Retrieval of the vacuolar H-ATPase from phagosomes revealed by live cell imaging.
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.


METHODS
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.


RESULTS
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
Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.
Clarke
Margaret
M
Program in Genetic Models of Disease, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States of America. clarkem@omrf.org
Maddera
Lucinda
L
Engel
Ulrike
U
Gerisch
Günther
G
D000199:Actins / Q000378:metabolism; D004023:Dictyostelium / Q000201:enzymology*; D005089:Exocytosis; D049452:Green Fluorescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D024461:Myosin Type I / Q000378:metabolism; D010587:Phagocytosis; D010588:Phagosomes / Q000201:enzymology*; D021381:Protein Transport; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D000199:Actins; D049452:Green Fluorescent Proteins; D024461:Myosin Type I; D025262:Vacuolar Proton-Translocating ATPases