Coronin and vacuolin identify consecutive stages of a late, actin-coated endocytic compartment in Dictyostelium
Coronin and vacuolin identify consecutive stages of a late, actin-coated endocytic compartment in Dictyostelium
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DOI：
10.1016/s0960-9822(97)70093-9
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发表时间：
1997-03-01
期刊：
影响因子：
9.2
通讯作者：
Maniak, M
中科院分区：
文献类型：
--
作者：
Rauchenberger, R;Hacker, U;Maniak, M
单细胞真核生物盘基网柄藻的细胞通过内吞作用吸收所有的营养物质，含有颗粒和液体的液泡都被细胞骨架外套短暂地包围[1，2]。当这层外壳解离时，发生囊泡内容物的酸化和消化，然后在60-90分钟后发生不可消化的残余物的胞吐作用。内吞转运的数学建模需要至少九个隔室[3]，这表明标记物仅与特定的内吞隔室关联几分钟。在已被鉴定为内吞囊泡组分的蛋白质中，V-H（+）ATP酶亚基和nab家族的小GTP结合蛋白[4-7]。使用针对盘基网柄菌内吞囊泡产生的单克隆抗体，我们分离出了与我们命名为vacuolin的新型蛋白质相对应的cDNA。为了确定沿着涉及vacuolin的内吞途径的精确沿着步骤，我们产生了绿色荧光蛋白（GFP）和vacuolin的融合蛋白，GFP-vacuolin-修饰的囊泡被鉴定为在胞吐之前不久获得内吞标记物的后溶酶体隔室。在早期阶段，通过标记的细胞骨架蛋白冠蛋白-GFP的结合来鉴定该溶酶体后区室。沿着整个后溶酶体途径，胞浆被丝状肌动蛋白沿着包被，并且肌动蛋白包被的完整性是胞吐作用所必需的。
Cells of the unicellular eukaryote Dictyostelium discoideum take up all their nutrients by endocytosis, Both particle- and fluid-containing vacuoles are transiently surrounded by a cytoskeletal coat [1,2]. When this coat has dissociated, acidification and digestion of the vesicle contents occur, followed by exocytosis of the indigestible remnants after 60-90 minutes, At least nine compartments are needed for mathematical modelling of endocytic transit [3], suggesting that markers associate for only a few minutes with a specific endocytic compartment Among the proteins that have been identified as components of endocytic vesicles are actin, subunits of the V-H(+)ATPase and small GTP-binding proteins of the nab family [4-7]. Using a monoclonal antibody produced against Dictyostelium endocytic vesicles, we have isolated a cDNA corresponding to a novel protein that we have named vacuolin. In order to determine the precise step along the endocytic pathway that involves vacuolin, we generated a fusion protein of the green fluorescent protein (GFP) and vacuolin, GFP-vacuolin-decorated vesicles were identified as a post-lysosomal compartment that acquires endocytic markers shortly before exocytosis. At earlier stages, this post-lysosomal compartment was identified by the binding of a tagged cytoskeletal protein, coronin-GFP. Vacuoles were coated with filamentous actin along the entire post-lysosomal pathway, and the integrity of the actin coat was required for exocytosis.