LOCALIZATION OF 5 ANNEXINS IN J774 MACROPHAGES AND ON ISOLATED PHAGOSOMES

Annexins are a family of structurally related proteins which bind phos pholipids in a calcium-dependent manner. Although the precise function s of annexins are unknown, there is an accumulating set of data arguin g for a role for some of them in vesicular transport and, specifically , in membrane-membrane or membrane-cytoskeletal interactions during th ese processes. Here we describe our qualitative and quantitative analy sis of the localization of annexins I-V in J774 macrophages that had i nternalized latex beads, both with and without IgG opsonization. Our r esults show that whereas all these annexins are present on both the pl asma membrane and on phagosomes, the localization on other organelles differs. Annexins I, II, III and V were detected on early endosomes, w hile only annexin V was seen on late endocytic organelles and mitochon dria. Annexins I and II distributed along the plasma membrane non-unif ormly and co-localized with F-actin at the sites of membrane protrusio ns. We also investigated by western blot analysis the association of a nnexins with purified phagosomes isolated at different time-points aft er latex bead internalization. While the amounts of annexins I, II, II I and V associated with phagosomes were similar at all times after the ir formation, the level of annexin IV was significantly higher on olde r phagosomes. Whereas annexins I, II, IV and V could be removed from p hagosome membranes with a Ca2+ chelator they remained membrane bound u nder low calcium conditions. In contrast, annexin III was removed unde r these conditions and needed a relatively high Ca2+ concentration to remain phagosome bound. Because of their purity and ease of preparatio n we suggest that phagosomes are a powerful system to study the potent ial role of annexins in membrane traffic.