RRC ID 29042
Author Clarke M, Köhler J, Arana Q, Liu T, Heuser J, Gerisch G.
Title Dynamics of the vacuolar H(+)-ATPase in the contractile vacuole complex and the endosomal pathway of Dictyostelium cells.
Journal J Cell Sci
Abstract The vacuolar H(+)-ATPase (V-ATPase) is a multi-subunit enzyme that plays important roles in eukaryotic cells. In Dictyostelium, it is found primarily in membranes of the contractile vacuole complex, where it energizes fluid accumulation by this osmoregulatory organelle and also in membranes of endolysosomes, where it serves to acidify the endosomal lumen. In the present study, a fusion was created between vatM, the gene encoding the 100 kDa transmembrane subunit of the V-ATPase, and the gene encoding Green Fluorescent Protein (GFP). When expressed in Dictyostelium cells, this fusion protein, VatM-GFP, was correctly targeted to contractile vacuole and endolysosomal membranes and was competent to direct assembly of the V-ATPase enzyme complex. Protease treatment of isolated endosomes indicated that the GFP moiety, located on the C-terminus of VatM, was exposed to the cytoplasmic side of the endosomal membrane rather than to the lumenal side. VatM-GFP labeling of the contractile vacuole complex revealed clearly the dynamics of this pleiomorphic vesiculotubular organelle. VatM-GFP labeling of endosomes allowed direct visualization of the trafficking of vacuolar proton pumps in this pathway, which appeared to be entirely independent from the contractile vacuole membrane system. In cells whose endosomes were pre-labeled with TRITC-dextran and then fed yeast particles, VatM-GFP was delivered to newly formed yeast phagosomes with the same time course as TRITC-dextran, consistent with transfer via a direct fusion of endosomes with phagosomes. Several minutes were required before the intensity of the VatM-GFP labeling of new phagosomes reached the level observed in older phagosomes, suggesting that this fusion process was progressive and continuous. VatM-GFP was retrieved from the phagosome membrane prior to exocytosis of the indigestible remnants of the yeast particle. These data suggest that vacuolar proton pumps are recycled by fusion of advanced with newly formed endosomes.
Volume 115(Pt 14)
Pages 2893-905
Published 2002-7-15
PMID 12082150
MeSH Animals Cell Compartmentation / physiology Dictyostelium / cytology Dictyostelium / enzymology* Endosomes / enzymology* Endosomes / ultrastructure Green Fluorescent Proteins Hydrogen-Ion Concentration Intracellular Membranes / metabolism* Luminescent Proteins Molecular Structure Phagocytosis / physiology Phagosomes / metabolism Phagosomes / ultrastructure Protein Structure, Tertiary / physiology Protein Transport / physiology Proton Pumps / metabolism* Proton-Translocating ATPases / metabolism* Recombinant Fusion Proteins / genetics Recombinant Fusion Proteins / metabolism Transport Vesicles / metabolism Transport Vesicles / ultrastructure Vacuoles / enzymology* Vacuoles / ultrastructure Water-Electrolyte Balance / physiology
IF 4.573
Times Cited 98
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