Functional characterization of protein-sorting machineries at the trans-Golgi network in Drosophila melanogaster
Functional characterization of protein-sorting machineries at the trans-Golgi network in Drosophila melanogaster
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DOI：
10.1242/jcs.055103
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发表时间：
2010-02-01
影响因子：
4
通讯作者：
Waguri, Satoshi
中科院分区：
文献类型：
--
作者：
Kametaka, Satoshi;Sawada, Naoki;Waguri, Satoshi
将蛋白质靶向其最终目的地是活细胞维持其稳态的先决条件。网格蛋白的功能是作为一个外壳，形成运输载体称为网格蛋白包被的囊泡（CCV）在质膜和后高尔基室。在这项研究中，我们建立了一个实验系统，使用施耐德S2细胞来自果蝇，黑腹果蝇，作为一个模型系统，研究网格蛋白衔接子的生理作用，并剖析CCV的形成过程。我们发现，网格蛋白适配器果蝇GGA（dGGA），哺乳动物GGA蛋白的同源物，定位到trans-Golgi网络（TGN），并能够招募网格蛋白从胞质到TGN膜。dGGA本身以ARF 1小GT3（dARF 79 F）依赖性方式从胞质溶胶募集到TGN。dGGA识别Lerp（溶酶体酶受体蛋白）的细胞质酸性簇双亮氨酸（ACLL）分选信号，Lerp是哺乳动物甘露糖6-磷酸受体的同系物。此外，dGGA和另一种类型的TGN-定位网格蛋白衔接子AP-1（衔接蛋白-1复合物），被证明参与Lerp从TGN到内体和/或溶酶体的运输。两者合计，我们的研究结果表明，在苍蝇细胞中的蛋白质分选机制是保守的相对于哺乳动物，使使用苍蝇细胞解剖CCV的生物发生和网格蛋白依赖的蛋白质运输在TGN的高等真核生物。
Targeting of proteins to their final destination is a prerequisite for living cells to maintain their homeostasis. Clathrin functions as a coat that forms transport carriers called clathrin-coated vesicles (CCVs) at the plasma membrane and post-Golgi compartments. In this study, we established an experimental system using Schneider S2 cells derived from the fruit fly, Drosophila melanogaster, as a model system to study the physiological roles of clathrin adaptors, and to dissect the processes of CCV formation. We found that a clathrin adaptor Drosophila GGA (dGGA), a homolog of mammalian GGA proteins, localizes to the trans-Golgi network (TGN) and is capable of recruiting clathrin from the cytosol onto TGN membranes. dGGA itself is recruited from the cytosol to the TGN in an ARF1 small GTPase (dARF79F)-dependent manner. dGGA recognizes the cytoplasmic acidic-cluster-dileucine (ACLL) sorting signal of Lerp ( lysosomal enzyme receptor protein), a homolog of mammalian mannose 6-phosphate receptors. Moreover, both dGGA and another type of TGN-localized clathrin adaptor, AP-1 (adaptor protein-1 complex), are shown to be involved in the trafficking of Lerp from the TGN to endosomes and/or lysosomes. Taken together, our findings indicate that the protein-sorting machinery in fly cells is well conserved relative to that in mammals, enabling the use of fly cells to dissect CCV biogenesis and clathrin-dependent protein trafficking at the TGN of higher eukaryotes.