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Specialized eukaryotic cells can ingest large particles and sequester them within membrane-delimited phagosomes. Many studies have described the delivery of lysosomal proteins to the phagosome, but little is known about membrane sorting during the early stages of phagosome formation. Here we used Dictyostelium discoideum amoebae to analyze the membrane composition of newly formed phagosomes. The membrane delimiting the closing phagocytic cup was essentially derived from the plasma membrane, but a subgroup of proteins was specifically excluded. Interestingly the same phenomenon was observed during the formation of macropinosomes, suggesting that the same sorting mechanisms are at play during phagocytosis and macropinocytosis. Analysis of mutant strains revealed that clathrin-associated adaptor complexes AP-1, -2 and -3 were not necessary for this selective exclusion and, accordingly, ultrastructural analysis revealed no evidence for vesicular transport around phagocytic cups. Our results suggest the existence of a new, as yet uncharacterized, sorting mechanism in phagocytic and macropinocytic cups

Dictyostelium discoideum cells continuously internalize extracellular material, which accumulates in well-characterized endocytic vacuoles. In this study, we describe a new endocytic compartment identified by the presence of a specific marker, the p25 protein. This compartment presents features reminiscent of mammalian recycling endosomes: it is localized in the pericentrosomal region but distinct from the Golgi apparatus. It specifically contains surface proteins that are continuously endocytosed but rapidly recycled to the cell surface and thus absent from maturing endocytic compartments. We evaluated the importance of each clathrin-associated adaptor complex in establishing a compartmentalized endocytic system by studying the phenotype of the corresponding mutants. In knockout cells for m3, a subunit of the AP-3 clathrin-associated complex, membrane proteins normally restricted to p25-positive endosomes were mislocalized to late endocytic compartments. Our results suggest that AP-3 plays an essential role in the compartmentalization of the endocytic pathway in Dictyostelium.Key words: adaptor protein complex, AP-3, clathrin, Dictyostelium discoideum, endosome, recycling Received, revised and accepted for publication 24 July 2006Eukaryotic cells can ingest components present in the extracellular medium through endocytosis, phagocytosis or macropinocytosis and transfer them to intracellular endocytic compartments delimited by a membrane. In mammalian cells, endocytosed material is transferred successively to early endosomes, late endosomes and then to lysosomes, where it is degraded. Transfer of extracellular medium to early endosomes is mediated by vesicles forming at the plasma membrane, and this is accompanied by the internalization of membrane proteins present at the cell surface. To maintain the composition of the cell surface, many internalized proteins are recycled rapidly to the plasma membrane (1). For this, they are transferred to a distinct juxtanuclear recycling endosome, from which they can be transported back to the cell surface (2,3).The transfer of membrane proteins from one endocytic compartment to another is mostly achieved by intracellular transport vesicles. The formation of transport vesicles is driven by the polymerization of cytosolic proteins on the cytosolic face of the membrane. This cytosolic coat of proteins also ensures the specificity of each transport step by interacting with a subset of transmembrane proteins and ensuring their incorporation into the forming vesicle. Many cytosolic coats have been identified, and several of them are composed of clathrin associated with various adaptor protein (AP) complexes. Briefly in mammalian cells, the AP-1 complex is mostly involved in transport between the trans Golgi network and the endosomes. The AP-2 complex participates in the formation of endocytic vesicles at the cell surface. The AP-3 and AP-4 complexes mostly participate in biogenesis of lysosomes. Each AP complex can apparently participate in several distinct transport events, and ...

Multivesicular endosomes are present in virtually every eucaryotic cell, where they arise by intra-endosomal budding of the limiting endosomal membrane. Some genetic diseases such as Chediak-Higashi syndrome are characterized by enlarged membrane-filled endosomes. The same altered endosomal morphology can be observed in cells exposed to certain drugs, for example U18666A. The mechanisms involved are still poorly characterized, partially because this atypical budding event is particularly difficult to observe in mammalian cells. Taking advantage of the simplicity of the endosomal structure in Dictyostelium discoideum, we could visualize intraendosomal budding at the ultrastructural level. In this model organism, the drug U18666A was shown to stimulate intra-endosomal budding, while an inhibitor of PI 3-kinase activity was found to have no effect on this process. Inactivation of a Dictyostelium gene with similarity to the gene responsible for Chediak-Higashi syndrome did not alter the intra-endosomal budding or the accumulation of intra-endosomal membranes. Thus, although treatment with U18666A and inactivation of the Chediak-Higashi gene cause similar morphological defects in mammalian cells, observations in a different model reveal that their respective modes of action are different