High-affinity binding of the AP-1 adaptor complex to trans-golgi network membranes devoid of mannose 6-phosphate receptors.
High-affinity binding of the AP-1 adaptor complex to trans-golgi network membranes devoid of mannose 6-phosphate receptors.
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AP-1 连接体复合物与不含 6-磷酸甘露糖受体的跨高尔基体网络膜高亲和力结合。
DOI：
10.1091/mbc.10.3.537
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发表时间：
1999
影响因子：
3.3
通讯作者：
Kornfeld,S
中科院分区：
文献类型：
--
作者：
Zhu,Y;Traub,LM;Kornfeld,S
GTP 结合蛋白 ADP 核糖基化因子 (ARF) 通过为 AP-1 接头复合物生成高亲和力膜结合位点，启动跨 Goli 网络 (TGN) 处的网格蛋白涂层组装。被分选到组装包被芽中的跨膜蛋白和新型对接蛋白均被认为与 GTP 结合的 ARF 一起生成 AP-1 对接位点。最具特征性的、也可能是分类到 TGN 上形成的网格蛋白包被囊泡中的主要跨膜分子是甘露糖 6-磷酸受体 (MPR)。在这里，我们通过比较来自正常或 MPR 阴性动物胚胎的成纤维细胞，研究了 MPR 在 AP-1 募集过程中的作用。尽管 MPR 缺陷细胞中的溶酶体区室发生了重大改变，但 AP-1 在 TGN 上的稳态分布与正常细胞相当。在 ARF 和 GTP 或 GTPγS 存在的情况下，由受体阴性细胞制备的富含高尔基体的膜也表现出明显正常的体外募集 AP-1 的能力。 AP-1 接头被特异性地募集到 TGN 上，而不是募集到 MPR 阴性成纤维细胞内积累的众多异常膜元件上。与来自正常或 MPR 阴性成纤维细胞的 TGN 膜结合的 AP-1 对 1 M Tris-HCl（pH 7）的化学提取具有完全抵抗力，表明该接头以高亲和力与两种膜类型结合。我们注意到，从 MPR 缺陷细胞制备的高尔基体与正常细胞之间的唯一区别是，在 ARF1·GTP 存在的情况下，招募到缺乏受体的膜上的 AP-1 始终对 Tris 提取具有更强的抵抗力。由于 Tris 提取的敏感性与核苷酸水解密切相关，这一发现可能表明 MPR 分选和 ARF GAP 调节之间可能存在联系。我们得出的结论是，MPR 并不是 AP-1 与 TGN 结合的初始步骤中的重要决定因素，而是可能通过影响 ARF·GTP 水解，在网格蛋白包被的囊泡形成中发挥调节作用。
The GTP-binding protein ADP-ribosylation factor (ARF) initiates clathrin-coat assembly at the trans-Goli network (TGN) by generating high-affinity membrane-binding sites for the AP-1 adaptor complex. Both transmembrane proteins, which are sorted into the assembling coated bud, and novel docking proteins have been suggested to be partners with GTP-bound ARF in generating the AP-1-docking sites. The best characterized, and probably the major transmembrane molecules sorted into the clathrin-coated vesicles that form on the TGN, are the mannose 6-phosphate receptors (MPRs). Here, we have examined the role of the MPRs in the AP-1 recruitment process by comparing fibroblasts derived from embryos of either normal or MPR-negative animals. Despite major alterations to the lysosome compartment in the MPR-deficient cells, the steady-state distribution of AP-1 at the TGN is comparable to that of normal cells. Golgi-enriched membranes prepared from the receptor-negative cells also display an apparently normal capacity to recruit AP-1 in vitro in the presence of ARF and either GTP or GTPγS. The AP-1 adaptor is recruited specifically onto the TGN and not onto the numerous abnormal membrane elements that accumulate within the MPR-negative fibroblasts. AP-1 bound to TGN membranes from either normal or MPR-negative fibroblasts is fully resistant to chemical extraction with 1 M Tris-HCl, pH 7, indicating that the adaptor binds to both membrane types with high affinity. The only difference we do note between the Golgi prepared from the MPR-deficient cells and the normal cells is that AP-1 recruited onto the receptor-lacking membranes in the presence of ARF1·GTP is consistently more resistant to extraction with Tris. Because sensitivity to Tris extraction correlates well with nucleotide hydrolysis, this finding might suggest a possible link between MPR sorting and ARF GAP regulation. We conclude that the MPRs are not essential determinants in the initial steps of AP-1 binding to the TGN but, instead, they may play a regulatory role in clathrin-coated vesicle formation by affecting ARF·GTP hydrolysis.