ARF1•GTP, tyrosine-based signals, and phosphatidylinositol 4,5-bisphosphate constitute a minimal machinery to recruit the AP-1 clathrin adaptor to membranes
ARF1•GTP, tyrosine-based signals, and phosphatidylinositol 4,5-bisphosphate constitute a minimal machinery to recruit the AP-1 clathrin adaptor to membranes
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DOI：
10.1091/mbc.e02-05-0309
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发表时间：
2002-10-01
影响因子：
3.3
通讯作者：
Spiess, M
中科院分区：
文献类型：
--
作者：
Crottet, P;Meyer, DM;Spiess, M
在反式高尔基网络中，含有AP-1接头复合物的网格蛋白外壳以arf1依赖的方式形成，产生将货物蛋白运送到核内体的囊泡。AP-1位点特异性靶向的机制和cargo的作用尚不清楚。我们已经开发了一种体外实验来研究纯化AP-1接头在化学上定义的脂质体上的募集，脂质体呈现与酪氨酸基分类基序相对应的肽。发现AP-1的募集依赖于豆芽糖酰化的ARF1、GTP或不可水解的GTP类似物、酪氨酸信号和少量磷酸肌醇，最突出的是磷脂酰肌醇4,5-二磷酸，在没有任何额外的细胞质或膜结合蛋白的情况下。胞浆中的AP-1可以独立于脂质组成被募集到酪氨酸信号中，但磷脂酰肌醇45-二磷酸增加了募集率。因此，结果表明，货物蛋白参与了外壳的招募，而局部脂质组成有助于确定囊泡形成的位置。
At the trans-Golgi network, clathrin coats containing AP-1 adaptor complexes are formed in an ARF1-dependent manner, generating vesicles transporting cargo proteins to endosomes. The mechanism of site-specific targeting of AP-1 and the role of cargo are poorly understood. We have developed an in vitro assay to study the recruitment of purified AP-1 adaptors to chemically defined liposomes presenting peptides corresponding to tyrosine-based sorting motifs. AP-1 recruitment was found to be dependent on myristoylated ARF1, GTP or nonhydrolyzable GTP-analogs, tyrosine signals, and small amounts of phosphoinositides, most prominently phosphatidylinositol 4,5-bisphosphate, in the absence of any additional cytosolic or membrane bound proteins. AP-1 from cytosol could be recruited to a tyrosine signal independently of the lipid composition, but the rate of recruitment was increased by phosphatidylinositol 45-bisphosphate. The results thus indicate that cargo proteins are involved in coat recruitment and that the local lipid composition contributes to specifying the site of vesicle formation.