| Summary: | 碩士 === 國立臺灣大學 === 分子醫學研究所 === 101 === ADP-ribosylation factor-like protein 4A (ARL4A) is a member of the ARF/ARL small GTPases family. The ARF/ARL family is well-known for its functions on vesicular trafficking, cytoskeleton reorganization and organelle morphogenesis. ARL4A has been found to participate in regulations of membrane ruffling, cytoskeleton remodeling and Golgi organization; however, whether ARL4A is directly involved in vesicular trafficking remains unclear. In order to explore the new functions of ARL4A, we used a more accurate inactive GDP-bound mutant ARL4A T51N as bait and performed yeast two-hybrid screening to identify its novel interacting proteins. We have identified 12 potential interacting proteins of ARL4A T51N, and one of them is the subunit of Endosomal Sorting Complex Required for Transport (ESCRT) complex. ESCRT complexes, including ESCRT-0, -I, -II, -III, and VPS4, are involved in sorting of ubiqutinated endosomal membrane proteins into the multivesicular body (MVB), thus are required for their degradation. We first demonstrated the interaction between ARL4A and ESCRT subunit in vitro using yeast two-hybrid assay and γGTP-, GDP-loaded GST pull-down assay. Next, we studied their co-localization in vivo by immunofluorescence staining assay, and followed with monitoring the MVB pathway using epidermal growth factor (EGF) -stimulated EGF receptor (EGFR) degradation assay. Finally, we also studied the MVB biogenesis using NRhPE labeling assay. We found that the ESCRT subunit prefers interact with GDP-bound ARL4A in vitro and co-localized with membrane-associated ARL4As in perinuclear region and plasma membrane in vivo. We also found that ARL4A is involved in MVB biogenesis and EGF internalization as well as EGFR turnover in HeLa cells. Depletion of ARL4A in HeLa cells speeds up the EGF-stimulated EGFR degradation. By contrast, overexpression of ARL4A in HeLa cells delays the degradation of EGFR and defects the formation of MVB. These results imply that ARL4A may play a negative regulatory role in MVB-dependent EGFR degradation pathway through interacting with the ESCRT subunit.
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