Identification and properties of an ATPase in vacuolar membranes of Neurospora crassa

Using a vacuolar preparation virtually free of contamination by other organelles, we isolated vacuolar membranes and demonstrated that they contain an ATPase. Sucrose density gradient profiles of vacuolar membranes show a single peak of ATPase activity at a density of 1.11 g/cm3. Comparison of this enzyme with the two well-studied proton-pumping ATPases of Neurospora plasma membranes and mitochondria shows that it is clearly distinct. The vacuolar membrane ATPase is insensitive to the inhibitors oligomycin, azide, and vanadate, but sensitive to N,N'-dicyclohexylcarbodiimide (Ki = 2 microM). It has a pH optimum of 7.5, requires a divalent cation (Mg2+ or Mn2+) for activity, and is remarkably unaffected (+/- 20%) by a number of monovalent cations, anions, and buffers. In its substrate affinity (Km for ATP = 0.2 mM), substrate preference (ATP greater than GTP, ITP greater than UTP greater than CTP), and loss of activity with repeated 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid washes, the vacuolar membrane ATPase resembles the F1F0 type of ATPase found in mitochondria and differs from the integral membrane type of ATPase in plasma membranes.     

The vacuolar ATPase ofNeurospora crassa

The filamentous fungusNeurospora crassa has many small vacuoles which, like mammalian lysosomes, contain hydrolytic enzymes. They also store large amounts of phosphate and basic amino acids. To generate an acidic interior and to drive the transport of small molecules, the vacuolar membranes are densely studded with a proton-pumping ATPase. The vacuolar ATPase is a large enzyme, composed of 8–10 subunits. These subunits are arranged into two sectors, a complex of peripheral subunits called V₁ and an integral membrane complex called V₀. Genes encoding three of the subunits have been isolated.vma-1 andvma-2 encode polypeptides homologous to the and subunits of F-type ATPases. These subunits appear to contain the sites of ATP binding and hydrolysis.vma-3 encodes a highly hydrophobic polypeptide homologous to the proteolipid subunit of vacuolar ATPases from other organisms. This subunit may form part of the proton-containing pathway through the membrane. We have examined the structures of the genes and attempted to inactivate them.     

Structure of the vacuolar ATPase from Neurospora crassa as determined by electron microscopy.

We have examined the structure of the vacuolar ATPase of Neurospora crassa using negatively stained preparations of vacuolar membranes and of detergent-solubilized and gradient-purified ATPase complexes. We also examined the peripheral sector (V1) of the enzyme after it had been removed and purified. Using different stains, vacuolar membranes displayed ball-and-stalk structures similar to those of the intact mitochondrial ATPase. However, the vacuolar ATPase was clearly different from the mitochondrial ATPase in both size and structural features. The vacuolar enzyme had a much larger head domain with a distinct cleft down the middle of the complex. This domain was held above the membrane by a prominent stalk. Most intriguing was the presence of basal components. These structures appeared to project from the vacuolar membrane near the base of the stalks. Detergent-solubilized, gradient-purified ATPases displayed the same head, stalk, and basal features as those found with the intact enzyme on vacuolar membranes. The mitochondrial ATPase was significantly smaller, and no clefted head domains or basal components were observed. When V1 and F1 particles were directly compared, a significant difference in size and shape between these two soluble ATPase sectors was apparent. V1 retained all of the features seen in the globular head of the intact complex: V-shaped, triangular, and square forms around a stain-filled core.     

Identification of an arginine carrier in the vacuolar membrane of Neurospora crassa

A number of arginine derivatives were tested for their ability to inhibit arginine uptake into vacuolar membrane vesicles of Neurospora crassa. The guanido side chain and L-configuration were found to be important for recognition by the arginine carrier. Based upon the specificity of recognition, a reactive arginine derivative (N alpha-p-nitrobenzyloxycarbonyl arginyl diazomethane) was synthesized which has an intact guanido side chain and a diazo group at the carboxyl end. The latter decomposes to a reactive carbene group. This derivative inhibited arginine uptake into vacuolar membrane vesicles at low concentrations. Radioactive N alpha-p-nitrobenzyloxycarbonyl arginyl diazomethane was covalently bound to vacuoles. Binding was specific for a single membrane protein with an approximate molecular weight of 40,000, saturable (2 pmol/mg vacuolar membrane protein), and inhibited by 100 mM L-arginine but not by 100 mM L-lysine. The results suggest that this protein is the arginine carrier.     

The Structure of the Vacuolar ATPase in Neurospora crassa

The filamentous fungus Neurospora crassa contains many smallvacuoles. These organelles contain high concentrations of polyphosphates andbasic amino acids, such as arginine and ornithine. Because of their size anddensity, the vacuoles can be separated from other organelles in the cell. TheATP-driven proton pump in the vacuolar membrane is a typical V-type ATPase.We examined the size and structure of this enzyme using radiationinactivation and electron microscopy. The vacuolar ATPase is a large andcomplex enzyme, which appears to contain at least thirteen different types ofsubunits. We have characterized the genes that encode eleven of thesesubunits. In this review, we discuss the possible function and structure ofthese subunits.     

Energetics of vacuolar compartmentation of arginine in Neurospora crassa

The energy requirements for the uptake and retention of arginine by vacuoles of Neurospora crassa have been studied. Exponentially growing mycelial cultures were treated with inhibitors of respiration or glycolysis or an uncoupler of respiration. Catabolism of arginine was monitored as urea production in urease-less strains. The rationale was that the rate and extent of such catabolism was indicative of the cytosolic arginine concentration. No catabolism was observed in cultures treated with an inhibitor or an uncoupler of respiration, but cultures treated with inhibitors of glycolysis rapidly degraded arginine. These differences could not be accounted for by alterations in the level or activity of arginase. Mycelia growing in arginine-supplemented medium and treated with an inhibitor or uncoupler of respiration degraded an amount of arginine equivalent to the cytosolic fraction of the arginine pool. The inhibitors and the uncoupler of respiration reduced the ATP pool and the energy charge. The inhibitors of glycolysis reduced the ATP pool but did not affect the energy charge. The results suggest that metabolic energy is required for the transport of arginine into the vacuoles but not for its retention. The latter is affected by inhibitors of glycolysis. The form of energy and the nature of the vacuolar transport mechanism(s) are discussed.     

The properties of arginine transport in vacuolar membrane vesicles of Neurospora crassa

We have measured the uptake of arginine into vacuolar membrane vesicles from Neurospora crassa. Arginine transport was found to be dependent on ATP hydrolysis, Mg2+, time, and vesicle protein with transported arginine remaining unmodified after entry into the vesicles. The Mg2+ concentration required for optimal arginine transport varied with the ATP concentration so that maximal transport occurred when the MgATP2- concentration was at a maximum and the concentrations of free ATP and Mg2+ were at a minimum. Arginine transport exhibited Michaelis-Menten kinetics when the arginine concentration was varied (Km = 0.4 mM). In contrast, arginine transport did not follow Michaelis-Menten kinetics when the MgATP2-concentration was varied (S0.5 = 0.12 mM). There was no inhibition of arginine transport when glutamine, ornithine, or lysine were included in the assay mixture. In contrast, arginine transport was inhibited 43% when D-arginine was present at a concentration 16-fold higher than that of L-arginine. Measurements of the internal vesicle volume established that arginine is concentrated 14-fold relative to the external concentration. Arginine transport was inhibited by dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl-hydrazone, and potassium nitrate (an inhibitor of vacuolar ATPase activity). Inhibitors of the plasma membrane or mitochondrial ATPase such as sodium vanadate or sodium azide did not affect arginine transport activity. In addition, arginine transport had a nucleoside triphosphate specificity similar to that of the vacuolar ATPase. These results suggest that arginine transport is dependent on vacuolar ATPase activity and an intact proton channel and proton gradient.     

Properties of Neurospora crassa plasma membrane ATPase.

A variety of the biochemical properties of the electrogenic plasma membrane ATPase of Neurospora crassa are described. The enzyme catalyzes the hydrolysis of ATP, resulting in the formation of ADP and inorganic phosphate. Optimal activity is observed between pH 6 and 6.5. ATP hydrolysis approaches a maximum rate at an Mg-ATP concentration of 10–20 mm with a half-maximal velocity around 2 mm Mg-ATP. The enzyme requires a divalent cation for activity in the following order of preference at 10 mm: Mg²⁺, Co²⁺ > Mn²⁺ > Zn²⁺ > Fe²⁺, Ca²⁺, Cu²⁺. The enzyme is quite specific for ATP compared to the other nucleotides tested. Treatment of the plasma membranes with sodium deoxycholate inactivates the ATPase and the inactivation can be prevented by the addition of certain acidic phospholipids with the deoxycholate. Other classes of lipids cannot prevent the deoxycholate inhibition. The organic mercurials parachloromercuribenzoate and parachloromercuriphenylsulfonate are potent inhibitors of the ATPase, but N-ethylmaleimide at a similar concentration is not inhibitory. The organic mercurial inhibition is not reversed by mercaptoethanol. Under appropriate conditions, the inhibitory effect of p-chloromercuribenzoate is suppressed in the presence of ATP. Treatment of the plasma membranes with trypsin leads to a marked inhibition of the ATPase activity and this inhibition can be prevented by Mg-ATP. Neither the organic mercurial reactive site(s) nor the trypsin-sensitive site(s) are accessible from the outer surface of the plasma membranes. Some of the implications of the above findings are discussed.     

A model for the proteolipid ring and bafilomycin/concanamycin-binding site in the vacuolar ATPase of Neurospora crassa

The vacuolar ATPase has been implicated in a variety of physiological processes in eukaryotic cells. Bafilomycin and concanamycin, highly potent and specific inhibitors of the vacuolar ATPase, have been widely used to investigate the enzyme. Derivatives have been developed as possible therapeutic drugs. We have used random mutagenesis and site-directed mutagenesis to identify 23 residues in the c subunit involved in binding these drugs. We generated a model for the structure of the ring of c subunits in Neurospora crassa by using data from the crystal structure of the homologous subunits of the bacterium Enterococcus hirae (Murata, T., Yamato, I., Kakinuma, Y., Leslie, A. G., and Walker, J. E. (2005) Science 308, 654-659). In the model 10 of the 11 mutation sites that confer the highest degree of resistance are closely clustered. They form a putative drug-binding pocket at the interface between helices 1 and 2 on one c subunit and helix 4 of the adjacent c subunit. The excellent fit of the N. crassa sequence to the E. hirae structure and the degree to which the structural model predicts the clustering of these residues suggest that the folding of the bacterial and eukaryotic polypeptides is very similar.     

Energy requirement for the mobilization of vacuolar arginine in Neurospora crassa

The bulk of the intracellular arginine pool in exponentially growing mycelia of Neurospora crassa is sequestered in the vacuoles. Vacuolar arginine effluxes from the vacuoles into the cytosol and is catabolized to ornithine and urea upon nitrogen starvation. The energy requirement for mobilization has been studied by treating nitrogen-starved mycelia with inhibitors or respiration or glycolysis or an uncoupler of respiration. Mobilization was inhibited by the inhibitors or the uncoupler of respiration, but not by the inhibitors of glycolysis. The inhibitors and the uncoupler of respiration reduced the ATP pool and the energy charge of the treated mycelia. The inhibitors of glycolysis reduced the ATP pool but had no effect on the energy charge. The results indicate that mobilization of arginine from the vacuoles requires metabolic energy. The forms of this energy and the mode of its association with the mobilization process are discussed.     

Biosynthetic pathway of mitochondrial ATPase subunit 9 in Neurospora crassa

Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.     

Two approaches to isolate cytoplasmic dynein ATPase from Neurospora crassa

Cytoplasmic dynein is a force-producing enzyme that, in association with dynactin, conducts minus-end directed transport of various organelles along microtubules. Biochemical analyses of cytoplasmic dynein and dynactin have been conducted primarily in vertebrate systems, whereas genetic analyses have been explored mainly in yeast and the filamentous fungi. To provide a complementary biochemical approach for the study of fungal dynein, we isolated/partially purified cytoplasmic dynein ATPase from the filamentous fungus Neurospora crassa. N. crassa dynein was partially purified by slightly modifying the existing procedures, described for mammalian cytoplasmic dynein that uses dynein-microtubule binding, followed by release with ATP and sucrose gradient fractionation. A novel approach was also used to isolate dynein-specific ATPase by gel filtration (Sepharose CL-4B). The K(m), ATP obtained by isolating dynein ATPase using gel filtration was similar to that obtained by using conventional method, suggests that contaminant proteins do not interfere with the dynein ATPase activity. Like vertebrate dynein, N. crassa dynein is a general NTPase with highest activity toward ATP, and only the ATPase activity is stimulated by microtubules. The K(m), ATP for N. crassa cytoplasmic dynein is 10- to 15-fold higher than that of the vertebrate enzyme.     

Purification and characterization of the plasma membrane ATPase of Neurospora crassa

The plasma membrane of Neurospora crassa contains a proton-translocating ATPase, which functions to generate a large membrane potential and thereby to drive a variety of H+-dependent co-transport systems. We have purified this ATPase by a three-step procedure in which 1) loosely bound membrane proteins are removed by treatment with 0.1% deoxycholate; 2) the ATPase is solubilized with 0.6% deoxycholate in the presence of 45% glycerol; and 3) the solubilized enzyme is purified by centrifugation through a glycerol gradient. This procedure typically yields approximately 30% of the starting ATPase activity in a nearly homogeneous enzyme preparation of high specific activity, 61-98 mumol/min/mg of protein. The membrane-bound and purified forms of the ATPase are very similar with respect to kinetic properties (pH optimum, nucleotide and divalent cation specificity, sigmoid dependence upon Mg-ATP concentration) and sensitivity to inhibitors (including N,N'-dicyclohexylcarbodiimide and vanadate). Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified ATPase displays a single major polypeptide band of Mr = 104,000, which is essentially identical in its electrophoretic mobility with the large subunit of [Na+, K+]-ATPase of animal cell membranes and [Ca2+]-ATPase of sarcoplasmic reticulum. The structural similarity of the fungal and animal cell ATPases, together with the fact that both are known to form acyl phosphate intermediates, suggests that they may share a common reaction mechanism.     

2-D structure of the Neurospora crassa plasma membrane ATPase as determined by electron cryomicroscopy.

Large, well-ordered 2-D crystals of the dodecylmaltoside complex of the Neurospora crassa plasma membrane H(+)-ATPase grow rapidly on the surface of a polyethylene glycol-containing mixture similar to that originally developed for growing 3-D crystals of this integral membrane transport protein. Negative stain electron microscopy of the crystals shows that many are single layers. Cryoelectron microscopy of unstained specimens indicates that the crystals have a p6 layer group with unit cell dimensions of a = b = 167 A. Image processing of selected electron micrographs has yielded a projection map at 10.3 A resolution. The repeating unit of the ATPase crystals comprises six 100 kDa ATPase monomers arranged in a symmetrical ring. The individual monomers in projection are shaped like a boot. These results provide the first indications of the molecular structure of the H(+)-ATPase molecule. They also establish the feasibility of precipitant-induced surface growth as a rapid, simple alternative to conventional methods for obtaining 2-D crystals of the integral membrane proteins useful for structure analysis.