Loss of P4 ATPases Drs2p and Dnf3p disrupts aminophospholipid transport and asymmetry in yeast post-Golgi secretory vesicles
Loss of P4 ATPases Drs2p and Dnf3p disrupts aminophospholipid transport and asymmetry in yeast post-Golgi secretory vesicles
复制标题
DOI：
10.1091/mbc.e05-10-0912
复制
发表时间：
2006-04-01
影响因子：
3.3
通讯作者：
Holthuis, JCM
中科院分区：
文献类型：
--
作者：
Alder-Baerens, N;Lisman, Q;Holthuis, JCM
真核生物的质膜通常显示不对称的脂质分布，氨基磷脂集中在细胞质小叶中。这种排列是由氨基磷脂转位酶（APLTs）维持的，它利用ATP水解将磷脂酰丝氨酸（PS）和磷脂酰乙醇胺（PE）从外部翻转到细胞质小叶。atp的身份尚未确定，但主要的候选者是p型atp酶的P4亚家族成员。从出芽酵母中去除P4 atp酶Dnf1p和Dnf2p可消除6-[（7-硝基苯-2-氧-1,3-二唑-4-酰基）氨基丙基]（NBD）标记的PS、PE和磷脂酰胆碱（PC）在质膜上的向内移位，并导致内源性PE的细胞表面暴露。在这里，我们发现酵母高尔基分泌囊泡（SVs）含有转位酶活性，将NBD-PS， NBD-PE和NBD-PC翻转到细胞质小叶。这种活性独立于Dnf1p和Dnf2p，但需要另外两个P4 atp酶，Drs2p和Dnf3p，它们主要存在于反式高尔基网络中。此外，sv具有不对称的PE排列，在去除Drs2p和Dnf3p后会丢失。我们的研究结果表明，当膜流过高尔基体时，会产生氨基磷脂不对称，而p4 - atp酶对这一过程至关重要。
Eukaryotic plasma membranes generally display asymmetric lipid distributions with the aminophospholipids concentrated in the cytosolic leaflet. This arrangement is maintained by aminophospholipid translocases (APLTs) that use ATP hydrolysis to flip phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external to the cytosolic leaflet. The identity of APLTs has not been established, but prime candidates are members of the P4 subfamily of P-type ATPases. Removal of P4 ATPases Dnf1p and Dnf2p from budding yeast abolishes inward translocation of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS, PE, and phosphatidylcholine (PC) across the plasma membrane and causes cell surface exposure of endogenous PE. Here, we show that yeast post-Golgi secretory vesicles (SVs) contain a translocase activity that flips NBD-PS, NBD-PE, and NBD-PC to the cytosolic leaflet. This activity is independent of Dnf1p and Dnf2p but requires two other P4 ATPases, Drs2p and Dnf3p, that reside primarily in the trans-Golgi network. Moreover, SVs have an asymmetric PE arrangement that is lost upon removal of Drs2p and Dnf3p. Our results indicate that aminophospholipid asymmetry is created when membrane flows through the Golgi and that P4-ATPases are essential for this process.