RabD, a Dictyostelium Rab14-related GTPase, regulates phagocytosis and homotypic phagosome and lysosome fusion

Authors

Ed Harris, University of Nebraska - LincolnFollow
James Cardelli, LSU Health Sciences Center, Shreveport, LAFollow

Document Type

Article

Date of this Version

2002

Comments

Published in Journal of Cell Science 115, 3703-3713 © 2002 The Company of Biologists Ltd doi:10.1242/jcs.00050 Used by permission.

Abstract

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 twofold in RabD^Q67L-expressing cells and reduced by 50% in RabD^N121l-expressing cells compared with control cells. To examine the role of RabD in the formation of muItiparticle 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^N121l-expressing cells as compared with control cells. GFP-RabD^Q67L was found to ring muItiparticle 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-Iysosome fusion.