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

Characterization of ADP-Ribosylation Factor-Like Protein 1 in vesicle trafficking

蔡佩秦(Pei-Chin Tsai)

指導教授 ： 李芳仁

國立臺灣大學/醫學院/分子醫學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.00188

摘要

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。

關鍵字

第一腺嘌呤核苷二磷酸核醣化因子相似蛋白 ； 囊泡運輸

並列摘要

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.

並列關鍵字

Arl1p ； vesicle trafficking

參考文獻

Alder-Baerens N, Lisman Q, Luong L, Pomorski T, Holthuis JC (2006) Loss of P4 ATPases Drs2p and Dnf3p disrupts aminophospholipid transport and asymmetry in yeast post-Golgi secretory vesicles. Mol Biol Cell 17: 1632-1642

Amor JC, Horton JR, Zhu X, Wang Y, Sullards C, Ringe D, Cheng X, Kahn RA (2001) Structures of yeast ARF2 and ARL1: distinct roles for the N terminus in the structure and function of ARF family GTPases. J Biol Chem 276: 42477-42484

Antonny B, Beraud-Dufour S, Chardin P, Chabre M (1997) N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange. Biochemistry 36: 4675-4684

Axelsen KB, Palmgren MG (1998) Evolution of substrate specificities in the P-type ATPase superfamily. J Mol Evol 46: 84-101

Barlowe C, Orci L, Yeung T, Hosobuchi M, Hamamoto S, Salama N, Rexach MF, Ravazzola M, Amherdt M, Schekman R (1994) COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77: 895-907