CELLULAR FUNCTION OF THE ADP-RIBOSYLATION FACTOR 6 GTP BINDING PROTEIN

adp核糖基化因子（Arfs）是一个GTP结合蛋白家族，调节细胞中的膜交通和细胞器结构。我们一直在研究Arf6的细胞功能，Arf6是一种影响质膜与内体间膜和肌动蛋白细胞骨架之间膜运动的Arf。在许多细胞中，这种途径是缺乏网格蛋白定位序列的膜蛋白所遵循的途径，因此通过非网格蛋白机制被内吞进入细胞。这些膜蛋白再循环回到质膜需要激活Arf6，而这些蛋白进入内体再循环系统需要水解gtp结合的Arf6。虽然这一途径已被证明影响吞噬、细胞扩散和迁移等质膜重塑事件，但参与介导Arf6反应的分子仍有待确定。膜磷酸肌醇，如磷脂酰肌醇4,5-二磷酸（PIP2），参与多种过程，如膜运输和肌动蛋白重塑。我们研究了PIP2的亚细胞分布及其在Arf6功能中的作用。我们发现Arf6调节PIP2在细胞内的定位。也有证据表明，当Arf6被激活时，可能会刺激一种酶的活性，磷脂酰肌醇磷酸5激酶，负责在膜上产生PIP2。事实上，我们发现细胞中这种酶的过表达重现了一种类似于激活的Arf6突变体在细胞中表达时所观察到的表型。值得注意的是，这些细胞在被肌动蛋白包裹的液泡状结构中积累了大量富含pip2的质膜。通常，Arf6的失活会阻止这种异常液泡积聚的形成。在与NCI的Paul Randazzo的合作中，我们已经确定了两个新的Arf GAP分子ACAP1和ACAP2，它们的功能是使Arf6失活。因此，Arf6的激活和失活可能介导PIP2合成和/或PIP2在特定膜位置的转运。实验室的另一个项目涉及通过Arf6核内体的蛋白质的命运。我们发现通过这一途径的蛋白质在溶酶体中被降解，并通过Arf6内体进入溶酶体。这种通往溶酶体的替代途径与通过网格蛋白介导的机制被内吞进入细胞的蛋白质所遵循的途径形成对比。然而，这两种途径确实在内体晚期间室汇合。我们正在进一步表征这一通往溶酶体的替代途径，因为许多重要分子，包括MHC I类，整合素，src和连环蛋白，已经被观察到通过这一途径运输。这种降解途径可能被已知影响溶酶体靶向机制的病毒或致病菌利用或破坏。

The ADP-ribosylation factors (Arfs) are a family of GTP binding proteins that regulate membrane traffic and organelle structure in the cell. We have been studying the cellular function of Arf6, an Arf that affects membrane movement between the plasma membrane and an endosomal compartment and the actin cytoskeleton. In many cells this pathway is the route followed by membrane proteins that lack clathrin localization sequences and hence are endocytosed into cells by non-clathrin mechanisms. Recycling of these membrane proteins back to the plasma membrane requires Arf6 activation whereas entry of these proteins into the endosomal recycling system requires hydrolysis of GTP-bound Arf6. While this pathway has been shown to influence plasma membrane remodeling events such as phagocytosis, and cell spreading and migration, the molecules involved in mediating Arf6 responses remain to be identified. Membrane phosphoinositides, such as phosphatidylinositol-4,5-bisphosphate (PIP2), have been implicated in multiple processes such as membrane trafficking and actin remodeling. We have investigated the subcellular distribution of PIP2 and its role in Arf6 function. We find that Arf6 regulates the localization of PIP2 within the cell. There is also evidence that Arf6, when activated, may stimulate the activity of an enzyme, phosphatidylinositol 4 phosphate 5-kinase, responsible for generating PIP2 on membranes. Indeed, we find that overexpression of this enzyme in cells recreates a phenotype similar to that observed when activated Arf6 mutants are expressed in cells. Notably, these cells accumulate massive amounts of PIP2-enriched plasma membrane in vacuolar-like structures that are coated with actin. Ordinarily, the inactivation of Arf6, would prevent such aberrant vacuole accumulations from forming. In a collaboration with Paul Randazzo at the NCI, we have identified two new Arf GAP molecules ACAP1 and ACAP2, that function to inactivate Arf6. Thus, Arf6 activation and inactivation may mediate the regulation of PIP2 synthesis and/or PIP2 trafficking at defined membrane locations. Another project in the lab concerns the fate of proteins that move through the Arf6 endosome. We find that proteins that traverse this pathway get degraded in lysosomes and gain access to the lysosomes via the Arf6 endosome. This alternative route to the lysosome contrasts to the route followed by proteins that are endocytosed into cells by clathrin-mediated mechanisms. Both pathways do converge, however, in a late endosomal compartment. We are in the process of further characterizing this alternative route to the lysosome since many important molecules, including MHC class I, integrins, src, and catenins, have been observed to traffic through this pathway. This route to degradation may be utilized or subverted by viruses or pathogenic bacteria known to influence lysosomal targeting mechanisms.