+ Retrieval of the Vacuolar H -ATPase from Phagosomes Revealed by Live Cell Imaging Margaret Clarke1*, Lucinda Maddera1, Ulrike Engel2, Gu¨ nther Gerisch3 1 Program in Genetic Models of Disease, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States of America, 2 Nikon Imaging Center at the University of Heidelberg, Bioquant, Heidelberg, Germany, 3 Max-Planck-Institut f u¨r Biochemie, Martinsried, Germany Abstract Background: The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized. Methodology: To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V- ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins. Principal Findings: We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved. Conclusions/Signficance: Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway. Citation: Clarke M, Maddera L, Engel U, Gerisch G (2010) Retrieval of the Vacuolar H+-ATPase from Phagosomes Revealed by Live Cell Imaging. PLoS ONE 5(1): e8585. doi:10.1371/journal.pone.0008585 Editor: Robin Charles May, University of Birmingham, United Kingdom Received August 27, 2009; Accepted December 7, 2009; Published January 5, 2010 Copyright: 2010 Clarke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grant MCB-0344541 from the National Science Foundation to MC, and by the Max Planck Society and grants in the SPP 1128 program of the Deutsche Forschungsgemeinschaft to GG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: clarkem@omrf.org Introduction The vacuolar H+-ATPase, or V-ATPase, is a multi-subunit enzyme conserved in all eukaryotes that uses the energy from ATP hydrolysis to move protons across membranes. The enzyme is divided into two sectors, each containing multiple subunits. The peripheral V1 complex is responsible for ATP hydrolysis, and the membrane- spanning V0 complex is responsible for proton translocation. The proton gradient generated by the V-ATPase is used to energize the uptake of small molecules such as neurotransmitters, to regulate extracellular pH, and to acidify the lumen of organelles. Acidification of endosomes and phagosomes by the V-ATPase mediates receptor recycling, activation of lysosomal enzymes, and killing of pathogens. There are several means of regulating V-ATPase activity, including control of V-ATPase subunit expression, intracellular targeting and recycling of V-ATPase-containing vesicles, reversible dissociation of the V0 and V1 domains, and modulation of the coupling ratio between ATP hydrolysis and proton pumping [1–4]. The professional phagocytes of Dictyostelium discoideum have proved to be an excellent system for analyzing the V-ATPase in the endocytic pathway [5–12]. Dictyostelium cells will ingest a wide range of particles including bacteria, yeast, latex beads, glass microspheres, and occasionally other cells. They can serve as hosts for a number of bacterial pathogens that also infect human cells [13,14]. Many aspects of the endocytic pathway are conserved between Dictyostelium and mammalian phagocytes, including mechanisms of particle recognition, the role of the cytoskeleton in uptake, the delivery of digestive enzymes, and membrane retrieval [11,15,16]. Here we add the localization of phosphati- dylinositol (3)-phosphate, or PI(3)P, to vesicle membranes in Dictyostelium cells as a common marker of the early endosomal compartment. PI(3)P is one of a group of phosphoinositides that specify the identity of compartments along the endocytic pathway by recruiting signaling proteins that carry specific phosphoinositide- binding modules [17,18,19]. Studies with mammalian phagocytes PLoS ONE | www.plosone.org 1 January 2010 | Volume 5 | Issue 1 | e8585