Identification and Functional Characterization of Human Arf-Like Proteins 1 (HArl1) Protein Complex in Vesicle Trafficking

ADP-ribosylation factors (ARFs) and ARF-like (ARL) proteins are a distinct subfamily of Ras small GTPases. ARFs, which have ~60% identity to each other, have been proposed to function through the direct activation of enzyme activities, the local alterations in lipid composition, or the recruitment of proteins to membranes, particularly vesicle-coat complexes or proteins. These proteins are regulated by guanine-nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) to cycle between active GTP-bound form and inactive GDT-bound form. ARL1 is a member of the ARF/ARL family of the Ras-like GTPase superfamily. Despite high similarity between ARLs and ARFs, among the ARL proteins only ARL1 has been shown to be involved in vesicle trafficking. ARL1 is enriched in the trans-Golgi network (TGN) and been proposed involved in endocytosis and exocytosis. According to our preliminary study, we have found particular human ARL1 interacting protein via yeast two-hybrid and provided its role in trafficking between TGN and plasma membrane. But limited knowledge about the functional and regulating specificity of these efforts. Neither GEFs nor GAPs have been characterized in vivo for their specificity to human ARL1 (hARL1). Base on our previous study and literature search, we expect there are undefined proteins/effectors interacting with ARL1, and the complexes were regulated to fit the needs of individual trafficking steps and particular physiological states. In this proposal, we will identification and characterization the ARL1 protein complex systemically and to understand the interaction networks involved in the trafficking regulation. In addition, according to our findings obtained from last year, we suggest that human type 4 subfamily of P-type ATPases will involve in the regulation of Golgi structure or golgins targeting to Golgi apparatus. We will combine molecular biology, proteomics, bioinformatics, biochemistry, and cell biology to reveal one of vesicle trafficking pathway regulated by human ARL1. Our specific aims are: 1. Identification of hARL1 protein complex by combination of affinity purification, chemical cross-linking and mass spectrometry. 2. Characterize the potential function and regulation of human type 4 subfamily of P-type ATPases in vesicular transportation. 3. Characterize the structure and function of human ARL1 and its interacting proteins. 4. Study the regulation of ARL1 and their interacting molecules in vesicular transport or membrane trafficking. The long-term goal of this project is demonstrate how the human ARL1 and its interacting proteins play an important role in vesicle trafficking. The proposed research is significant in that: 1) It will offer an opportunity to achieve the comprehensive discovery of human ARL1 protein complex 2) It will provide the fundamental knowledge to explore the interacting network of ARL1. 3) It will contribute to molecular mechanism of ARL1 function in the vesicle trafficking.

Project ID:PC9801-2496
External Project ID:NSC97-2320-B182-026-MY3