Retrieval of the Vacuolar H+-ATPase from Phagosomes Revealed by Live Cell Imaging

Abstract

BackgroundThe 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.MethodologyTo 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 FindingsWe 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/SignficanceThus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.

The Nramp (Slc11) proteins regulate development, resistance to pathogenic bacteria and iron homeostasis in Dictyostelium discoideum

“…This is in sharp contrast with Nramp1 or the VatA subunit of the vacuolar ATPase, which were transiently recruited from internal stores to TRITC-containing vesicles shortly after uptake (Fig. 4C,D) Clarke et al, 2010;Peracino et al, 2006). Cells were also incubated with neutral red, which stains acidic vesicles.…”

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

“…These proteins include Rab GTPases, such as Rab14 and Rab7, lysosomal hydrolytic enzymes, and the vATPase proton pump (Rupper and Cardelli, 2001;Gotthardt et al, 2002;. Many of these shared proteins are delivered to phagosomes through fusion and intermingling of endo-lysosomal compartments with nascent and maturing phagosomes (Rupper and Cardelli, 2001;Clarke et al, 2010). Under these conditions, the fission defect model described above would predict that delayed progression of The LvsB-null cell line was compared with its parental NC4A2 wild-type cell line and the WASH-null line was compared with its parental AX2 wild-type cell line.…”

Defective lysosome maturation and Legionella pneumophila replication in Dictyostelium ArfGAP ACAP-A mutant cells

“…This result strongly suggests that ACAP-A might only partially control the retrieval of H + -ATPase from lysosomes leading to pH neutralization. The retrieval of H + -ATPase from maturing lysosomes relies on vesicles (Clarke et al, 2010) whose biogenesis depends on actin polymerization driven by WASH (Carnell et al, 2011). Because ACAP-A has been shown to regulate the actin cytoskeleton dynamics in an ArfA-dependent manner , we propose that ArfA and ACAP-A could also participate in the control of actin polymerization on these recycling vesicles by regulating the activity of actin nucleation-promoting factors such as WASH.…”

“…One key step in lysosome maturation is the depletion of H + -ATPase from lysosomes leading to pH neutralization. H + -ATPase retrieval is mediated by small actin-coated vesicles budding from lysosomes (Carnell et al, 2011;Clarke et al, 2010). The role of actin is essential in this process.…”