The Cellular Biology of Proton-Motive Force Generation by V-ATPases

Abstract

The vacuolar H(+)-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force, V-ATPases function exclusively as ATP-dependent proton pumps. The proton-motive force generated by V-ATPases in organelles and across plasma membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. The enzyme is also vital for the proper functioning of endosomes and the Golgi apparatus. In contrast to yeast vacuoles, which maintain an internal pH of approximately 5. 5, it is believed that the vacuoles of lemon fruit may have a pH as low as 2. Similarly, some brown and red algae maintain an internal pH as low as 1 in their vacuoles. It was yeast genetics that allowed the identification of the special properties of individual subunits and the discovery of the factors that are involved in V-ATPase biogenesis and assembly. Null mutations in genes encoding V-ATPase subunits of Saccharomyces cerevisiae result in a phenotype that is unable to grow at high pH and is sensitive to high and low metal-ion concentrations. Treatment of these null mutants with ethyl methanesulphonate causes mutations that suppress the V-ATPase null phenotype, and these cells are able to grow at pH 7.5. The suppressor mutants were denoted as svf (Suppressor of V-ATPase Function). The svf mutations are recessive: crossing the svf mutants with their corresponding V-ATPase null mutants resulted in diploid strains that were not able to grow at pH 7.5. A novel gene family in which null mutations cause pleiotropic effects on metal-ion resistance or on the sensitivity and distribution of membrane proteins in different targets was discovered. We termed this gene family VTC (Vacuolar Transporter Chaperon) and discovered four genes in S. cerevisiae that belong to the family. Inactivation of one of them, VTC1, in the background of V-ATPase null mutations resulted in an svf phenotype that was able to grow at pH 7.5. Apparently, Vtc1p is one of a few membrane organizers that determine the relative amounts of different membrane proteins in the various cellular membranes. We utilize the numerous yeast mutants generated in our laboratory to identify the specific organelle whose acidification is vital. The interaction between V-ATPase and the secretory pathway is investigated.

Methodology for the Simulation of Molecular Motors at Different Scales

“…A second example showcasing the simulation protocol presented here is provided by the pathway of ADP inhibition in the hexameric ring of the molecular motor V 1 -ATPase . Vacuolar ATPases, or V-ATPases, are thought to originate from an ancestral enzyme in common with the more ubiquitous F-ATPases .…”

“…After outlining the theoretical underpinnings of the computational strategy, we provide a workflow for modeling chemomechanical coupling in molecular motors. The strategy is then showcased in two concrete examples, namely ADP inhibition of vacuolar V 1 -ATPase motor action, 59,60 and the compression− decompression stroke in the 6A-deoxy-6A-(N-methyl-3-phenylpropionamido)-β-cyclodextrin motor. 22 ■ METHODS Theoretical Underpinnings.…”

Fluorescent Indicators For Biological Imaging of Monatomic Ions

“…The pH values vary in different cellular compartments: 7.0–7.4 in the cytoplasm, 7.2 for ER, 6.4 in the Golgi apparatus, 5.0 in lysosomes, 5.4 in secretory granules, 6.2 in early endosomes, and 5.3 in late endosomes ( Futai et al, 2000 ). The pH values of organelles are maintained mainly by vacuolar H + -ATPase (V-ATPase), which actively pumps H + against its concentration gradient with the energy from ATP hydrolysis ( Nelson et al, 2000 ). The pH environment is important for the functions of the organelles.…”

Simultaneous Monitoring of Mitochondrial Temperature and ATP Fluctuation Using Fluorescent Probes in Living Cells

“…The synthesis of ATP is an endergonic process, which requires an input of energy; the flow of electrons from electron donors to electron acceptors through the electron transport chain is an exergonic process, which releases energy. 5 In simple terms, if the ADP is converted to ATP, the energy could be consumed; if the reaction is blocked and the ADP could not be converted to ATP, the energy could be released as heat, which would make the temperature increase in the mitochondria. Therefore, the synthesis of ATP is significantly affected by the temperature at which oxidative phosphorylation occurs.…”

“…Intracellular energy release supports the function of many organelles and subcellular organelles. In mitochondria, oxidative phosphorylation (OXPHOS) is an important metabolic pathway by which enzymes oxidize metabolites, producing energy in the form of ATP. − During the OXPHOS reaction, the ATP synthase (complex V) is the final enzyme in the OXPHOS pathway, and the reaction is shown below: …”