Coronin and vacuolin identify consecutive stages of a late, actin-coated endocytic compartment in Dictyostelium.
Cells of the unicellular eukaryote Dictyostelium discoideum take up all their nutrients by endocytosis. Both particle- and fluid-containing vacuoles are transiently surrounded by a cytoskeletal coat [1] [2]. When this coat has dissociated, acidification and digestion of the vesicle contents occur, followed by exocytosis of the indigestible remnants after 60-90 minutes. At least nine compartments are needed for mathematical modelling of endocytic transit [3], suggesting that markers associate for only a few minutes with a specific endocytic compartment. Among the proteins that have been identified as components of endocytic vesicles are actin, subunits of the V-H+ ATPase and small GTP-binding proteins of the Rab family [4] [5] [6] [7]. Using a monoclonal antibody produced against Dictyostelium endocytic vesicles, we have isolated a cDNA corresponding to a novel protein that we have named vacuolin. In order to determine the precise step along the endocytic pathway that involves vacuolin, we generated a fusion protein of the green fluorescent protein (GFP) and vacuolin. GFP-vacuolin-decorated vesicles were identified as a post-lysosomal compartment that acquires endocytic markers shortly before exocytosis. At earlier stages, this post-lysosomal compartment was identified by the binding of a tagged cytoskeletal protein, coronin-GFP. Vacuoles were coated with filamentous actin along the entire post-lysosomal pathway, and the integrity of the actin coat was required for exocytosis.
Rauchenberger
R
R
Abteilung Zellbiologie, Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany.
Hacker
U
U
Murphy
J
J
Niewöhner
J
J
Maniak
M
M
D000199:Actins / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015415:Biomarkers; D003598:Cytoskeletal Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D018076:DNA, Complementary / Q000235:genetics; D004023:Dictyostelium / Q000737:chemistry / Q000648:ultrastructure*; D004705:Endocytosis; D011992:Endosomes / Q000737:chemistry / Q000648:ultrastructure*; D005456:Fluorescent Dyes; D005656:Fungal Proteins / Q000032:analysis*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008840:Microfilament Proteins / Q000032:analysis*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014617:Vacuoles / Q000737:chemistry / Q000648:ultrastructure
D000199:Actins; D015415:Biomarkers; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C105803:vac A protein, Dictyostelium discoideum; C072061:coronin proteins; D049452:Green Fluorescent Proteins