scholarly journals Functional complementation reveals that 9 of the 13 human V-ATPase subunits can functionally substitute for their yeast orthologs

2019 ◽  
Vol 294 (20) ◽  
pp. 8273-8285
Author(s):  
Michiko Abe ◽  
Mayu Saito ◽  
Ayana Tsukahara ◽  
Shuka Shiokawa ◽  
Kazuma Ueno ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Sequence Similarity ◽  
Proton Translocation ◽  
Functional Complementation ◽  
Growth Defects ◽  
Vacuolar Acidification ◽  
Atpase Subunits ◽  
Intracellular Organelles ◽  
Yeast Genes ◽  
Yeast Mutants

Vacuolar-type H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for acidification of intracellular organelles and potential drug target. It is a multisubunit complex comprising a cytoplasmic V1 domain responsible for ATP hydrolysis and a membrane-embedded Vo domain that contributes to proton translocation across the membrane. Saccharomyces cerevisiae V-ATPase is composed of 14 subunits, deletion of any one of which results in well-defined growth defects. As the structure of V-ATPase and the function of each subunit have been well-characterized in yeast, this organism has been recognized as a preferred model for studies of V-ATPases. In this study, to assess the functional relatedness of the yeast and human V-ATPase subunits, we investigated whether human V-ATPase subunits can complement calcium- or pH-sensitive growth, acidification of the vacuolar lumen, assembly of the V-ATPase complex, and protein sorting in yeast mutants lacking the equivalent yeast genes. These assessments revealed that 9 of the 13 human V-ATPase subunits can partially or fully complement the function of the corresponding yeast subunits. Importantly, sequence similarity was not necessarily correlated with functional complementation. We also found that besides all Vo domain subunits, the V1 F subunit is required for proper assembly of the Vo domain at the endoplasmic reticulum. Furthermore, the human H subunit fully restored the level of vacuolar acidification, but only partially rescued calcium-sensitive growth, suggesting a specific role of the H subunit in V-ATPase activity. These findings provide important insights into functional homologies between yeast and human V-ATPases.

scholarly journals Hph1 and Hph2 Are Novel Components of the Sec63/Sec62 Posttranslational Translocation Complex That Aid in Vacuolar Proton ATPase Biogenesis

2010 ◽  
Vol 10 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Francisco J. Piña ◽  
Allyson F. O'Donnell ◽  
Silvere Pagant ◽  
Hai Lan Piao ◽  
John P. Miller ◽  
...  
Keyword(s):  
Environmental Stress ◽  
Complex Function ◽  
Content Type ◽  
Growth Defects ◽  
Proton Atpase ◽  
Vacuolar Acidification ◽  
Atpase Subunits ◽  
Specific Proteins ◽  
A Subunit ◽  
Split Ubiquitin

ABSTRACT Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for Saccharomyces cerevisiae survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63. hph1 Δ hph2 Δ cells display reduced vacuole acidification; increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase); and growth defects similar to those of mutants lacking V-ATPase activity. sec71 Δ cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in hph1 Δ hph2 Δ and hph1 Δ hph2 Δ sec71 Δ cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates hph1 Δ hph2 Δ phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.

CRISPR/Cas9-Mediated Gene Editing of Vacuolar ATPase Subunit D Mediates Phytohormone Biosynthesis and Virus Resistance in Rice

2021 ◽  
Author(s):  
Qinghua Lu ◽  
Xiangwen Luo ◽  
Xiao Yang ◽  
Tong Zhou ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Virus Resistance ◽  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Resistance Breeding ◽  
Rice Stripe Virus ◽  
Proton Pumps ◽  
Wild Type ◽  
Knockout Mutant ◽  
Atpase Subunit ◽  
Vacuolar Atpases

Abstract Background: Vacuolar ATPases (v-ATPases) are proton pumps for proton translocation across membranes that utilize energy derived from ATP hydrolysis; Previous research revealed Osv-ATPases mediates phytohormes levels and resistance in rice. Osv-ATPase subunit d (Osv-ATPase d) is part of an integral, membrane-embedded V0 complex of V-ATPases complex, whether Osv-ATPase d involves in phytohormes biosynthesis and resistance in rice remains unknown.Finding: The knockout mutant line (line 5) of Osv-ATPase d was generated using the CRISPR/Cas9 system, mutation of Osv-ATPase d did not show any detrimental effect on plant growth or yield productivity. Transcriptomic results showed Osv-ATPase d probably involved in mediating the biosynthesis of plant hormones and resistance in rice. Mutation of Osv-ATPase d significantly increased JA and ABA biosynthesis than wild type. Compared to wild type, mutation of Osv-ATPase d increased the resistance against Southern rice black-streaked dwarf virus (SRBSDV), however, decreased the resistance against Rice stripe virus (RSV) in rice. Conclusion: Taken together, our data reveal the Osv-ATPase d mediates phytohormone biosynthesis and virus resistance in rice, which can be selected as a potential target for resistance breeding in rice.

scholarly journals Structure of a bacterial ATP synthase

2018 ◽  
Author(s):  
Hui Guo ◽  
Toshiharu Suzuki ◽  
John L. Rubinstein
Keyword(s):  
Atp Synthase ◽  
Biochemical Analysis ◽  
Atp Hydrolysis ◽  
Genetic Manipulation ◽  
Proton Translocation ◽  
Atp Synthesis ◽  
Mitochondrial Complex ◽  
Rotational States ◽  
Membrane Region ◽  
Atp Synthases

AbstractATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed theBacillusPS3 ATP synthase inEschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunitεshows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

Characterization of yeast V-ATPase mutants lacking Vph1p or Stv1p and the effect on endocytosis

2002 ◽  
Vol 205 (9) ◽  
pp. 1209-1219 ◽  
Author(s):  
Natalie Perzov ◽  
Vered Padler-Karavani ◽  
Hannah Nelson ◽  
Nathan Nelson
Keyword(s):  
Sucrose Gradient ◽  
Ph Sensor ◽  
Wild Type ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Vacuolar Acidification ◽  
Atpase Subunits ◽  
Immunological Assays ◽  
Gradient Fractionation

SUMMARYSubunit a of V-ATPase in the yeast Saccharomyces cerevisiae, in contrast to its other subunits, is encoded by two genes VPH1 and STV1. While disruption of any other gene encoding the V-ATPase subunits results in growth arrest at pH 7.5, null mutants of Vph1p or Stv1p can grow at this pH. We used a polyclonal antibody to yeast Stv1p and a commercially available monoclonal antibody to Vph1p for analysis of yeast membranes by sucrose gradient fractionation, and two different vital dyes to characterize the phenotype of vph1 ▵ and stv1 ▵mutants as compared to the double mutant and the wild-type cells. Immunological assays of sucrose gradient fractions revealed that the amount of Stv1p was elevated in the vph1 ▵ strain, and that vacuoles purified by this method with no detectable endosomal contamination contain an assembled V-ATPase complex, but with much lower activity than the wild type. These results suggest that Stv1p compensates for the loss of Vph1p in the vph1 ▵ strain. LysoSensor Green DND-189 was used as a pH sensor to demonstrate unexpected changes in vacuolar acidification in stv1▵ as the Vph1p-containing V-ATPase complex is commonly considered to acidify the vacuoles. In the vph1 ▵ strain, the dye revealed slight but definite acidification of the vacuole as well. The lipophilic dye FM4-64 was used as an endocytic marker. We show that the null V-ATPase mutants, as well as the vph1 ▵ one, markedly slow down endocytosis of the dye.

scholarly journals Prodigiosins uncouple lysosomal vacuolar-type ATPase through promotion of H+/Cl− symport

1998 ◽  
Vol 334 (3) ◽  
pp. 731-741 ◽  
Author(s):  
Shoji OHKUMA ◽  
Tomohiko SATO ◽  
Masayuki OKAMOTO ◽  
Hidekazu MATSUYA ◽  
Kunizo ARAI ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Dependent Manner ◽  
Lysosomal Ph ◽  
Vacuolar Acidification ◽  
Ic50 Values ◽  
Atp Inhibition ◽  
Glycoprotein Processing ◽  
Type V ◽  
Micro Organisms ◽  
Lysosomal Acidification

We reported previously [Kataoka, Muroi, Ohkuma, Waritani, Magae, Takatsuki, Kondo, Yamasaki and Nagai (1995) FEBS Lett. 359, 53–59] that prodigiosin 25-C (one of the red pigments of the prodigiosin group produced by micro-organisms like Streptomycesand Serratia) uncoupled vacuolar H+-ATPase, inhibited vacuolar acidification and affected glycoprotein processing. In the present study we show that prodigiosin, metacycloprodigiosin and prodigiosin 25-C, all raise intralysosomal pH through inhibition of lysosomal acidification driven by vacuolar-type (V-)ATPase without inhibiting ATP hydrolysis in a dose-dependent manner with IC50 values of 30–120 pmol/mg of protein. The inhibition against lysosomal acidification was quick and reversible, showing kinetics of simple non-competitive (for ATP) inhibition. However, the prodigiosins neither raised the internal pH of isolated lysosomes nor showed ionophoric activity against H+ or K+ at concentrations where they strongly inhibited lysosomal acidification. They required Cl- for their acidification inhibitory activity even when driven in the presence of K+ and valinomycin, suggesting that their target is not anion (chloride) channel(s). In fact, the prodigiosins inhibited acidification of proteoliposomes devoid of anion channels that were reconstituted from lysosomal vacuolar-type (V-)ATPase and Escherichia coli phospholipids. However, they did not inhibit the formation of an inside-positive membrane potential driven by lysosomal V-ATPase. Instead, they caused quick reversal of acidified pH driven by lysosomal V-ATPase and, in acidic buffer, produced quick acidification of lysosomal pH, both only in the presence of Cl-. In addition, they induced swelling of liposomes and erythrocytes in iso-osmotic ammonium salt of chloride but not of gluconate, suggesting the promotion of Cl- entry by prodigiosins. These results suggest that prodigiosins facilitate the symport of H+ with Cl- (or exchange of OH- with Cl-) through lysosomal membranes, resulting in uncoupling of vacuolar H+-ATPase.

scholarly journals The Inhibitory Effect of Celangulin V on the ATP Hydrolytic Activity of the Complex of V-ATPase Subunits A and B in the Midgut of Mythimna separata

Toxins ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 130
Author(s):  
Liwen Ding ◽  
Zongxin Guo ◽  
Hang Xu ◽  
Tie Li ◽  
Yuanyuan Wang ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Insect Pests ◽  
Feedback Inhibition ◽  
Hydrolytic Activity ◽  
Inhibitory Effect ◽  
Toxic Activity ◽  
Atp Binding Site ◽  
Insecticidal Effect ◽  
Atpase Subunits ◽  
Mechanism Of Interaction

Celangulin V (CV) is a compound isolated from Celastrus angulatus Max that has a toxic activity against agricultural insect pests. CV can bind to subunits a, H, and B of the vacuolar ATPase (V-ATPase) in the midgut epithelial cells of insects. However, the mechanism of action of CV is still unclear. In this study, the soluble complex of the V-ATPase A subunit mutant TSCA which avoids the feedback inhibition by the hydrolysate ADP and V-ATPase B subunit were obtained and then purified using affinity chromatography. The H+K+-ATPase activity of the complex and the inhibitory activity of CV on ATP hydrolysis were determined. The results suggest that CV inhibits the ATP hydrolysis, resulting in an insecticidal effect. Additionally, the homology modeling of the AB complex and molecular docking results indicate that CV can competitively bind to the AB complex at the ATP binding site, which inhibits ATP hydrolysis. These findings suggest that the AB subunits complex is one of the potential targets for CV and is important for understanding the mechanism of interaction between CV and V-ATPase.

Transhydrogenase and the anaerobic mitochondrial metabolism of adult Hymenolepis diminuta

Parasitology ◽  
2009 ◽  
Vol 137 (3) ◽  
pp. 395-410 ◽  
Author(s):  
C. F. FIORAVANTI ◽  
K. P. VANDOCK
Keyword(s):  
Electron Transport ◽  
Malic Enzyme ◽  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Phosphorylation Site ◽  
Hymenolepis Diminuta ◽  
Intermembrane Space ◽  
Hydride Ion ◽  
Matrix Surface ◽  
Atp Generation

SUMMARYThe adult cestode, Hymenolepis diminuta, is essentially anaerobic energetically. Carbohydrate dissimilation results in acetate, lactate and succinate accumulation with succinate being the major end product. Succinate accumulation results from the anaerobic, mitochondrial, ‘malic’ enzyme-dependent utilization of malate coupled to ATP generation via the electron transport-linked fumarate reductase. A lesser peroxide-forming oxidase is apparent, however, fumarate reduction to succinate predominates even in air. The H. diminuta matrix-localized ‘malic’ enzyme is NADP-specific whereas the inner membrane (IM)-associated electron transport system prefers NADH. This dilemma is circumvented by the mitochondrial, IM-associated NADPH→NAD+ transhydrogenase in catalyzing hydride ion transfer from NADPH to NAD+ on the IM matrix surface. Hydride transfer is reversible and phospholipid-dependent. NADP+ reduction occurs as a non energy-linked and energy-linked reaction with the latter requiring electron transport NADH utilization or ATP hydrolysis. With NAD+ reduction, the cestode transhydrogenase also engages in concomitant proton translocation from the mitochondrial matrix to the intermembrane space and supports net ATP generation. Thus, the cestode NADPH→NAD+ system can serve not only as a metabolic connector, but an additional anaerobic phosphorylation site. Although its function(s) is unknown, a separate IM-associated NADH→ NAD+ transhydrogenation, catalyzed by the lipoamide and NADH dehydrogenases, is noted.

scholarly journals Structures of an intact yeast V-ATPase alone and in complex with bacterial effector VopQ

2020 ◽  
Author(s):  
Wei Peng ◽  
Jessie Fernandez ◽  
Amanda K. Casey ◽  
Lisa N. Kinch ◽  
Diana R. Tomchick ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Regulatory Subunit ◽  
Cellular Membranes ◽  
Catalytic Subunits ◽  
Catalysis Mechanism

AbstractVacuolar-type ATPase (V-ATPase) is a rotary protein pump involved in proton translocation across various cellular membranes using the energy of ATP hydrolysis. Despite previous studies on bacterial and eukaryotic V-ATPases, information on the intact structure of a eukaryotic V-ATPase is missing. Here we report cryo-EM structures of the intact yeast V-ATPase and this complex bound to a bacterial effector. We reveal the interaction of the elusive regulatory subunit H with its neighboring subunits. Insight for the catalysis mechanism is gained by determining conformations of the catalytic subunits either empty or bound with nucleotides.

scholarly journals Structure and activation mechanism of the hexameric plasma membrane H+-ATPase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Peng Zhao ◽  
Chaoran Zhao ◽  
Dandan Chen ◽  
Caihong Yun ◽  
Huilin Li ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Conformational Changes ◽  
Liquid Crystalline ◽  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Proton Pumping ◽  
Antifungal Drugs ◽  
Activation Mechanism ◽  
Activation Process ◽  
Α Helix

AbstractThe S. cerevisiae plasma membrane H+-ATPase, Pma1, is a P3A-type ATPase and the primary protein component of the membrane compartment of Pma1 (MCP). Like other plasma membrane H+-ATPases, Pma1 assembles and functions as a hexamer, a property unique to this subfamily among the larger family of P-type ATPases. It has been unclear how Pma1 organizes the yeast membrane into MCP microdomains, or why it is that Pma1 needs to assemble into a hexamer to establish the membrane electrochemical proton gradient. Here we report a high-resolution cryo-EM study of native Pma1 hexamers embedded in endogenous lipids. Remarkably, we found that the Pma1 hexamer encircles a liquid-crystalline membrane domain composed of 57 ordered lipid molecules. The Pma1-encircled lipid patch structure likely serves as the building block of the MCP. At pH 7.4, the carboxyl-terminal regulatory α-helix binds to the phosphorylation domains of two neighboring Pma1 subunits, locking the hexamer in the autoinhibited state. The regulatory helix becomes disordered at lower pH, leading to activation of the Pma1 hexamer. The activation process is accompanied by a 6.7 Å downward shift and a 40° rotation of transmembrane helices 1 and 2 that line the proton translocation path. The conformational changes have enabled us to propose a detailed mechanism for ATP-hydrolysis-driven proton pumping across the plasma membrane. Our structures will facilitate the development of antifungal drugs that target this essential protein.

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