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: 6817646
• 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/6817646
• 关键词: ADP ribosylation; actins; binding proteins; cell morphology; cell motility; clathrin; cytoskeleton; 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. 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.

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。通过这种不依赖网格蛋白的途径内吞的PM蛋白可以被再循环回PM或与“经典”早期内体途径聚合并在溶酶体中降解。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）。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。我们发现一些脂筏相关蛋白，那些通过糖基磷脂酰肌醇（GPI）片段锚定在膜上的蛋白，也通过这种不依赖网格蛋白的机制进入细胞，并与Rab5早期内体汇合或与MHCI一起循环回到PM。目前正在开发检测方法，以在体外重建这些膜运输事件。