title: Biochemical characterization of APPL endosomes: the role of annexin A2 in APPL membrane recruitment.
abstract: 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.
authors.affiliation: 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.
authors.forename: Anna
authors.initials: A
authors.lastname: Urbanska
authors.forename: Lukasz
authors.initials: L
authors.lastname: Sadowski
authors.forename: Yannis
authors.initials: Y
authors.lastname: Kalaidzidis
authors.forename: Marta
authors.initials: M
authors.lastname: Miaczynska
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017306:Annexin A2 / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D011992:Endosomes / Q000737:chemistry* / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D010716:Phosphatidylinositols / Q000378:metabolism; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C464801:APPL1 protein, human; C528555:APPL2 protein, human; D048868:Adaptor Proteins, Signal Transducing; D017306:Annexin A2; D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins; D020696:rab5 GTP-Binding Proteins; D002118:Calcium