Ma Hui, Lou Ying, Lin Wen Hui, Xue Hong Wei
National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Science (SIBS), Chinese Academy of Sciences, 200032 Shanghai, China.
Cell Res. 2006 May;16(5):466-78. doi: 10.1038/sj.cr.7310058.
Multiple repeats of membrane occupation and recognition nexus (MORN) motifs were detected in plant phosphatidylinositl monophosphate kinase (PIPK), a key enzyme in PI-signaling pathway. Structural analysis indicates that all the MORN motifs (with varied numbers at ranges of 7-9), which shared high homologies to those of animal ones, were located at N-terminus and sequentially arranged, except those of OsPIPK1 and AtPIPK7, in which the last MORN motif was separated others by an approximately 100 amino-acid "island" region, revealing the presence of two kinds of MORN arrangements in plant PIPKs. Through employing a yeast-based SMET (sequence of membrane-targeting) system, the MORN motifs were shown being able to target the fusion proteins to cell plasma membrane, which were further confirmed by expression of fused MORN-GFP proteins. Further detailed analysis via deletion studies indicated the MORN motifs in OsPIPK1, together with the 104 amino-acid "island" region are involved in the regulation of differential subcellular localization, i.e. plasma membrane or nucleus, of the fused proteins. Fat Western blot analysis of the recombinant MORN polypeptide, expressed in Escherichia coli, showed that MORN motifs could strongly bind to PA and relatively slightly to PI4P and PI(4,5)P2. These results provide informative hints on mechanisms of subcellular localization, as well as regulation of substrate binding, of plant PIPKs.
在植物磷脂酰肌醇单磷酸激酶（PIPK，磷脂酰肌醇信号通路中的关键酶）中检测到多个膜占据与识别连接点（MORN）基序重复序列。结构分析表明，所有的MORN基序（数量在7 - 9个之间变化）与动物的MORN基序具有高度同源性，它们位于N端且顺序排列，但水稻PIPK1和拟南芥PIPK7除外，在这两个蛋白中，最后一个MORN基序被一个约100个氨基酸的“岛”状区域与其他基序隔开，这揭示了植物PIPK中存在两种MORN排列方式。通过使用基于酵母的SMET（膜靶向序列）系统，发现MORN基序能够将融合蛋白靶向到细胞质膜，融合的MORN - GFP蛋白的表达进一步证实了这一点。通过缺失研究进行的进一步详细分析表明，水稻PIPK1中的MORN基序与104个氨基酸的“岛”状区域共同参与调控融合蛋白在细胞质膜或细胞核中的差异亚细胞定位。对在大肠杆菌中表达的重组MORN多肽进行的荧光脂质免疫印迹分析表明，MORN基序能与磷脂酸（PA）强烈结合，与磷脂酰肌醇-4-磷酸（PI4P）和磷脂酰肌醇-4,5-二磷酸（PI(4,5)P2）的结合相对较弱。这些结果为植物PIPK的亚细胞定位机制以及底物结合调控提供了有价值的线索。
