A Role for Adaptor Protein‐3 Complex in the Organization of the Endocytic Pathway in Dictyostelium

Abstract

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 ...

WASH drives early recycling from macropinosomes and phagosomes to maintain surface phagocytic receptors

“…The rapid depletion of surface proteins from macropinosomes has previously been described in Dictyostelium (46,47). Both the plasma membrane and a perinuclear recycling pool are marked by the protein p25, which is stripped from macropinosomes within the first minutes of internalization-at the same time that WASH and retromer are active.…”

“…S1A) (48). Interestingly, the p25 recycling compartment is also accessible to proteins lacking any cytosolic sorting signals, indicating nonspecific bulk recycling (46).…”

Lipid Composition of Multilamellar Bodies Secreted by Dictyostelium discoideum Reveals Their Amoebal Origin

“…The H161, H72, and H36 mouse monoclonal antibodies have been described previously (21,23,24) and were generously furnished by P.…”

Comparison of Dictyostelium LvsB and endosomal fission defect mutants support a fusion regulatory role for LvsB

“…AP3 is a clathrin adaptor complex found on endosomal compartments (Bonifacino and Traub, 2003). Absence of Dictyostelium m3 results in fissionmediated recycling defects during the early stages of endosome maturation (Charette et al, 2006;Charette and Cosson, 2008). The WASH protein is required for the removal of the vATPase from late lysosomes that are transitioning to the post-lysosomal stage.…”