Modification of the F0 portion of ECF1-F0 by the water-soluble carbodiimide EDC and effect on the proton channeling function

V-ATPases are ubiquitous proton pumps responsible for compartment acidification in all eukaryotic cells (1, 2). These pumps couple hydrolysis of cytosolic ATP to proton transport into the lysosome/vacuole, endosomes, Golgi apparatus, clathrin-coated vesicles, and synaptic vesicles. Through their role in organelle acidification, V-ATPases are linked to cellular functions as diverse as protein sorting and targeting, zymogen activation, cytosolic pH homeostasis, and resistance to multiple types of stress (3). They are also recruited to the plasma membrane of certain cells, where they catalyze proton export (4, 5).V-ATPases are evolutionarily related to ATP synthases of bacteria and mitochondria and consist of two multisubunit complexes, V 1 and V 0 , which contain the sites for ATP hydrolysis and proton transport, respectively. Like the ATP synthase (F-ATPase), V-ATPases utilize a rotational catalytic mechanism. ATP binding and hydrolysis in the three catalytic subunits of the V 1 sector generate sequential conformational changes that drive rotation of a central stalk (6 -8). The central stalk subunits are connected to a ring of proteolipid subunits in the V 0 sector that bind protons to be transported. The actual transport is believed to occur at the interface of the proteolipids and V 0 subunit a. Rotational catalysis will be productive in proton transport only if V 0 subunit a is held stationary, whereas the proteolipid ring rotates (8). This "stator function" resides in a single peripheral stalk in F-ATPases (9, 10), but is distributed among up to three peripheral stalks in V-ATPases (11-13). The peripheral stator stalks link V 0 subunit a to the catalytic headgroup and ensures that there is rotation of the central stalk complex relative to the V 0 a subunit and catalytic headgroup.Eukaryotic V-ATPases are highly conserved in both their overall structure and the sequences of individual subunits. Although homologs of most subunits of eukaryotic V-ATPases are present in archaebacterial V-ATPases (also known as A-ATPases), the C and H subunits are unique to eukaryotes. Both subunits have been localized at the interface of the V 1 and V 0 sectors, suggesting that they are positioned to play a critical role in structural and functional interaction between the two sectors (14 -16). The yeast C and H subunits are the only eukaryotic V-ATPase subunits for which x-ray crystal structures are available (17,18). The structure of the C subunit revealed an elongated "dumbbell-shaped" molecule, with foot, head, and neck domains (18). The structure of the H subunit indicated two domains. The N-terminal 348 amino acids fold into a series of HEAT repeats and are connected by a 4-amino acid linker to a C-terminal domain containing amino acids 352-478 (17). These two domains have partially separable functions in the context of the assembled V-ATPase (19). Complexes containing only the N-terminal domain of the H subunit (H-NT) 2 supported some ATP hydrolysis but little or no proton pumping in isolated vacuolar vesicles (19,20). The...

Section: Methodsmentioning

confidence: 99%

Subunit Interactions and Requirements for Inhibition of the Yeast V1-ATPase

Diab

Ohira

Liu

et al. 2009

“…JWY1/2-vma4-1 ts cells and SF838 -5A␣ cells transformed with YEp24 were grown in supplemented minimal medium lacking uracil to an optical density at 600 nm (OD 600 ) of 0.8 -1.0 before harvesting. The measurement of vacuolar ATPase activity was performed on a Beckman DU640 spectrophotometer using a coupled enzyme assay at 25°C (38). To examine the thermal stability of the V-ATPase, both types of vesicles were incubated at 25 and 37°C for 30 and 60 min, then the activity was assayed at 25°C and samples were taken for Western blotting.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a Temperature-sensitive Yeast Vacuolar ATPase Mutant with Defects in Actin Distribution and Bud Morphology

Zhang

Parra

Liu

et al. 1998

The 27-kDa E subunit, encoded by the VMA4 gene, is a peripheral membrane subunit of the yeast vacuolar H ؉ -ATPase. We have randomly mutagenized the VMA4 gene in order to examine the structure and function of the 27-kDa subunit. Cells lacking a functional VMA4 gene are unable to grow at pH > 7 or in elevated concentrations of CaCl 2 . Plasmid-borne, mutagenized vma4 genes were screened for failure to complement these phenotypes. Mutants producing Vma4 proteins detectable by immunoblot were selected; one (vma4 -1 ts ) is temperature conditional, exhibiting the Vma ؊ phenotype only at elevated temperature (37°C). Sequencing revealed that a single point mutation, D145G, was responsible for the phenotypes of the vma4-1 ts allele. The unassembled 27-kDa subunit made in the vma4-1 ts cells is rapidly degraded, particularly at 37°C, but can be protected from degradation by prior assembly into the V-ATPase complex. In purified vacuolar vesicles from the mutant cells, the peripheral subunits are localized to the vacuolar membrane at decreased levels and a comparably decreased level of ATPase activity (14% of the activity in wild-type vesicles) is observed. When vma4-1 ts mutant cells are shifted to pH 7.5 medium at 37°C, the cells become enlarged and exhibit multiple large buds, elongated buds, and other abnormal morphologies, together with delocalization of actin and chitin, within 4 h. These phenotypes suggest connections between the vacuolar ATPase, bud morphology, and cytokinesis that had not been recognized previously.Acidification of the intracellular compartments comprising the vacuolar network plays an important role in membrane trafficking, protein sorting, protein degradation, ion homeostasis, and nutrient storage (reviewed in Refs. 1-3). Vacuolar-type ATPases (V-ATPases) 1 are present in the vacuole/lysosome, Golgi apparatus, coated vesicles, chromaffin granules, and synaptic membrane vesicles (1, 3). The yeast vacuolar protontranslocating ATPase (H ϩ -ATPase) is a multisubunit complex that acidifies the yeast vacuole (4, 5), and biochemical and genetic characterization indicates that it consists of at least 13 polypeptides (3). The enzyme complex is divided into a peripheral cytoplasmic domain, V 1 , which contains the sites for ATP hydrolysis, and an integral membrane domain, V o , which forms the proton channel. With the exception of the vph1⌬ and stv1⌬ mutants, which lack two different isoforms of the 100-kDa subunit (6, 7), disruption of any of the ATPase subunit genes in yeast results in an identical set of Vma Ϫ (vacuolar membrane H ϩ -ATPase) phenotypes, including a complete loss of vacuolar acidification and bafilomycin A 1 -sensitive ATPase activity in isolated vacuoles, an inability to grow in medium buffered higher than pH 7 (with optimal growth at pH 5), sensitivity to high extracellular Ca 2ϩ concentration, and a number of other physiological alterations (8 -10).The overall physiological consequences of loss of vacuolar acidification have recently been examined in a number of organisms other than...

“…ATPase activity was measured spectrophotometrically at 37°C using the coupled enzyme assay of Lotscher et al (36). One unit of ATPase activity is defined as 1 mol of ATP hydrolyzed/min.…”

Section: Methodsmentioning

confidence: 99%

Wild-type and Mutant Vacuolar Membranes Support pH-dependent Reassembly of the Yeast Vacuolar H+-ATPase in Vitro

Parra

Kane

1996

Treatment of the yeast vacuolar proton-translocatingATPase (H ؉ -ATPase) with 300 mM KI in the presence of 5 mM MgATP results in a 90% inhibition of ATPase activity accompanied by removal of at least five of the peripheral subunits of the enzyme from the membrane. Functional reassembly of the enzyme, as indicated by reattachment of the peripheral subunits and a partial (30 -70%) recovery of ATPase activity, could be achieved by dialysis of the stripped wild-type membranes to remove the KI and MgATP, but proved to be strongly pH-dependent, with optimal reassembly and recovery of activity occurring after dialysis at pH 5.5. Vacuolar membranes isolated from vma2⌬ mutants, which lack one of the peripheral subunits of the enzyme, do not contain any of the peripheral subunits but are shown to contain assembled membrane (V o ) complexes. The vma2⌬ mutant vacuoles are demonstrated to be competent for attachment of KI-stripped peripheral subunits and reactivation of ATPase activity. The results indicate that previously assembled V o complexes are capable of inducing assembly of the peripheral subunits, both with each other and with the membrane subunits, and of activating the ATPase activity that resides in the peripheral subunits in a pH-dependent manner. Acidification of intracellular compartments by vacuolar Hϩ -ATPases 1 (V-type ATPases) is crucial for a variety of processes in eukaryotic cells, including receptor-mediated endocytosis and receptor recycling, intracellular transport of newly synthesized lysosomal enzymes, coupled transport in lysosomes and secretory organelles, and proteolytic processing in vacuoles and lysosomes (reviewed in Refs. 1 and 2).On the basis of copurification, immunoprecipitations, and genetic studies, the yeast vacuolar H ϩ -ATPase closely resembles the vacuolar H ϩ -ATPases of other fungi, animals, and plants (reviewed in Refs. 3 and 4). The yeast enzyme is a multisubunit complex composed of at least 11 subunits that includes both integral and peripheral membrane proteins (3-8). Seven of the subunits (69, 60, 54,42,32,27, and 14 kDa) are part of the peripherally bound V 1 sector which, although not separately active as an ATPase, contains both catalytic and noncatalytic ATP binding sites (located on the 69-and 60-kDa subunits, respectively) (5, 6, 9 -12). The remaining four subunits (100, 36, 17, and 13 kDa) are thought to be part of the pore-forming V o sector (8,13,14).One characteristic feature of vacuolar-type ATPases is their inhibition by relatively low (50 -300 mM) concentrations of chaotropic anions such as iodide, nitrate, and isothiocyanate (15). Inhibition of enzyme activity is usually coupled with removal of the V 1 sector from the membrane (11, 16 -20). Both inhibition and subunit stripping appear to be favored by the presence of ATP. For the yeast vacuolar H ϩ -ATPase, loss of the V 1 subunits is greatest in the presence of MgATP, the physiological substrate, suggesting that catalysis may induce a conformation that is particularly susceptible to stripping (11). There is som...