scholarly journals The signaling lipid PI(3,5)P2 stabilizes V1–Vo sector interactions and activates the V-ATPase

2014 ◽  
Vol 25 (8) ◽  
pp. 1251-1262 ◽  
Author(s):  
Sheena Claire Li ◽  
Theodore T. Diakov ◽  
Tao Xu ◽  
Maureen Tarsio ◽  
Wandi Zhu ◽  
...  
Keyword(s):  
Salt Stress ◽  
Atpase Activity ◽  
Human Disease ◽  
Proton Pumps ◽  
Disease Phenotypes ◽  
Reversible Dissociation ◽  
Signaling Mechanisms ◽  
Vacuolar Acidification ◽  
Direct Binding

Vacuolar proton-translocating ATPases (V-ATPases) are highly conserved, ATP-driven proton pumps regulated by reversible dissociation of its cytosolic, peripheral V1 domain from the integral membrane Vo domain. Multiple stresses induce changes in V1-Vo assembly, but the signaling mechanisms behind these changes are not understood. Here we show that certain stress-responsive changes in V-ATPase activity and assembly require the signaling lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). V-ATPase activation through V1-Vo assembly in response to salt stress is strongly dependent on PI(3,5)P2 synthesis. Purified Vo complexes preferentially bind to PI(3,5)P2 on lipid arrays, suggesting direct binding between the lipid and the membrane sector of the V-ATPase. Increasing PI(3,5)P2 levels in vivo recruits the N-terminal domain of Vo-sector subunit Vph1p from cytosol to membranes, independent of other subunits. This Vph1p domain is critical for V1-Vo interaction, suggesting that interaction of Vph1p with PI(3,5)P2-containing membranes stabilizes V1-Vo assembly and thus increases V-ATPase activity. These results help explain the previously described vacuolar acidification defect in yeast fab1∆ and vac14∆ mutants and suggest that human disease phenotypes associated with PI(3,5)P2 loss may arise from compromised V-ATPase stability and regulation.

scholarly journals SOS2 Promotes Salt Tolerance in Part by Interacting with the Vacuolar H+-ATPase and Upregulating Its Transport Activity

2007 ◽  
Vol 27 (22) ◽  
pp. 7781-7790 ◽  
Author(s):  
Giorgia Batelli ◽  
Paul E. Verslues ◽  
Fernanda Agius ◽  
Quansheng Qiu ◽  
Hiroaki Fujii ◽  
...  
Keyword(s):  
Salt Stress ◽  
Salt Tolerance ◽  
Atpase Activity ◽  
Ion Transport ◽  
Affinity Purification ◽  
Tandem Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Transport Activity ◽  
Sos Pathway

ABSTRACT The salt overly sensitive (SOS) pathway is critical for plant salt stress tolerance and has a key role in regulating ion transport under salt stress. To further investigate salt tolerance factors regulated by the SOS pathway, we expressed an N-terminal fusion of the improved tandem affinity purification tag to SOS2 (NTAP-SOS2) in sos2-2 mutant plants. Expression of NTAP-SOS2 rescued the salt tolerance defect of sos2-2 plants, indicating that the fusion protein was functional in vivo. Tandem affinity purification of NTAP-SOS2-containing protein complexes and subsequent liquid chromatography-tandem mass spectrometry analysis indicated that subunits A, B, C, E, and G of the peripheral cytoplasmic domain of the vacuolar H+-ATPase (V-ATPase) were present in a SOS2-containing protein complex. Parallel purification of samples from control and salt-stressed NTAP-SOS2/sos2-2 plants demonstrated that each of these V-ATPase subunits was more abundant in NTAP-SOS2 complexes isolated from salt-stressed plants, suggesting that the interaction may be enhanced by salt stress. Yeast two-hybrid analysis showed that SOS2 interacted directly with V-ATPase regulatory subunits B1 and B2. The importance of the SOS2 interaction with the V-ATPase was shown at the cellular level by reduced H+ transport activity of tonoplast vesicles isolated from sos2-2 cells relative to vesicles from wild-type cells. In addition, seedlings of the det3 mutant, which has reduced V-ATPase activity, were found to be severely salt sensitive. Our results suggest that regulation of V-ATPase activity is an additional key function of SOS2 in coordinating changes in ion transport during salt stress and in promoting salt tolerance.

NaCl-altered oxygen flux profiles and H+-ATPase activity in roots of two contrasting poplar species

2020 ◽  
Author(s):  
Xiuying Ma ◽  
Jinke Li ◽  
Chen Deng ◽  
Jian Sun ◽  
Jian Liu ◽  
...  
Keyword(s):  
Salt Stress ◽  
Atpase Activity ◽  
Mitochondrial Respiration ◽  
Populus Euphratica ◽  
Nacl Stress ◽  
Proton Pumps ◽  
O2 Uptake ◽  
Transient Kinetics ◽  
Root Cells ◽  
Cytochemical Analysis

Abstract Maintaining mitochondrial respiration is crucial for proving ATP for H+ pumps to continuously exclude Na+ under salt stress. NaCl-altered O2 uptake, mitochondrial respiration, and the relevance to H+-ATPase activity were investigated in two contrasting poplar species, Populus euphratica (salt-tolerant) and P. popularis 35–44 (salt-sensitive). Compared with P. popularis, P. euphratica roots exhibited a greater capacity to extrude Na+ under NaCl stress (150 mM). The cytochemical analysis with Pb(NO3)2 staining revealed that P. euphratica root cells retained higher H+ hydrolysis activity than the salt-sensitive poplar during a long-term (LT) of increasing salt stress (50 to 200 mM NaCl, 4 weeks). Long-sustained activation of proton pumps require long-lasting supply of energy (ATP), delivered by aerobic respiration. Taking advantage of the vibrating-electrodes technology combined with the use of membrane-tipped, polarographic oxygen microelectrodes, the species, spatial, and temporal differences in root O2 uptake were characterized under conditions of salt stress. Oxygen uptake upon NaCl shock (150 mM) was less declined in P. euphratica than in P. popularis, although the salt-induced transient kinetics were distinct from the drastic drop of O2 caused by hyperosmotic shock (255 mM mannitol). Short-term (ST) treatment (150 mM NaCl, 24 h) stimulated O2 influx in P. euphratica roots, and LT-treated P. euphratica displayed an increased O2 influx along root axis, whereas O2 influx declined with increasing salinity in P. popularis roots. The spatial localization of O2 influxes revealed that the apical zone was more susceptible than elongation region upon high NaCl (150, 200 mM) during ST and LT stress. Pharmacological experiments showed that the Na+ extrusion and H+-ATPase activity in salinized roots were correspondingly suppressed when O2 uptake was inhibited by a mitochondrial respiration inhibitor, NaN3. Therefore, we conclude that the stable mitochondrial respiration energized H+-ATPase of P. euphratica root cells for maintaining Na+ homeostasis under salt environments.

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.

Changes in Na+/K+-ATPase expression during adaptive cell differentiation in avian nasal salt gland

1997 ◽  
Vol 200 (13) ◽  
pp. 1895-1904 ◽  
Author(s):  
JP Hildebrandt
Keyword(s):  
Salt Stress ◽  
Cyclic Amp ◽  
Atpase Activity ◽  
Time Course ◽  
Atp Hydrolysis ◽  
Salt Gland ◽  
Secretory Cells ◽  
Salt Glands ◽  
Atpase Subunit

Chronic salt stress in ducklings (Anas platyrhynchos) resulted in a sustained accumulation of cyclic AMP in the secretory cells of the nasal salt glands. Adaptive increases in the activity of the Na+/K+-ATPase, measured as ATP hydrolysis rates in freshly isolated tissue, were observed after 12 h of salt stress. This change in enzyme activity was associated with increases in protein abundance in the - as well as in the ss-subunit of Na+/K+-ATPase and an increase in ss-subunit glycosylation. We investigated whether the increase in the cytosolic cyclic AMP concentration and the adaptive changes in Na+/K+-ATPase activity were causally related. Using an organotypic tissue culture system for salt gland slices from unstressed (naive) ducklings, we produced similar changes in Na+/K+-ATPase activity and subunit abundance by treating cultured tissue with drugs that elevate cytosolic cyclic AMP levels (forskolin, 8-CPT-cAMP) during a 15 h culture period. Protein synthesis assays using cultured tissue revealed that elevations in cytosolic cyclic AMP level mediate increases in Na+/K+-ATPase subunit abundance by slowing down the degradation of ATPase subunits. This increase in the amount of enzyme protein was associated with a significant increase in Na+/K+-ATPase activity in tissue homogenates. The time course of these changes in cyclic-AMP-treated cultured tissue resembled that observed in salt-stressed intact animals, indicating that the elevation in cyclic AMP level in salt gland tissue may constitute a portion of the signalling events ultimately leading to the adaptive increase in Na+/K+-ATPase activity in vivo.

scholarly journals Mutations in the Yeast KEX2 Gene Cause a Vma−-Like Phenotype: a Possible Role for the Kex2 Endoprotease in Vacuolar Acidification

1998 ◽  
Vol 18 (3) ◽  
pp. 1534-1543 ◽  
Author(s):  
Yemisi E. Oluwatosin ◽  
Patricia M. Kane
Keyword(s):  
Atpase Activity ◽  
Intact Cell ◽  
Full Range ◽  
Carbon Sources ◽  
Calcium Sensitivity ◽  
Proton Pumping ◽  
Wild Type ◽  
Vacuolar Acidification ◽  
Null Mutants

ABSTRACT Mutants of Saccharomyces cerevisiae that lack vacuolar proton-translocating ATPase (V-ATPase) activity show a well-defined set of Vma− (stands for vacuolar membrane ATPase activity) phenotypes that include pH-conditional growth, increased calcium sensitivity, and the inability to grow on nonfermentable carbon sources. By screening based on these phenotypes and the inability ofvma mutants to accumulate the lysosomotropic dye quinacrine in their vacuoles, five new vma complementation groups (vma41 to vma45) were identified. TheVMA45 gene was cloned by complementation of the pH-conditional growth of the vma45-1 mutant strain and shown to be allelic to the previously characterized KEX2gene, which encodes a serine endoprotease localized to the late Golgi compartment. Both vma45-1 mutants and kex2 null mutants exhibit the full range of Vma− growth phenotypes and show no vacuolar accumulation of quinacrine, indicating loss of vacuolar acidification in vivo. However, immunoprecipitation of the V-ATPase from both strains under nondenaturing conditions revealed no defect in assembly of the enzyme, vacuolar vesicles isolated from akex2 null mutant showed levels of V-ATPase activity and proton pumping comparable to those of wild-type cells, and the V-ATPase complex purified from kex2 null mutants was structurally indistinguishable from that of wild-type cells. The results suggest that kex2 mutations exert an inhibitory effect on the V-ATPase in the intact cell but that the ATPase is present in the mutant strains in a fully assembled state, potentially capable of full enzymatic activity. This is the first time a mutation of this type has been identified.

Spectroscopic imaging of human disease

1996 ◽  
Vol 54 ◽  
pp. 884-885
Author(s):  
D.J. Meyerhoff
Keyword(s):  
Magnetic Field ◽  
Magnetic Resonance ◽  
Field Gradient ◽  
Human Disease ◽  
Morphological Changes ◽  
Magnetic Field Gradient ◽  
Spectroscopic Imaging ◽  
Resonance Spectroscopy ◽  
Tissue Water

Magnetic Resonance Imaging (MRI) observes tissue water in the presence of a magnetic field gradient to study morphological changes such as tissue volume loss and signal hyperintensities in human disease. These changes are mostly non-specific and do not appear to be correlated with the range of severity of a certain disease. In contrast, Magnetic Resonance Spectroscopy (MRS), which measures many different chemicals and tissue metabolites in the millimolar concentration range in the absence of a magnetic field gradient, has been shown to reveal characteristic metabolite patterns which are often correlated with the severity of a disease. In-vivo MRS studies are performed on widely available MRI scanners without any “sample preparation” or invasive procedures and are therefore widely used in clinical research. Hydrogen (H) MRS and MR Spectroscopic Imaging (MRSI, conceptionally a combination of MRI and MRS) measure N-acetylaspartate (a putative marker of neurons), creatine-containing metabolites (involved in energy processes in the cell), choline-containing metabolites (involved in membrane metabolism and, possibly, inflammatory processes),

scholarly journals Potassium depletion increases proton pump (H+-ATPase) activity in intercalated cells of cortical collecting duct

2000 ◽  
Vol 279 (1) ◽  
pp. F195-F202 ◽  
Author(s):  
Randi B. Silver ◽  
Sylvie Breton ◽  
Dennis Brown
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Collecting Duct ◽  
Proton Pumps ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Collecting Ducts ◽  
Acid Load ◽  
Collecting Tubules ◽  
Atpase Staining

Intercalated cells (ICs) from kidney collecting ducts contain proton-transporting ATPases (H+-ATPases) whose plasma membrane expression is regulated under a variety of conditions. It has been shown that net proton secretion occurs in the distal nephron from chronically K+-depleted rats and that upregulation of tubular H+- ATPase is involved in this process. However, regulation of this protein at the level of individual cells has not so far been examined. In the present study, H+-ATPase activity was determined in individually identified ICs from control and chronically K+-depleted rats (9–14 days on a low-K+ diet) by monitoring K+- and Na+-independent H+ extrusion rates after an acute acid load. Split-open rat cortical collecting tubules were loaded with the intracellular pH (pHi) indicator 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and pHiwas determined by using ratiometric fluorescence imaging. The rate of pHi recovery in ICs in response to an acute acid load, a measure of plasma membrane H+-ATPase activity, was increased after K+ depletion to almost three times that of controls. Furthermore, the lag time before the start of pHirecovery after the cells were maximally acidified fell from 93.5 ± 13.7 s in controls to 24.5 ± 2.1 s in K+-depleted rats. In all ICs tested, Na+- and K+-independent pHi recovery was abolished in the presence of bafilomycin (100 nM), an inhibitor of the H+-ATPase. Analysis of the cell-to-cell variability in the rate of pHi recovery reveals a change in the distribution of membrane-bound proton pumps in the IC population of cortical collecting duct from K+-depleted rats. Immunocytochemical analysis of collecting ducts from control and K+-depleted rats showed that K+-depletion increased the number of ICs with tight apical H+ATPase staining and decreased the number of cells with diffuse or basolateral H+-ATPase staining. Taken together, these data indicate that chronic K+ depletion induces a marked increase in plasma membrane H+ATPase activity in individual ICs.

scholarly journals Dissection of Swa2p/Auxilin Domain Requirements for Cochaperoning Hsp70 Clathrin-uncoating Activity In Vivo

2006 ◽  
Vol 17 (7) ◽  
pp. 3281-3290 ◽  
Author(s):  
Jing Xiao ◽  
Leslie S. Kim ◽  
Todd R. Graham
Keyword(s):  
Atpase Activity ◽  
Point Mutation ◽  
Mutational Analysis ◽  
Weak Interactions ◽  
Tetratricopeptide Repeat ◽  
Uba Domain ◽  
J Domain ◽  
Tpr Domain

The auxilin family of J-domain proteins load Hsp70 onto clathrin-coated vesicles (CCVs) to drive uncoating. In vitro, auxilin function requires its ability to bind clathrin and stimulate Hsp70 ATPase activity via its J-domain. To test these requirements in vivo, we performed a mutational analysis of Swa2p, the yeast auxilin ortholog. Swa2p is a modular protein with three N-terminal clathrin-binding (CB) motifs, a ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal J-domain. In vitro, clathrin binding is mediated by multiple weak interactions, but a Swa2p truncation lacking two CB motifs and the UBA domain retains nearly full function in vivo. Deletion of all CB motifs strongly abrogates clathrin disassembly but does not eliminate Swa2p function in vivo. Surprisingly, mutation of the invariant HPD motif within the J-domain to AAA only partially affects Swa2p function. Similarly, a TPR point mutation (G388R) causes a modest phenotype. However, Swa2p function is abolished when these TPR and J mutations are combined. The TPR and J-domains are not functionally redundant because deletion of either domain renders Swa2p nonfunctional. These data suggest that the TPR and J-domains collaborate in a bipartite interaction with Hsp70 to regulate its activity in clathrin disassembly.

scholarly journals The Direct Binding of Mrc1, a Checkpoint Mediator, to Mcm6, a Replication Helicase, Is Essential for the Replication Checkpoint against Methyl Methanesulfonate-Induced Stress

2009 ◽  
Vol 29 (18) ◽  
pp. 5008-5019 ◽  
Author(s):  
Makiko Komata ◽  
Masashige Bando ◽  
Hiroyuki Araki ◽  
Katsuhiko Shirahige
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Terminal Region ◽  
Methyl Methanesulfonate ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Hydroxyurea Treatment ◽  
Dna Replication Checkpoint ◽  
Direct Binding

ABSTRACT Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific Mrc1-binding protein. The central portion of Mrc1, containing a conserved coiled-coil region, was found to be essential for interaction with the 168-amino-acid C-terminal region of Mcm6, and introduction of two amino acid substitutions in this C-terminal region abolished the interaction with Mrc1 in vivo. An mcm6 mutant bearing these substitutions showed a severe defect in DNA replication checkpoint activation in response to stress caused by methyl methanesulfonate. Interestingly, the mutant did not show any defect in DNA replication checkpoint activation in response to hydroxyurea treatment. The phenotype of the mcm6 mutant was suppressed when the mutant protein was physically fused with Mrc1. These results strongly suggest for the first time that an Mcm helicase acts as a checkpoint sensor for methyl methanesulfonate-induced DNA damage through direct binding to the replication checkpoint mediator Mrc1.

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