RabD, a Dictyostelium Rab14-related GTPase, regulates phagocytosis and homotypic phagosome and lysosome fusion.
RabD, a Dictyostelium Rab14-related GTPase, localizes in the endo-lysosomal pathway and contractile vacuole system of membranes. Cell lines expressing dominant-negative RabD were defective in endocytosis, endosomal membrane flow and homotypic lysosome fusion. In support of a role for RabD in fusion, cells overexpressing constitutively active RabD(Q67L) accumulated enlarged hydrolase-rich acidic vesicles ringed with GFP-RabD, consistent with RabD directly regulating lysosome fusion. To determine whether RabD also regulated phagocytosis and/or homotypic phagosome fusion (a process stimulated by many intracellular pathogens), cells overexpressing dominant-active (RabD(Q67L)) or dominant-negative (Rab(N121I)) RabD were analyzed microscopically and biochemically. The rate of phagocytosis was increased two-fold in RabD(Q67L)-expressing cells and reduced by 50% in RabD(N121I)-expressing cells compared with control cells. To examine the role of RabD in the formation of multiparticle phagosomes, we performed a series of pulse-chase experiments using fluorescently labeled bacteria and fluorescent latex beads. The rate of fusion of newly formed phagosomes was five times higher in the RabD(Q67L)-expressing cells and reduced by over 50% in RabD(N121I)-expressing cells as compared with control cells. GFP-RabD(Q67L) was found to ring multiparticle spacious phagosomes, which supports a direct role for this protein in regulating fusion. Inhibition of PI 3-kinase activity, which is known to regulate phagosome fusion in the wild-type cells, reduced the rate of phagosome fusion in RabD(Q67L+) cells, indicating that RabD acted upstream of or parallel with PI 3-kinase. We hypothesize that RabD and, possibly, Rab14, a related GTPase that associates with phagosomes in mammalian cells, are important regulators of homotypic phagosome and endo-lysosome fusion.
Department of Microbiology and Immunology and Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA 71130, USA.
Edward
E
Harris
James
J
Cardelli
D000818:Animals; D002478:Cells, Cultured; D004023:Dictyostelium / Q000166:cytology / Q000201:enzymology*; D011992:Endosomes / Q000378:metabolism; D005456:Fluorescent Dyes; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D008247:Lysosomes / Q000201:enzymology* / Q000648:ultrastructure; D008863:Microspheres; D008969:Molecular Sequence Data; D010587:Phagocytosis / Q000502:physiology*; D010588:Phagosomes / Q000201:enzymology* / Q000648:ultrastructure; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction / Q000502:physiology; D022161:Transport Vesicles / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D005456:Fluorescent Dyes; D019869:Phosphatidylinositol 3-Kinases; D020691:rab GTP-Binding Proteins