Miller Sharon E, Mathiasen Signe, Bright Nicholas A, Pierre Fabienne, Kelly Bernard T, Kladt Nikolay, Schauss Astrid, Merrifield Christien J, Stamou Dimitrios, Höning Stefan, Owen David J
Cambridge Institute for Medical Research and Department of Clinical Biochemistry, University of Cambridge, Cambridge Biomedical Campus, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK.
Bionanotechnology and Nanomedicine Laboratory, Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark.
Dev Cell. 2015 Apr 20;33(2):163-75. doi: 10.1016/j.devcel.2015.03.002.
The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of "open" clathrin-coated pits (CCPs) to "necked"/"closed" CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake.
内吞网格蛋白包被小泡（CCV）的大小非常均匀，这表明其大小经过优化，以实现适当水平的货物和脂质内化。哺乳动物内吞CCV中含量最丰富的三种蛋白质是网格蛋白以及两种货物选择型网格蛋白衔接蛋白CALM和AP2。在此我们证明，CALM的缺失会导致“开放”网格蛋白包被小窝（CCP）与“缢缩”/“封闭”CCV的比例大幅增加，且CCP/CCV直径加倍，而AP2的缺失则产生相反的效果。然而，任一衔接蛋白的缺失都会显著抑制转铁蛋白和表皮生长因子的内吞作用。CALM缺失的表型效应可通过野生型CALM的重新表达得以挽救，但不能通过缺乏功能性N端、膜插入、曲率感知/驱动两亲螺旋的CALM来挽救，本文证明了该螺旋的存在及其特性。因此，CALM是控制CCV大小和成熟的主要因素，从而也是决定内吞货物摄取速率的主要因素。
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