Cellular Function Of The ADP-ribosylation Factor 6 (Arf6

Arf6 GTP酶调节质膜(PM)和内小体室之间的膜运输，并影响皮质肌动蛋白细胞骨架的动力学。在许多细胞中，这条途径是PM蛋白遵循的路线，这些蛋白缺乏分子筛定位序列，因此独立于分子筛进入细胞内。通过这种不依赖于笼蛋白的途径被内吞的PM蛋白可以被循环回到PM，或者被引导到与“经典的”早期内体途径汇合，进而在溶酶体中降解。在通过这种非网状蛋白途径进入细胞的PM蛋白中，主要组织相容性复合体I类(MHCI)是其中之一。将MHCI循环回PM的管状内膜与含有Eps15同源结构域的蛋白EHD1相关，当过表达时，EHD1可增强MHCI的循环。表达具有结构性活性的Arf6突变体Arf6Q67L，在富含PIP2的空泡中隔离非笼蛋白货物，并阻止它们转运到Rab5/EEA1内体和降解，但不影响笼蛋白衍生货物的运输。这些观察表明，Arf6途径产生的内体与Rab5/EEA1早期内体系统的融合需要Arf6的失活、PIP2的周转和PI3P的获得。我们发现，一些脂筏相关蛋白，那些由糖基磷脂酰肌醇(GPI)部分锚定在膜上的蛋白，也通过这种不依赖于笼蛋白的机制进入细胞，并与Rab5早期的内体收敛或通过MHCI循环回到PM。正在开发在体外重建这些膜转运事件的分析方法。

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. PM proteins that are endocytosed via this clathrin-independent pathway can either be recycled back to the PM or routed to convergence with the "classical" early endosomal pathway and on to degradation in lysosomes. Among the PM proteins that enter cells through this non-clathrin pathway is major histocompatibility complex class I (MHCI). 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. We have found that some lipid raft-associated proteins, those anchored to the membrane by a glycosylphosphatidyl inositol (GPI) moiety, also enter cells via this clathrin-independent mechanism and converge with the Rab5 early endosome or recycle back to the PM with MHCI. Assays are being developed to reconstitute in vitro these membrane trafficking events.