Inhibition of calcium-independent mannose 6-phosphate receptor incorporation into trans-Golgi network-derived clathrin-coated vesicles by Wortmannin
Inhibition of calcium-independent mannose 6-phosphate receptor incorporation into trans-Golgi network-derived clathrin-coated vesicles by Wortmannin
复制标题
DOI：
10.1074/jbc.272.39.24170
复制
发表时间：
1997-09-26
影响因子：
4.8
通讯作者：
Clague, MJ
中科院分区：
文献类型：
--
作者：
Gaffet, P;Jones, AT;Clague, MJ
catepsin D前体从反式高尔基体网络（TGN）转运至内体途径依赖于与钙非依赖性甘露糖6-磷酸受体（ci-M6 PR）的结合，该受体被整合到TGN衍生的网格蛋白包被的转运囊泡（CCV）中。渥曼青霉素对该转运步骤的抑制已经导致了这样的建议，即它依赖于ci-M6 PR募集到TGMs衍生的CCV中或形成那些囊泡所必需的磷酸肌醇3-激酶活性（Brown，W.，J，，DeWald，D，B，，Emr，S. D，，Plutner，H.，和Balch，W，E，（1995）J. Cell Biol.130，781-796; Davidson，H，W.（1995）J. Cell Biol.130，781-796）。在该研究中，我们已经讨论了渥曼青霉素对ci-M6 PR循环的TGN步骤的影响。在平衡密度梯度上纯化来自H562细胞的CCV，用250 nM渥曼青霉素预处理或未预处理。通过纯化的囊泡级分中的γ-适应蛋白含量评估的TGN衍生的CCV的定量显示，在用药物处理20分钟后，囊泡的形成仅略微减少，而对于相同的渥曼青霉素处理，与对照相比，募集到那些囊泡中的ci-M6 PR的药物减少了70%。在稍后的时间点（2小时），还观察到CCV级分中γ-适应蛋白的量减少。这些发现表明，抑制c1-M6 PR募集到CCV中而不是抑制囊泡形成是观察到渥曼青霉素处理后组织蛋白酶D转运缺陷的主要原因。
The transport of pro-catepsin D from the trans-Golgi network (TGN) to the endosomal pathway is dependent on binding to the calcium-independent mannose 6-phosphate receptor (ci-M6PR), which is incorporated into TGN-derived clathrin-coated transport vesicles (CCVs). Inhibition of this transport step by wortmannin has led to the proposal that it is dependent upon a phosphoinositide 3-kinase activity necessary for ci-M6PR recruitment into TGM-derived CCVs or in the formation of those vesicles (Brown, W., J,, DeWald, D, B,, Emr, S. D,, Plutner, H., and Balch, W, E, (1995) J. Cell Biol. 130, 781-796; Davidson, H, W. (1995) J. Cell Biol. 130, 781-796), In this study we have addressed the effect of wortmannin on the TGN step of the ci-M6PR, cycle, CCVs from H562 cells, pretreated or not with 250 nM wortmannin, were purified on equilibrium density gradients. Quantification of TGN-derived CCVs, assessed by gamma-adaptin content in purified vesicle fractions, showed that the formation of the vesicles was only marginally decreased after 20 min of treatment with the drug, while for the same wortmannin treatment, the drug of ci-M6PR recruited into those vesicles was decreased by 70% compared with control, At a later time point (2 h), a reduction in the amount of gamma-adaptin in CCV fractions was also observed, These findings demonstrate that inhibition of ci-M6PR recruitment into CCVs but not of vesicle formation is the primary reason for the observed defect in cathepsin D transport following wortmannin treatment.