Dynamics of the vacuolar H+-ATPase in the contractile vacuole complex and the endosomal pathway ofDictyosteliumcells

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.

Free-Living Amoebas in Extreme Environments: The True Survival in our Planet

“…The expression of V-ATPase (vacuolar H + -ATPase) in the vacuolar membrane has been reported. V-ATPases move H + inside, lowering the vacuole's pH relative to the cytosol, promoting an osmosis gradient across the vacuole membrane, facilitating water entry through aquaporins in the vacuole membrane [ 5 , 6 ].…”

Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

“…Despite numerous studies on phagosomal dynamics, the terminal fate of the phagolysosome remains obscure. For years, phagocytosis was assumed to end with the exocytosis of the phagolysosome and egestion of debris, a notion that likely arose from studies on phagotrophic protozoans ( Clarke et al, 2002 ; Maniak, 2003 ; Stewart and Weisman, 1972 ; Gotthardt et al, 2002 ). Yet, recent evidence suggests that mammalian phagosomes containing degradable cargo, like apoptotic cells or RBCs, undergo shrinkage and/or fission ( Krajcovic et al, 2013 ; Levin-Konigsberg et al, 2019 ).…”

“…Phagolysosome exocytosis was assumed to expel indigestible material and return membrane to the surface of phagocytes ( Stewart and Weisman, 1972 ; Maniak, 2003 ; Clarke et al, 2002 ; Gotthardt et al, 2002 ). Yet, we failed to observe exocytosis of phagosomes containing either bacteria or beads.…”

Deletion of a Putative GPI-Anchored Protein-Encoding Gene Aog185 Impedes the Growth and Nematode-Trapping Efficiency of Arthrobotrys oligospora by Disrupting Transmembrane Transport Homeostasis

“…In the fusion of phagosome with endosomes or autophagosome with vacuolar, vATPase is the critical rate-limiting enzyme [33,34]. The enzyme vATPase transports protons across biological membranes by utilizing the energy from ATP hydrolysis [35][36][37]. In addition, ATPase is a critical enzyme involved in the oxidative phosphorylation system and plays a vital role in regulating proton transmembrane transport [38].…”