相关脂质转移结构域。CERT PH 结构域特异性识别磷酸肌醇 4-单磷酸（PtdIns（4）P），这是高尔基体膜中的一种特征磷酸肌醇，对于 CERT 介导的神经酰胺的内质网到高尔基体运输是不可或缺的。在这项研究中，我们使用溶液 NMR 技术确定了 CERT PH 结构域的三维结构。该结构揭示了在典型的 PtdIns（4）P 识别位点附近存在特征性的碱性凹槽。使用 NMR 和其他生物物理技术的广泛相互作用研究表明，碱性凹槽协调 CERT PH 结构域，以实现有效的 PtdIns（4）P 识别和在高尔基体中的定位。这一观点也得到了活细胞中 CERT 突变体高尔基体定位异常的支持。独特的结合模式反映了 PH 结构域的功能，因为碱性凹槽仅存在于与 PtdIns（4）P 依赖性脂质转运活性相关的 PH 结构域中，而不存在于与信号转导活性相关的 PH 结构域中。
