International Institute of Molecular and Cell Biology, Laboratory of Cell Biology, International Institute of Molecular and Cell Biology, 4 Ks. Trojdena Street, 02-109 Warsaw, Poland.
Traffic. 2011 Sep;12(9):1227-41. doi: 10.1111/j.1600-0854.2011.01226.x. Epub 2011 Jul 3.
APPL endosomes are a recently identified subpopulation of early endosomes characterized by the presence of two homologous Rab5 effector proteins APPL1 and APPL2. They exhibit only limited colocalization with EEA1, another Rab5 effector and a marker of the canonical early endosomes. Although APPL endosomes appear to play important roles in cargo trafficking and signal transduction, their protein composition and biochemical properties remain largely unknown. Here we employed membrane fractionation methods to characterize APPL endosomes biochemically. We demonstrate that they represent heterogeneous membrane structures which can be discriminated from the canonical EEA1-positive early endosomes by their partly different physical properties and a distinct migration pattern in the continuous density gradients. In search for other potential markers of APPL endosomes we identified Annexin A2 as an interacting partner of both APPL1 and APPL2. Annexin A2 is a Ca(2+) and phosphatidylinositol 4,5-bisphosphate binding protein, previously implicated in several endocytic steps. We show that Annexin A2 co-fractionates and colocalizes with APPL endosomes. Moreover, silencing of its expression causes solubilization of APPL2 from endosomes. Although Annexin A2 is not an exclusive marker of APPL endosomes, our data suggest that it has an important function in membrane recruitment of APPL proteins, acting in parallel to Rab5.
APPL 内涵体是最近被鉴定的早期内涵体亚群，其特征是存在两种同源 Rab5 效应蛋白 APPL1 和 APPL2。它们与另一种 Rab5 效应蛋白 EEA1 （经典早期内涵体的标志物）的共定位有限。尽管 APPL 内涵体在货物运输和信号转导中似乎发挥着重要作用，但它们的蛋白质组成和生化特性在很大程度上仍然未知。在这里，我们采用膜分离方法对 APPL 内涵体进行了生化特性分析。我们证明它们代表了异构的膜结构，通过其部分不同的物理性质和在连续密度梯度中的独特迁移模式，可以与经典的 EEA1 阳性早期内涵体区分开来。在寻找 APPL 内涵体的其他潜在标志物时，我们发现 Annexin A2 是 APPL1 和 APPL2 的相互作用伙伴。Annexin A2 是一种 Ca（2+）和磷脂酰肌醇 4,5-二磷酸结合蛋白，先前被认为参与了几个内吞步骤。我们表明 Annexin A2 与 APPL 内涵体共分离和共定位。此外，沉默其表达会导致 APPL2 从内涵体中溶解。虽然 Annexin A2 不是 APPL 内涵体的唯一标志物，但我们的数据表明，它在 APPL 蛋白的膜募集中具有重要功能，与 Rab5 平行作用。
