第一腺嘌呤核苷二磷酸核醣化因子相似蛋白在 囊泡運輸中的功能性探討

Abstract

Drs2p 為一類的P 型三磷酸腺脢，位在高機氏體上且具有翻轉磷脂質絲胺酸(PS) 的能力。Gea2p 為腺嘌呤核苷二磷酸核醣化因子交換因子，會與Drs2p 結合並且 影響Drs2p 翻轉酶的活性。本文我們發現第一腺嘌呤核苷二磷酸核醣化因子相 似蛋白(Arl1p)會和Gea2p 共同去調控Drs2p 的翻轉酶活性。我們發現Drs2p 和 Gea2p 都會去影響Arl1p 的下游基因Imh1p 在細胞中的分佈。Drs2p 會和鳥糞呤 核苷三磷酸 (GTP) 形式的Arl1p 結合，當Drs2p 無法與Arl1p 結合，會影響其翻 轉酶的活性。Gea2p 用利用其N 端與Arl1p 結合，當其無法與Arl1p 結合時，也 會影響高基氏體上磷酸絲氨酸的轉位，但是不會影響到Gea2p 腺嘌呤核苷二磷 酸核醣化因子交換因子的活性。此外，Drs2p 和Gea2p 的結合需要有Arl1p 的存 在。因此我們的研究發現在特定高基氏體位置的Drs2p 活性會被Arl1p 以及Gea2p 所調控並且影響到Imh1p 在高基氏體上的分佈。

Drs2p, a resident type 4 P-type ATPase (P4-ATPase), requires for a phosphatidylserine (PS) flippase activity in the yeast trans Golgi network (TGN) and plays essential roles in protein transport in the secretory and endocytic pathways. The Arf activator Gea2p interacts with Drs2p and stimulates its flippase activity in yeast TGN. Here we show that the ARF-like (ARL) protein, Arl1p, acts with Gea2p to modulate Drs2p activity at the TGN. We found that gea2- and drs2-null mutants, like arl1-null, exhibits severe defects in recruitment of Imh1p to the Golgi. Arl1p directly interacts with N-terminus of Drs2p in a GTP-dependent manner. Deletion of the Arl1p-interacting region of Drs2p results in a significant decrease of its flippase activity. In addition, the active form of Arl1p directly interacts with N-terminus of Gea2p. Deletion of the Arl1p-interacting region GEA2 impaired PS translocation on the TGN membranes, but appears to keep its GEF function for Arf. Deletion of ARL1 impairs Gea2p-Drs2p interaction. Thus, we infer that subcellular spatial regulation of flippase Drs2p by Arl1p and Arf-GEF Gea2p controls membrane dynamics at the trans-Golgi network.