The cellular biology of proton-motive force generation by V-ATPases

2000 ◽  
Vol 203 (1) ◽  
pp. 89-95 ◽  
Author(s):  
N. Nelson ◽  
N. Perzov ◽  
A. Cohen ◽  
K. Hagai ◽  
V. Padler ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Gene Family ◽  
Metal Ion ◽  
Proton Motive Force ◽  
Transport Processes ◽  
Motive Force ◽  
Eukaryotic Cells ◽  
Proton Pumps ◽  
Internal Ph ◽  
Null Mutants

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.

scholarly journals Energizing porters by proton-motive force.

1994 ◽  
Vol 196 (1) ◽  
pp. 7-13 ◽  
Author(s):  
N Nelson
Keyword(s):  
Proton Motive Force ◽  
Transport Processes ◽  
Motive Force ◽  
Common Mechanism ◽  
Proton Pumps ◽  
High Proton ◽  
The World ◽  
Common Coupling ◽  
Proton Movement ◽  
Life On Earth

It is generally accepted that the chemistry of water was the most crucial determinant in shaping life on earth. Among the more important chemical features of water is its dissociation into protons and hydroxyl ions. The presence of relatively high proton concentrations in the ambient solution resulted in the evolution of proton pumps during the dawn of life on earth. These proton pumps maintained neutral pH inside the cells and generated electrochemical gradients of protons (proton-motive force) across their membranes. The existence of proton-motive force enabled the evolution of porters driven by it that are most probably among the more primitive porters in the world. The directionality of the substrate transport by the porters could be to both sides of the membranes because they can serve as proton symporters or antiporters. One of the most important subjects of this meeting is the mechanism by which proton-motive and other ion-motive forces drive the transport processes through porters. Is there a common mechanism of action for all proton-driven porters? Is there some common partial reaction by which we can identify the way that porters are energized by proton-motive force? Is there a common coupling between proton movement and uptake or secretion of certain molecules? Even a partial answer to one of these questions would advance our knowledge... or confusion. As my mentor Efraim Racker used to say: 'If you are not totally confused you do not understand the issue'.

Vacuolar and Plasma Membrane Proton-Adenosinetriphosphatases

1999 ◽  
Vol 79 (2) ◽  
pp. 361-385 ◽  
Author(s):  
Nathan Nelson ◽  
William R. Harvey
Keyword(s):  
Driving Force ◽  
Plasma Membranes ◽  
Reproductive Tract ◽  
Transport Processes ◽  
Transport Systems ◽  
Eukaryotic Cells ◽  
Proper Function ◽  
Proton Pumps ◽  
Atpase Subunit ◽  
Internal Ph

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. V-ATPases have similar structure and mechanism of action with F-ATPase and several of their subunits evolved from common ancestors. In eukaryotic cells, F-ATPases are confined to the semi-autonomous organelles, chloroplasts, and mitochondria, which contain their own genes that encode some of the F-ATPase subunits. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the proton-motive force (pmf), V-ATPases function exclusively as ATP-dependent proton pumps. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. The mechanistic and structural relations between the two enzymes prompted us to suggest similar functional units in V-ATPase as was proposed to F-ATPase and to assign some of the V-ATPase subunit to one of four parts of a mechanochemical machine: a catalytic unit, a shaft, a hook, and a proton turbine. It was the yeast genetics that allowed the identification of special properties of individual subunits and the discovery of factors that are involved in the enzyme biogenesis and assembly. The V-ATPases play a major role as energizers of animal plasma membranes, especially apical plasma membranes of epithelial cells. This role was first recognized in plasma membranes of lepidopteran midgut and vertebrate kidney. The list of animals with plasma membranes that are energized by V-ATPases now includes members of most, if not all, animal phyla. This includes the classical Na+absorption by frog skin, male fertility through acidification of the sperm acrosome and the male reproductive tract, bone resorption by mammalian osteoclasts, and regulation of eye pressure. V-ATPase may function in Na+uptake by trout gills and energizes water secretion by contractile vacuoles in Dictyostelium. V-ATPase was first detected in organelles connected with the vacuolar system. It is the main if not the only primary energy source for numerous transport systems in these organelles. The driving force for the accumulation of neurotransmitters into synaptic vesicles is pmf generated by V-ATPase. The acidification of lysosomes, which are required for the proper function of most of their enzymes, is provided by V-ATPase. The enzyme is also vital for the proper function of endosomes and the Golgi apparatus. In contrast to yeast vacuoles that maintain an internal pH of ∼5.5, it is believed that the vacuoles of lemon fruit may have a pH as low as 2. Similarly, some brown and red alga maintain internal pH as low as 0.1 in their vacuoles. One of the outstanding questions in the field is how such a conserved enzyme as the V-ATPase can fulfill such diverse functions.

Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H(+)-ATPases

2000 ◽  
Vol 203 (1) ◽  
pp. 71-80 ◽  
Author(s):  
M. Forgac
Keyword(s):  
Molecular Mass ◽  
Proton Transport ◽  
Atp Hydrolysis ◽  
Transport Processes ◽  
Site Directed Mutagenesis ◽  
Eukaryotic Cells ◽  
Coated Vesicle ◽  
Proton Pumps ◽  
Reversible Dissociation ◽  
Vacuolar Acidification

The vacuolar H(+)-ATPases (or V-ATPases) are a family of ATP-dependent proton pumps that carry out acidification of intracellular compartments in eukaryotic cells. This review is focused on our work on the V-ATPases of clathrin-coated vesicles and yeast vacuoles. The coated-vesicle V-ATPase undergoes trafficking to endosomes and synaptic vesicles, where it functions in receptor recycling and neurotransmitter uptake, respectively. The yeast V-ATPase functions to acidify the central vacuole and is necessary both for protein degradation and for coupled transport processes across the vacuolar membrane. The V-ATPases are multisubunit complexes composed of two functional domains. The V(1) domain is a 570 kDa peripheral complex composed of eight subunits of molecular mass 73–14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V(o) domain is a 260 kDa integral complex composed of five subunits of molecular mass 100-17 kDa (subunits a, d, c, c' and c”) that is responsible for proton translocation. To explore the function of individual subunits in the V-ATPase complex as well as to identify residues important in proton transport and ATP hydrolysis, we have employed a combination of chemical modification, site-directed mutagenesis and in vitro reassembly. A central question concerns the mechanism by which vacuolar acidification is controlled in eukaryotic cells. We have proposed that disulfide bond formation between conserved cysteine residues at the catalytic site of the V-ATPase plays an important role in regulating V-ATPase activity in vivo. Other regulatory mechanisms that are discussed include reversible dissociation and reassembly of the V-ATPase complex, changes in the tightness of coupling between proton transport and ATP hydrolysis, differential targeting of V-ATPases within the cell and control of the Cl(−) conductance that is necessary for vacuolar acidification.

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