Blocker A, Severin F F, Habermann A, Hyman A A, Griffiths G, Burkhardt J K
Cell Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
J Biol Chem. 1996 Feb 16;271(7):3803-11. doi: 10.1074/jbc.271.7.3803.
In macrophages, phagosome movement is microtubule-dependent. Microtubules are a prerequisite for phagosome maturation because they facilitate interactions between phagosomes and organelles of the endocytic pathway. We have established an in vitro assay that measures the binding of purified phagosomes to microtubules. This binding depends on the presence of membrane proteins, most likely integral to the surface of phagosomes, and on macrophage cytosol. The cytosolic binding factor can interact with microtubules prior to the addition of phagosomes to the assay, suggesting that it is a microtubule-associated protein (MAP). Consistent with this, depletion of MAPs from the cytosol by microtubule affinity removes all binding activity. Microtubule motor proteins show no binding activity, whereas a crude MAP preparation is sufficient to support binding and to restore full binding activity to MAP-depleted cytosol. We show that the activating MAP factor is a heat-sensitive protein(s) that migrates at around 150 kDa by gel filtration.
在巨噬细胞中，吞噬体的移动依赖于微管。微管是吞噬体成熟的先决条件，因为它们促进了吞噬体与内吞途径细胞器之间的相互作用。我们建立了一种体外测定法，用于测量纯化的吞噬体与微管的结合。这种结合依赖于膜蛋白的存在，膜蛋白很可能是吞噬体表面的整合蛋白，并且依赖于巨噬细胞胞质溶胶。胞质结合因子在将吞噬体添加到测定中之前就能与微管相互作用，这表明它是一种微管相关蛋白（MAP）。与此一致的是，通过微管亲和作用从胞质溶胶中去除MAP会消除所有结合活性。微管运动蛋白没有结合活性，而粗制的MAP制剂足以支持结合并将完全的结合活性恢复到MAP耗尽的胞质溶胶中。我们表明，激活MAP因子是一种热敏感蛋白，通过凝胶过滤法其迁移分子量约为150 kDa。
