第五腺嘌呤核苷二磷酸核糖化相似因子與其結合蛋白EB1之特性探討

Abstract

腺嘌呤核苷二磷酸核醣化因子相似蛋白 (ARL) 為腺嘌呤核苷二磷酸核醣化因子 (ARF) 家族成員之一。目前對於人類ARL蛋白質群中ARL5蛋白質的功能仍不清楚。因此，在本論文研究中，我們將探討此蛋白質之生物特性以及其功能。ARL5蛋白質廣泛地表現於許多哺乳動物細胞株中。在整個細胞週期中，除了胞質分裂時期座落於中體 (midbody) 之外，COS-7細胞株中內生性 (endogenous) ARL5以及與綠色螢光蛋白質 (GFP) 融合之GFP-ARL5皆座落於中心體 (centrosome) 上。藉由酵母菌雙雜合篩檢 (yeast two-hybrid screen) 和活體內蛋白質結合反應試驗 (in vivo protein interaction) 證實ARL5可與一微小管結合蛋白EB1結合。利用RNA干擾 (RNAi) 技術調降（knockdown）ARL5蛋白質表達後，COS-7細胞株微小管 (microtubule) 自中心粒再生 (regrowth) 以及錨定 (anchoring) 之能力均受到影響。該影響與調降EB1蛋白質表達後之現象雷同。此外，大量表現之野生型 (wild type) ARL5或者其活化型突變蛋白質 (ARL5Q80L) 皆均勻散佈於細胞中，且部分座落於內小體 (endosome)；而ARL5非活型突變蛋白質 (ARL5T35N) 在COS-7細胞株中，則有許多不同的分佈型式，包括座落於內小體、粒線體，或者形成聚集體 (aggresome)。座落於粒線體上之ARL5T35N會透過其C端造成粒線體膜電位差消失。綜合以上結果，我們推測ARL5在細胞中參與調控：微小管再生與錨定、囊泡運輸，以及蛋白質降解等作用。

ADP-ribosylation factor like (ARL) proteins are one subfamily of ADP-ribosylation factor (ARF) small GTP binding protein family. Little is known about a human ARL protein, ARL5. Here we characterized the biologic properties and functions of hARL5 and described several novel observations. ARL5 expressed ubiquitously in many mammalian cell lines. In COS-7 cells, endogenous ARL5 and overexpressed GFP-ARL5 localized to centrosomes through out the cell cycle with exception of midbody localization during cytokinesis. By yeast two-hybrid screening, a microtubule plus-end binding protein EB1 was identified as an interacting partner of ARL5. ARL5 also interacted with EB1 in vivo. To investigate the biologic functions of ARL5, RNAi knockdown was performed. In ARL5 knockdown COS-7 cells, the microtubule regrowth from and anchoring at centrosomes were delayed and disordered similar to the defects caused by EB1 knockdown. Furthermore, overexpressed wild type and constitutively active form (ARL5Q80L) of ARL5 were diffused and partially localized to endosomes in COS-7 cells. The heterogeneous distributions of ARL5 constitutively inactive form (ARL5T35N) at endosomes, mitochondria, and aggresome-like compartments provided a distinct feature of ARL5. In addition, ARL5T35N diminished mitochondria membrane potential through its C-terminal region when it localized to mitochondria. Taken together, we infer that ARL5 might regulate different cellular processes, including centrosome-mediated microtubule nucleation and anchoring, vesicle trafficking, mitochondrial function, and protein degradation.