Mochizuki Shinya, Miki Harukata, Zhou Ruyun, Noda Yasuko
Dept. of Anatomy, Bioimaging and Neuro-cell Science, Jichi Medical University, Japan.
Biochem Biophys Rep. 2022 Apr 25;30:101257. doi: 10.1016/j.bbrep.2022.101257. eCollection 2022 Jul.
Oxysterol-binding protein (OSBP)-related protein (ORP) 6, a member of subfamily III in the ORP family, localizes to membrane contact sites between the endoplasmic reticulum (ER) and other organelles and functions in non-vesicular exchange of lipids including phosphatidylinositol-4-phosphate (PI4P) in neurons. In this study, we searched for the lipid counter-transported in exchange for PI4P by using molecular cell biology techniques. Deconvolution microscopy revealed that knockdown of ORP6 partially shifted localization of a phosphatidylserine (PS) marker but not filipin in primary cultured cerebellar neurons. Overexpression of ORP6 constructs lacking the OSBP-related ligand binding domain (ORD) resulted in the same shift of the PS marker. A PI4KⅢα inhibitor specifically inhibiting the synthesis and plasma membrane (PM) localization of PI4P, suppressed the localization of ORP6 and the PS marker at the PM. Overexpression of mutant PS synthase 1 (PSS1) inhibited transport of the PS marker to the PM and relocated the PI4P marker to the PM in Neuro-2A cells. Introduction of ORP6 but not the dominant negative ORP6 constructs, shifted the localization of PS back to the PM. These data collectively suggest the involvement of ORP6 in the counter-transport of PI4P and PS.
氧化甾醇结合蛋白（OSBP）相关蛋白（ORP）6是ORP家族III亚家族的成员，定位于内质网（ER）与其他细胞器之间的膜接触位点，并在神经元中参与包括磷脂酰肌醇-4-磷酸（PI4P）在内的脂质非囊泡交换过程。在本研究中，我们运用分子细胞生物学技术寻找与PI4P进行交换的反向转运脂质。解卷积显微镜显示，在原代培养的小脑神经元中，敲低ORP6会使磷脂酰丝氨酸（PS）标记物的定位部分发生改变，但并不会影响制霉菌素的定位。缺乏OSBP相关配体结合域（ORD）的ORP6构建体的过表达导致PS标记物出现相同的定位改变。一种特异性抑制PI4P合成和质膜（PM）定位的PI4KⅢα抑制剂，可抑制ORP6和PS标记物在质膜上的定位。突变型PS合酶1（PSS1）的过表达抑制了PS标记物向质膜的转运，并使PI4P标记物在Neuro-2A细胞中重新定位于质膜。引入ORP6而非显性负性ORP6构建体，可使PS的定位恢复到质膜。这些数据共同表明ORP6参与了PI4P和PS的反向转运。
