Oligomerization and dissociation of AP-1 adaptors are regulated by cargo signals and by ArfGAP1-induced GTP hydrolysis
Oligomerization and dissociation of AP-1 adaptors are regulated by cargo signals and by ArfGAP1-induced GTP hydrolysis
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DOI：
10.1091/mbc.e05-06-0568
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发表时间：
2005-10-01
影响因子：
3.3
通讯作者：
Spiess, M
中科院分区：
文献类型：
--
作者：
Meyer, DM;Crottet, P;Spiess, M
使用纯化的衔接蛋白和呈现酪氨酸分选信号的合成脂质体分析AP-1/网格蛋白外壳形成的机制。在Arf 1中心点GTP和分选信号的存在下招募的AP-1衔接子被发现即使在没有网格蛋白的情况下也会寡聚成高分子量复合物。AP-1衔接素的附属结构域不需要寡聚化。在GTP酶激活蛋白ArfGAP 1诱导的GTP水解中，复合物被分解，AP-1从膜上解离。AP-1刺激ArfGAP 1活性，提示AP-1在调节Arf 1-GT β计时器中的作用。“在胞质溶胶的存在下，AP-1可以在没有分选信号的情况下被招募到脂质体中，这与胞质溶胶中存在的对接因子一致。然而，在这些条件下，AP-1保持单体，在GTP的存在下的招聘是短暂的。分选信号允许稳定的招聘和寡聚化，也在胞质溶胶的存在下。这些结果表明，AP-1与Arf 1中心点GTP和ArfGAP 1在膜上的初始组装刺激Arf 1 GTP酶活性，而与货物的相互作用诱导寡聚化并降低GTP水解速率，从而有助于有效的货物分选的机制。
The mechanism of AP-1/clathrin coat formation was analyzed using purified adaptor proteins and synthetic liposomes presenting tyrosine sorting signals. AP-1 adaptors recruited in the presence of Arf1 center dot GTP and sorting signals were found to oligomerize to high-molecular-weight complexes even in the absence of clathrin. The appendage domains of the AP-1 adaptins were not required for oligomerization. On GTP hydrolysis induced by the GTPase-activating protein ArfGAP1, the complexes were disassembled and AP-1 dissociated from the membrane. AP-1 stimulated ArfGAP1 activity, suggesting a role of AP-1 in the regulation of the Arf1 "GTPase timer." In the presence of cytosol, AP-1 could be recruited to liposomes without sorting signals, consistent with the existence of docking factors in the cytosol. Under these conditions, however, AP-1 remained monomeric, and recruitment in the presence of GTP was short-lived. Sorting signals allowed stable recruitment and oligomerization also in the presence of cytosol. These results suggest a mechanism whereby initial assembly of AP-1 with Arf1 center dot GTP and ArfGAP1 on the membrane stimulates Arf1 GTPase activity, whereas interaction with cargo induces oligomerization and reduces the rate of GTP hydrolysis, thus contributing to efficient cargo sorting.