MOLECULAR CHARACTERIZATION OF PHAGOSOMES

The transformation of newly formed phagosomes into mature phagolysosom es is a process that involves a complex series of interactions between phagosomes and other vacuolar organelles. The machinery required by p hagosomes to mediate these interactions is poorly understood. In this study, we allowed human and various rodent cells to take up latex bead s whose density facilitates a simple purification of phagosomes using discontinuous sucrose gradients. With this system, we initiated a syst ematic study of phagosome proteins using two-dimensional gel electroph oresis and the currently available two-dimensional gel protein data ba ses. By this approach, we were able to recognize a group of polypeptid es associated with mouse J774 phagosomes-phagolysosomes including anne xin II, annexin VI, the beta-1 and beta-2 subunits of trimeric G prote ins, and a group of actin-binding proteins. While the amount of annexi n II associated to phagosomes was similar at all times of latex intern alization, the levels of annexin VI were higher on late phagosomes. Ph ospholipid analysis of J774 phagosomes isolated at early and late time points during phagolysosome formation also revealed significant diffe rences in their lipid composition. In the human phagosomes, we resolve d over 200 polypeptides on the two-dimensional gels. These included th e proteins described in the mouse, as well as 32 polypeptides that wer e found to be highly enriched in phagosomes, 15 of which are not prese nt in the current data bases. The results demonstrate that the use of latex bead phagosomes is a powerful system to identify key molecules i nvolved in phagolysosome biogenesis.