Clathrin assembly lymphoid myeloid leukemia (CALM) protein: Localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated traffic
Clathrin assembly lymphoid myeloid leukemia (CALM) protein: Localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated traffic
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DOI：
10.1091/mbc.10.8.2687
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发表时间：
1999-08-01
影响因子：
3.3
通讯作者：
Sorkin, A
中科院分区：
文献类型：
--
作者：
Tebar, F;Bohlander, SK;Sorkin, A
网格蛋白组装淋巴样髓性白血病（CALM）基因编码网格蛋白组装突触蛋白AP 180的推定同源物。因此，CALM蛋白的生化特性，亚细胞定位，和内吞作用的研究。在体外结合和免疫共沉淀表明，网格蛋白重链是CALM的主要结合伴侣。大部分细胞CALM与细胞的膜组分相关，并定位于质膜的网格蛋白包被区域。在膜级分中，CALM相对于网格蛋白以接近化学计量的量存在。为了进行CALM的结构-功能分析，我们设计了CALM及其片段与绿色荧光蛋白（GFP）的嵌合融合蛋白。GFP-CALM靶向质膜包被的凹坑，也发现与网格蛋白共定位在高尔基体区域。高水平表达GFP-CALM或其具有网格蛋白结合活性的片段抑制转铁蛋白和表皮生长因子受体的内吞作用，并改变细胞中甘露糖-6-磷酸受体的稳态分布。此外，GFP-CALM过表达导致trans-Golgi网络区域中网格蛋白积累的损失，而trans-Golgi网络中网格蛋白衔接蛋白复合物1的定位保持不受影响。GFP标记的CALM片段影响网格蛋白介导的过程的能力与片段靶向网格蛋白包被区域及其网格蛋白结合能力相关。网格蛋白-CALM相互作用似乎受到多个接触界面的调节。CALM的C-末端部分结合网格蛋白重链，尽管全长蛋白表现出最大的相互作用能力。总之，数据表明，CALM是一个重要的组成部分，包衣坑内化机制，可能参与调节网格蛋白招聘的膜和/或形成的包衣坑。
The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. in the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure-function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP-CALM was targeted to the plasma membrane-coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP-CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. Ln addition, GFP-CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin-CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.