Rab10介导ATP8A1的TGN输出及其对内吞体运输影响的机制研究

Intracellular membrane transport is tightly regulated by an array of proteins and molecular machineries. The P4 subfamily of P-type ATPases (P4-ATPases) are responsible for the flipping of aminophospholipids to the cytosolic leaflet, which is evolutionarily highly conserved. The human genome contains 14 P4-ATPases, and mutations in some P4-ATPases cause inherited genetic diseases. Research in yeast suggested that P4-ATPase is intensively involved in various steps of membrane trafficking along the exocytic and the endocytic pathway. Obviously the correct subcellular localization promises the normal physiological functions of P4-ATPases. CDC50 proteins are recognized as accessary subunits and are essential for the maturation of P4-ATPases and ER exit. However, less is known of what other proteins regulate the subcellular localization of P4-ATPases in mammalian cells. In addition, to what extent does the effect of P4-ATPases on the membrane transport depend on their functional regulation for asymmetric distribution of phospholipids? In our previous work, we found that ATP8A1, a P4-ATPase member, was co-immunoprecipitated by Rab10 GTPase in Hela cells. In contrast to the peripheral puncta and perinuclear Golgi-like structures of ATP8A1 in control cells, expression of constitutive active Rab10 (Rab10Q68L) resulted in the profound reduced peripheral puncta of ATP8A1 and only showed localization on perinulear structures. We propose that Rab10 GTPase probably mediates the post-Golgi transport of ATP8A1. In this project, we plan to: 1) further demonstrate the interaction between Rab10 GTPase and ATP8A1, and to identiy whether this interaction relies on the activity of Rab10 and map the crucial cytosolic region on ATP8A1 responsible for ATP8A1-Rab10 binding. 2) dissect how the Rab10 activity affects the post-Golgi transport and the subcellular localization of ATP8A1 at various endosomal membranes; 3) investigate the effect of ATP8A1 depletion on endodomal transport and the relationship with the PS flippase function of ATP8A1. Our studies will shed light on regulation of Rab GTPases on the P4-ATPases proteins and deepen our understanding of establishment and maintainance of the Golgi-endosomes interaction network.

细胞器稳态和功能的维持依赖彼此间的相互接触和物质运输。P4-ATPase通过调节磷脂内翻维持细胞膜和内膜中磷脂的不对称分布。在酵母上发现P4-ATPases参与分泌或内吞途径膜转运的不同环节，而哺乳动物中P4-ATPases对不同囊泡运输路径有何影响及与其磷脂翻转功能的关系尚不清楚。虽然已知CDC50蛋白作为辅助亚基调节P4-ATPase的成熟和离开内质网，更多有关P4-ATPases的亚细胞定位和功能调节的机制尚有待研究。我们依据前期在HeLa细胞上的工作提出Rab10 GTPase可能介导PS内翻酶ATP8A1的后高尔基体输出。据此，我们拟深入研究二者间的作用机制，解析Rab10如何影响ATP8A1在内吞体膜上的定位，探究ATP8A1的亚细胞定位异常及功能缺失对内吞体系统物质运输的影响及其与ATP8A1的PS翻转功能的关系，阐明Rab10在高尔基体-内吞体互作网络建立与调控中的功能。

细胞器稳态和功能的维持具有重要生理意义。小GTPase家族成员Rabs是维护细胞内膜转运系统正常运行的重要调节器。细胞膜磷脂的不对称分布对囊泡形成和物质运输的影响机制，目前认识尚不十分清楚。在本项目中，我们主要从以下4个方面开展研究，并取得了一些创新性发现：1）发现P4-ATPase家族成员磷脂酰丝氨酸翻转酶ATP8A1对细胞内吞后降解途径物质运输的负调控作用，并初步解析其功能机制。研究结果有助于拓宽认识磷脂对细胞膜转运系统正常运行的维护功能。2）发现Rab10调控ATP8A1的后高尔基运输并尝试阐明其作用机制及生理意义。研究结果初步显示可能代表了一类新型的反式高尔基体-内体的新生膜蛋白运输路径。3）发现Rab10-ACAP1-Arf6 GTPase级联通路调控M4毒蕈碱乙酰胆碱受体激活后转运及信号传导，并阐明Rab10的分子制动器作用。该研究首次揭示了Rab家族成员精确调控GPCR成员的胞内运输路径及动态过程进而影响其信号传导的分子制动器机制。上述研究为阿尔兹海默症和精神分裂症等与M4受体信号失调密切相关的精神类疾病的干预治疗手段的发展提供了新的思路。4）提出SNX-3以Retromer复合物不依赖的方式介导循环内体膜管生成及其工作机制。我们的结果首次揭示仅含PX结构域的SNX-3对于一类clathrin非依赖内吞途径的货物蛋白进入内吞体膜管进行循环具有重要作用。以及该类型运输载体的形成出乎意料的不需要Retromer，促使我们重新认识SNXs与Retromer在早期内体分选平台精密调控货物蛋白分选及其运输路径的功能以及二者之间功能关系的传统观点。