Cellular Function Of The Adp-ribosylation Factor 6 (arf6

Arf6 GTPase调节质膜（PM）和内体间的膜交通，并影响皮质肌动蛋白细胞骨架的动力学。在许多细胞中，这一途径是缺乏网格蛋白定位序列的PM蛋白遵循的途径，因此被独立于网格蛋白被内吞入细胞。我们已经证明，通过这种arf6调节的、不依赖网格蛋白的途径被内吞的PM蛋白可以被回收或与“经典”早期内体途径汇合，并在溶酶体中降解。我们跟踪了非网格蛋白货物分子的运输和命运，并将其与通过网格蛋白介导的内吞作用内化的PM蛋白进行了比较。通过非网格蛋白途径进入细胞的PM蛋白包括主要组织相容性复合体I类（MHCI）。像MHCI这样的非网格蛋白载货分子最初在Arf6和磷脂酰肌醇4,5-二磷酸（PIP2）相关的核内体中被观察到，这些核内体与含有网格蛋白载货的核内体不同。在5到10分钟之间，含有非网格蛋白货物的早期核内体的一部分获得磷脂酰肌醇3-磷酸（PI3P）和早期核内体自身抗原1 (EEA1)，使其与经典的Rab5、EEA1相关的早期核内体融合。Arf6早期核内体的另一部分被循环回PM。将MHCI再循环回PM的管状内体膜与含有Eps15同源结构域的蛋白EHD1相关，该蛋白在过表达时增强MHCI的再循环。本构活性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，阻断它们向Rab5/EEA1内体的运输和降解，但不影响网格蛋白衍生货物的运输。这些观察结果表明，Arf6失活、PIP2周转和PI3P的获得是Arf6途径产生的核内体与Rab5/EEA1早期核内体系统融合所必需的。目前正在开发检测方法，以在体外重建这些膜运输事件。

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.