Department of Cell Biology, Emory University, Atlanta, GA 30322, USA.
Mol Biol Cell. 2013 Aug;24(15):2378-88. doi: 10.1091/mbc.E12-12-0860. Epub 2013 Jun 12.
A role for clathrin in AP-3-dependent vesicle biogenesis has been inferred from biochemical interactions and colocalization between this adaptor and clathrin. The functionality of these molecular associations, however, is controversial. We comprehensively explore the role of clathrin in AP-3-dependent vesicle budding, using rapid chemical-genetic perturbation of clathrin function with a clathrin light chain-FKBP chimera oligomerizable by the drug AP20187. We find that AP-3 interacts and colocalizes with endogenous and recombinant FKBP chimeric clathrin polypeptides in PC12-cell endosomes. AP-3 displays, however, a divergent behavior from AP-1, AP-2, and clathrin chains. AP-3 cofractionates with clathrin-coated vesicle fractions isolated from PC12 cells even after clathrin function is acutely inhibited by AP20187. We predicted that AP20187 would inhibit AP-3 vesicle formation from endosomes after a brefeldin A block. AP-3 vesicle formation continued, however, after brefeldin A wash-out despite impairment of clathrin function by AP20187. These findings indicate that AP-3-clathrin association is dispensable for endosomal AP-3 vesicle budding and suggest that endosomal AP-3-clathrin interactions differ from those by which AP-1 and AP-2 adaptors productively engage clathrin in vesicle biogenesis.
网格蛋白在 AP-3 依赖的囊泡生物发生中的作用是通过生化相互作用和该衔接蛋白与网格蛋白的共定位推断出来的。然而，这些分子相互作用的功能存在争议。我们使用化学遗传方法快速干扰网格蛋白的功能，用药物 AP20187 聚合网格蛋白轻链-FKBP 嵌合体，全面研究了网格蛋白在 AP-3 依赖的囊泡出芽中的作用。我们发现 AP-3 在 PC12 细胞内体中与内源性和重组 FKBP 嵌合网格蛋白多肽相互作用并共定位。然而，AP-3 的行为与 AP-1、AP-2 和网格蛋白链不同。AP-3 与从小鼠 PC12 细胞中分离的网格蛋白包被小泡级分共洗脱，即使在 AP20187 急性抑制网格蛋白功能后也是如此。我们预测 AP20187 在布雷菲德菌素 A 阻断后会抑制内体 AP-3 囊泡的形成。然而，尽管 AP20187 损害了网格蛋白的功能，但 AP-3 囊泡的形成在布雷菲德菌素 A 洗脱后仍继续进行。这些发现表明 AP-3-网格蛋白的关联对于内体 AP-3 囊泡的出芽不是必需的，并表明内体 AP-3-网格蛋白的相互作用与 AP-1 和 AP-2 衔接蛋白在囊泡生物发生中有效地与网格蛋白结合的相互作用不同。
