Vol. 13, Issue 10, 3508-3520, October 2002

High-Resolution Dissection of Phagosome Maturation Reveals Distinct Membrane Trafficking Phases

Daniel Gotthardt,^* Hans Jörg Warnatz,^* Oliver Henschel, Franz Brückert, Michael Schleicher,^§ and Thierry Soldati^*¶

Departments of  ^*Molecular Cell Research and  Cell Physiology, Max-Planck-Institute for Medical Research, D-69120 Heidelberg, Germany;  Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, UMR5092-CNRS, CEA, 38054 Grenoble, France;  ^§Institute of Cell Biology, Ludwig-Maximilians University, D-80336 Munich, Germany; and  Department of Biological Sciences, Alexander Fleming Building, Imperial College of Science Technology and Medicine, London SW7 2AZ, UK

Molecular mechanisms of endocytosis in the genetically and biochemically tractable professional phagocyte Dictyostelium discoideum reveal a striking degree of similarity to higher eukaryotic cells. Pulse-chase feeding with latex beads allowed purification of phagosomes at different stages of maturation. Gentle ATP stripping of an actin meshwork entrapping contaminating organelles resulted in a 10-fold increase in yield and purity, as confirmed by electron microscopy. Temporal profiling of signaling, cytoskeletal, and trafficking proteins resulted in a complex molecular fingerprint of phagosome biogenesis and maturation. First, nascent phagosomes were associated with coronin and rapidly received a lysosomal glycoprotein, LmpB. Second, at least two phases of delivery of lysosomal hydrolases (cathepsin D [CatD] and cysteine protease [CPp34]) were accompanied by removal of plasma membrane components (PM4C4 and biotinylated surface proteins). Third, a phase of late maturation, preparing for final exocytosis of undigested material, included quantitative recycling of hydrolases and association with vacuolin. Also, lysosomal glycoproteins of the Lmp family showed distinct trafficking kinetics. The delivery and recycling of CatD was directly visualized by confocal microscopy. This heavy membrane traffic of cargos was precisely accompanied by regulatory proteins such as the Rab7 GTPases and the endosomal SNAREs Vti1 and VAMP7. This initial molecular description of phagocytosis demonstrates the feasibility of a comprehensive analysis of phagosomal lipids and proteins in genetically modified strains.