Engstler Markus, Weise Frank, Bopp Karoline, Grünfelder Christoph G, Günzel Mark, Heddergott Niko, Overath Peter
Ludwig-Maximilians-Universität, Department Biologie I, Genetik, Maria-Ward-Strasse 1a, München, 80638, Germany.
J Cell Sci. 2005 May 15;118(Pt 10):2105-18. doi: 10.1242/jcs.02327. Epub 2005 Apr 26.
In the parasitic protozoan Trypanosoma brucei, endocytosis and exocytosis occur exclusively at an invagination of the plasma membrane around the base of the flagellum, called the flagellar pocket, which actively communicates by vesicular membrane flow with cisternal/tubulovesicular endosomes. The division of the cell surface into three morphologically distinct sub-domains and the rapid plasma membrane turnover establishes T. brucei as an interesting model for investigations on the sorting and recycling of membrane proteins. In this study we show that the type I membrane protein TbMBAP1, an L-(+)-tartrate-sensitive acid phosphatase, is present in all endosomal membranes but is virtually absent from the lysosome membrane (where this type of protein is mainly found in other organisms) and is not detectable at the cell surface. The endosomal localization of TbMBAP1 is a function of protein abundance. Moderate overexpression (three- to fourfold) leads to an increased appearance within the flagellar pocket membrane. At higher levels the protein is found in the flagellum, and routing to the pellicular plasma membrane is observed at levels 10- to 25-fold above that of wild type. In other organisms L-(+)-tartrate-sensitive acid phosphatases appear to be dispensable but TbMBAP1 is essential, as shown by RNA interference, which causes growth arrest followed by cell death. Comparison of the phenotype of TbMBAP1-depleted cells with that of cells in which endocytosis or exocytosis has been specifically inhibited by RNAi against clathrin of RAB11, reveals that TbMBAP1 is essential for both incoming and recycling membrane traffic. During differentiation of the organism from bloodstream to insect stage, TbMBAP1 is down-regulated and differentially modified in parallel with a 10-fold decrease in the rate of endocytosis.
在寄生原生动物布氏锥虫中，内吞作用和外排作用仅发生在围绕鞭毛基部的质膜内陷处，即所谓的鞭毛袋，它通过囊泡膜流与池状/管状小泡内体进行活跃的通讯。细胞表面分为三个形态上不同的亚结构域，且质膜快速更新，这使得布氏锥虫成为研究膜蛋白分选和循环利用的有趣模型。在本研究中，我们发现I型膜蛋白TbMBAP1，一种L-(+)-酒石酸敏感酸性磷酸酶，存在于所有内体膜中，但在溶酶体膜中几乎不存在（在其他生物体中这类蛋白主要存在于溶酶体膜），且在细胞表面无法检测到。TbMBAP1在内体中的定位是蛋白质丰度的函数。适度过表达（三到四倍）会导致在鞭毛袋膜中的出现增加。在更高水平时，该蛋白存在于鞭毛中，并且在高于野生型水平10到25倍时可观察到其转运至表膜质膜。在其他生物体中，L-(+)-酒石酸敏感酸性磷酸酶似乎是可有可无的，但TbMBAP1是必需的，如RNA干扰所示，RNA干扰会导致生长停滞随后细胞死亡。将TbMBAP1缺失细胞的表型与通过针对网格蛋白或RAB11的RNAi特异性抑制内吞作用或外排作用的细胞的表型进行比较，发现TbMBAP1对于内吞和循环膜运输都是必需的。在生物体从血液阶段向昆虫阶段分化过程中，TbMBAP1与内吞作用速率下降10倍同时被下调并发生差异修饰。
