Cellular Function Of The Adp-ribosylation Factor 6 (arf6

Adp-核糖基化因子 6 (arf6) 的细胞功能

• 批准号: 6690456
• 负责人: Julie G Donaldson
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6690456
• 关键词: ADP ribosylation; actins; cell morphology; cell motility; endocytosis; guanine nucleotide binding protein; guanosinetriphosphatases; intracellular transport; membrane activity; membrane fusion; phosphatidylinositols; protein structure function; protein transport; tissue /cell culture; transfection

The Arf6 GTPase regulates membrane traffic between the plasma membrane (PM) and an endosomal compartment and influences the dynamics of the cortical actin cytoskeleton. In many cells this pathway is the route followed by PM proteins that lack clathrin localization sequences and hence are endocytosed into cells independently of clathrin. We have demonstrated that PM proteins that are endocytosed via this Arf6-regulated, clathrin-independent pathway can either be recycled or routed to convergence with the "classical" early endosomal pathway and on to degradation in lysosomes. We followed the trafficking and fate of non-clathrin cargo molecules and compared it to PM proteins internalized by clathrin-mediated endocytosis. Among the PM proteins that enter cells through this non-clathrin pathway is major histocompatibility complex class I (MHCI). Non-clathrin cargo molecules like MHCI are initially observed in Arf6 and phosphatidylinositol 4,5-bisphosphate (PIP2)-associated endosomes that are distinct from the endosomes that contain clathrin cargo. Between 5 and 10 minutes, a fraction of the early endosomes containing non-clathrin cargo acquires phosphatidylinositol 3-phosphate (PI3P) and the early endosomal autoantigen 1 (EEA1) allowing it to fuse with the classical, Rab5, EEA1-associated early endosome. Another fraction of the Arf6 early endosome is recycled back to the PM. . The tubular endosomal membranes that recycle MHCI back to the PM have associated with them the Eps15 homology domain-containing protein, EHD1, which enhances MHCI recycling when overexpressed. Expression of constitutively active Arf6 mutant, Arf6Q67L, sequesters non-clathrin cargo in vacuoles that are enriched in PIP2 and blocks their trafficking to Rab5/EEA1 endosomes and degradation but does not affect trafficking of clathrin-derived cargo. These observations suggest that inactivation of Arf6, PIP2 turnover and acquisition of PI3P are required for fusion of endosomes arising from the Arf6 pathway with the Rab5/EEA1 early endosome system. Assays are being developed to reconstitute in vitro these membrane trafficking events.

Arf6 GTPase 调节质膜 (PM) 和内体区室之间的膜运输，并影响皮质肌动蛋白细胞骨架的动力学。在许多细胞中，该途径是缺乏网格蛋白定位序列的 PM 蛋白所遵循的途径，因此独立于网格蛋白被内吞到细胞中。我们已经证明，通过这种 Arf6 调节的、不依赖于网格蛋白的途径内吞的 PM 蛋白可以被回收，也可以与“经典”早期内体途径汇合，并在溶酶体中降解。我们跟踪非网格蛋白货物分子的运输和命运，并将其与网格蛋白介导的内吞作用内化的 PM 蛋白进行比较。通过这种非网格蛋白途径进入细胞的 PM 蛋白之一是主要组织相容性复合物 I 类 (MHCI)。像 MHCI 这样的非网格蛋白货物分子最初是在 Arf6 和磷脂酰肌醇 4,5-二磷酸 (PIP2) 相关的内体中观察到的，这些内体与含有网格蛋白货物的内体不同。 5 到 10 分钟之间，一部分含有非网格蛋白货物的早期内体获得磷脂酰肌醇 3-磷酸 (PI3P) 和早期内体自身抗原 1 (EEA1)，使其与经典的 Rab5、EEA1 相关早期内体融合。 Arf6 早期内体的另一部分被回收回 PM。 。将 MHCI 回收回 PM 的管状内体膜与包含 Eps15 同源结构域的蛋白质 EHD1 相关联，EHD1 在过度表达时可增强 MHCI 回收。组成型活性 Arf6 突变体 Arf6Q67L 的表达将非网格蛋白货物隔离在富含 PIP2 的液泡中，并阻止其运输到 Rab5/EEA1 内体和降解，但不影响网格蛋白衍生货物的运输。这些观察结果表明，Arf6 途径的内体与 Rab5/EEA1 早期内体系统的融合需要 Arf6 的失活、PIP2 更新和 PI3P 的获得。正在开发检测方法以在体外重建这些膜运输事件。