Changes induced in the permeability barrier of the yeast plasma membrane by cupric ion.

A specific effect of Cu2+ eliciting selective changes in the permeability of intact Saccharomyces cerevisiae cells is described. When 100 microM CuCl2 was added to a cell suspension in a buffer of low ionic strength, the permeability barrier of the plasma membranes of the cells was lost within 2 min at 25 degrees C. The release of amino acids was partial, and the composition of the amino acids released was different from that of those retained in the cells. Mostly glutamate was released, but arginine was mainly retained in the cells. Cellular K+ was released rapidly after CuCl2 addition, but 30% of the total K+ was retained in the cells. These and other observations suggested that Cu2+ caused selective lesions of the permeability barrier of the plasma membrane but did not affect the permeability of the vacuolar membrane. These selective changes were not induced by the other divalent cations tested. A novel and simple method for differential extraction of vacuolar and cytosolic amino acid pools by Cu2+ treatment was established. When Ca2+ was added to Cu2+-treated cells, a large amount of Ca2+ was sequestered into vacuoles, with formation of an inclusion of a Ca2+-polyphosphate complex in the vacuoles. Cu2+-treated cells also showed enhanced uptake of basic amino acids and S-adenosylmethionine. The transport of these substrates showed saturable kinetics with low affinities, reflecting the vacuolar transport process in situ. With Cu2+ treatment, selective leakage of K+ from the cytosolic compartment appears to create a large concentration gradient of K+ across the vacuolar membrane and generates an inside-negative membrane potential, which may provide a driving force of uptake of positively charged substances into vacuoles. Cu2+ treatment provides a useful in situ method for investigating the mechanisms of differential solute pool formation and specific transport phenomena across the vacuolar membrane.     

Characterization of Vacuolar Arginine Uptake and Amino Acid Efflux inNeurospora crassaUsing Cupric Ion to Permeabilize the Plasma Membrane

Treatment ofNeurospora crassamycelia with cupric ion has been shown to permeabilize the plasma and mitochondrial membranes. Permeabilized mycelia were shown to take up arginine into the vacuoles. Uptake was ATP-independent and appeared to be driven by an existing K⁺-gradient. The kinetic characteristics of the observed uptake were similar to those observed using vacuolar membrane vesicles: theK_mfor arginine uptake was found to be 4.2–4.5 mM. Permeabilized mycelia were used to study the regulation of arginine uptake into vacuoles. The results suggest that uptake is relatively indifferent to the contents of the vacuoles and is not affected by growth of mycelia in amino acid-supplemented medium. Efflux of arginine, lysine, and ornithine from vacuoles was also measured using mycelia permeabilized with cupric ion. Arginine release was shown to be specifically enhanced by cytosolic ornithine and/or increases in the vacuolar pool of arginine or ornithine. Lysine efflux was shown be indifferent to the presence of other amino acids. These observations emphasize the importance of vacuolar compartmentation in controlling arginine and ornithine metabolism and suggest that vacuolar compartmentation may play an important role in nitrogen homeostasis of filamentous fungi.     

Mutants of Saccharomyces cerevisiae with defective vacuolar function.

Mutants of the yeast Saccharomyces cerevisiae that have a small vacuolar lysine pool were isolated and characterized. Mutant KL97 (lys1 slp1-1) and strain KL197-1A (slp1-1), a prototrophic derivative of KL97, did not grow well in synthetic medium supplemented with 10 mM lysine. Genetic studies indicated that the slp1-1 mutation (for small lysine pool) is recessive and is due to a single chromosomal mutation. Mutant KL97 shows the following pleiotropic defects in vacuolar functions. (i) It has small vacuolar pools for lysine, arginine, and histidine. (ii) Its growth is sensitive to lysine, histidine, Ca2+, heavy metal ions, and antibiotics. (iii) It has many small vesicles but no large central vacuole. (iv) It has a normal amount of the vacuolar membrane marker alpha-mannosidase but shows reduced activities of the vacuole sap markers proteinase A, proteinase B, and carboxypeptidase Y.     

Polyphosphate-cation interaction in the amino acid-containing vacuole of Neurospora crassa

The vacuoles of Neurospora crassa, grown in minimal medium, contain a 1:1 ratio of basic amino acids and phosphate, the latter in the form of long-chain, inorganic polyphosphate-P. Vacuoles isolated from cells depleted of polyphosphate retain basic amino acids despite the absence of over 90% of their polyphosphate. Thus, vacuolar retention of basic amino acids is not dependent upon binding to or charge neutralization by polyphosphate. Polyphosphate was found to be the only macromolecular polyanion in vacuoles of normal or phosphate-depleted cells. Gel filtration experiments revealed that about half the polyphosphate of normal vacuoles is bound strongly by vacuolar spermidine, Mg2+, and Ca2+. The polyphosphate thus occupied was not available for basic amino acid binding. We have identified about 90% of the cations of isolated vacuoles; in addition to spermidine, Mg2+, and Ca2+, the cation pool consists mainly of arginine, ornithine, histidine, lysine, and Na+, with a small amount of K+. Isolated vacuoles appear to be almost wholly impermeable to all these ions, and in vivo, vacuoles appear to be highly selective in ion uptake by an active process. The interaction of basic amino acid with the available polyphosphate was found to reduce the chemical activity of the former. In keeping with this effect, cells with abnormally high basic amino acid-polyphosphate ratios displayed greatly swollen vacuoles, indicating considerable osmotic activity of the basic amino acids and their counterions under these conditions.     

Isolation from Cigar Tobacco Leaves of Tetrahydroactinidiolide

Basic amino acids (lysine, histidine and arginine) accumulated in Saccharomyces cerevisiae vacuoles should be mobilized to cytosolic nitrogen metabolism under starvation. We found that the decrease of vacuolar basic amino acids in response to nitrogen starvation was impaired by the deletion of AVT4 gene encoding a vacuolar transporter. In addition, overexpression of AVT4 reduced the accumulation of basic amino acids in vacuoles under nutrient-rich condition. In contrast to AVT4, the deletion and overexpression of AVT3, which encodes the closest homologue of Avt4p, did not affect the contents of vacuolar basic amino acids. Consistent with these, arginine uptake into vacuolar membrane vesicles was decreased by Avt4p-, but not by Avt3p-overproduction, whereas various neutral amino acids were excreted from vacuolar membrane vesicles in a manner dependent on either Avt4p or Avt3p. These results suggest that Avt4p is a vacuolar amino acid exporter involving in the recycling of basic amino acids.     

Alteration in the Amino Acid Content of Yeast During Growth Under Various Nutritional Conditions

Yeast cells grown under optimal and suboptimal concentrations of biotin were analyzed for the amino acid content of their soluble pool and cellular protein. Optimally grown yeast cells exhibited a maximum amino acid content after 18 hr of growth. Biotin-deficient cells were depleted of all amino acids at 26 and 43 hr, with alanine, arginine, aspartate, cysteine, glutamate, isoleucine, leucine, lysine, methionine, serine, threonine, and valine being present in less than half the concentration observed in biotin-optimal cells. At early time intervals, the amino acid pool of biotin-deficient yeast contained lower concentrations of all amino acids except alanine. After more prolonged incubation, several amino acids accumulated in the pool of biotin-deficient yeast, but citrulline and ornithine accumulated to appreciable levels. The addition of aspartate to the growth medium resulted in a decrease in the amino acid content of biotin-optimal cells but caused a marked increase in the concentration of amino acids in biotin-deficient cells. The pools of biotin-deficient yeast grown in the presence of aspartate displayed a marked reduction in every amino acid with the exception of aspartate itself. These data provide evidence that the amino acid content of yeast cells and their free amino acid pools are markedly affected by biotin deficiency as well as by supplementation with aspartate, indicating that aspartate plays a major role in the nitrogen economy of yeast under both normal as well as abnormal nutritional conditions.     

Differential extraction of soluble pools from the cytosol and the vacuoles of yeast (Candida utilis) using DEAE-dextran

The plasma membrane of Candida utilis cells was rapidly disrupted by a small dose of DEAE-dextran. The vacuolar membranes, in contrast, remained intact under isotonic conditions. Therefore, the cytosolic pool could be extracted in a first step, and in a second step, after disruption of the vacuoles, the vacuolar pool. The two extracts were studied in cells grown on different nitrogen sources, namely ammonium, arginine, ornithine, citrulline, glycine, and proline.The amount of soluble amino acids in Candida cells varies considerably depending on the nitrogen source. This is largely caused by the variation in size of the vacuolar pool (0.8–2.4 mmol per g protein) containing nearly all nitrogen-rich amino acids such as arginine and ornithine, whereas the size of the cytoplasmic pool, holding most of the glutamic acid, is fairly constant (1.3 mmol per g protein). Upon nitrogen starvation the vacuolar pool was reduced much more than the cytosolic pool. A storage and buffer function of the vacuolar pool was also indicated by the much slower turnover of the vacuolar than of the cytosolic glutamine in an isotope labelling experiment. Potassium, sodium, orthophosphate, ATP, and other substances absorbing at 260 nm were found predominantly in the cytosolic extracts. Extraction of uniformly ¹⁴C-labelled cells showed that the total soluble pool of the cells contained about 10% of the total carbon. Of this about 45% was in the vacuolar the rest in the cytosolic extract. The labelled extracts were further characterized by ion exchange chromatography.Non-Standard Abbreviations DEAE-dextran diethylaminoethyl-dextran - MES 2-(N-morpholino)ethane sulfonic acid - PIPES piperazine-N,N-bis-2-ethane sulfonic acid - c-extract cytosolic extract - v-extract vacuolar extract     

Mobilization of vacuolar arginine in Neurospora crassa. Mechanism and role of glutamine

Nitrogen starvation has been shown to increase the cytosolic arginine concentration and to accelerate protein turnover in mycelia of Neurospora crassa. The cytosolic arginine is derived from a metabolically inactive vacuolar pool. Redistribution of arginine between cytosolic and vacuolar compartments is the result of mobilization of this metabolite in response to nitrogen starvation. Mobilization of arginine (and purines) also occurred in response to glutamine limitation, but arginine accumulated upon proline starvation. These observations indicate that mobilization is a consequence of glutamine limitation rather than a general response to amino acid starvation (or limitation). Analysis of the amino acid pools in mycelia subjected to starvation or limitation suggests that glutamine (or a metabolite derived from glutamine) provides a signal which determines the metabolic fate of vacuolar arginine. The results are consistent with the hypothesis that vacuolar compartmentation provides a readily available store of nitrogen-rich compounds to be utilized during differentiation or under conditions of nutritional stress.     

Basic amino acids and inorganic polyphosphates in Neurospora crassa: independent regulation of vacuolar pools

At least 78%, and perhaps all, of inorganic polyphosphate is shown to be contained within the vesicles (vacuoles) of Neurospora crassa, where over 97% of the soluble arginine, lysine, and ornithine pools are known to accumulate. Furthermore, synthetic polyphosphate can concentrate arginine up to 400-fold from dilute (0.01 mM) solutions in equilibrium dialysis. For these reasons and because the molar ratio of basic amino acids and polyphosphate phosphorus is approximately 1, we tested the hypothesis that there was an obligate physiological relationship between them. Experiments in which nitrogen starvation and arginine excess were imposed upon cells showed that polyphosphate content was insensitive to changes in the basic amino acid content. Experiments involving phosphate starvation and restoration showed that basic amino acid content was almost wholly independent of polyphosphate pools. Moreover, the normal high degree of compartmentation of arginine in vesicles was maintained despite polyphosphate depletion, and arginine was still exchanged across the vesicular membrane. We conclude that N. crassa, like yeasts, can regulate polyphosphates and basic amino acids independently, and that the accumulation of basic amino acids in vesicles may depend upon an energy-requiring mechanism in addition to the demonstrated charge interaction with polyphosphate.     

Restoration of aerial mycelium and antibiotic production in a Streptomyces griseoflavus arginine auxotroph

An arginine auxotrophic mutant was obtained from Streptomyces griseoflavus (bicozamycin-producing strain). The mutant grew on synthetic agar supplemented with either arginine, ornithine, citrulline or argininosuccinate, but produced massive aerial mycelium and bicozamycin only with citrulline. In liquid culture, citrulline also completely restored the ability of the mutant to produce bicozamycin. Culture with arginine or ornithine markedly changed intracellular pools of these ornithine-cycle amino acids, but did not affect the other amino acid pools. The ability to produce antibiotic (but not that to form aerial mycelium) was partially restored by certain mutations to ethionine resistance (Eth-1 and Eth-2). These mutations caused decreased or increased S-adenosylmethionine synthetase activity, but both resulted in a 4.5-8-fold increase in the intracellular S-adenosylmethionine pool. Exogenous addition of S-adenosylmethionine (0.5-3 mM) also partially restored the antibiotic-producing ability of the arginine auxotroph. No difference in the S-adenosylmethionine pool was observed in organisms grown with arginine and citrulline. It was suggested that citrulline and S-adenosylmethionine are somehow involved in the initiation of differentiation and secondary metabolism of S. griseoflavus.     

Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction

For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.     

Intracellular distribution of amino acids in an slp1 vacuole-deficient mutant of the yeast Saccharomyces cerevisiae

Amino acid pools were compared in a constructed diploid strain of Saccharomyces cerevisiae , SKD1, and a closely related strain, SKD2, carrying the slp1 mutation characterized by low pools of lysine and lacking a central vacuole. Cells of SKD2 grew more poorly than SKD1 but took up the same total amount of amino acids from the medium per cell although the profile differed between the two strains. Initially, the total pool was much higher in SKD1 than in SKD2 but the overall relative distribution between cytosol and vacuole was identical and mainly cytosolic even though the composition differed between the two strains. At the end of growth the amino acid concentration had increased and become predominantly vacuolar. Two days later the total pool in SKD1 had declined to the starting level but the intracellular distribution remained identical to that at the end of fermentation. The total concentration of amino acids in SKD2 continued to increase, particularly in the cytosol.     

Characterization of amino acid pools in the vacuolar compartment of Saccharomyces cerevisiae

Intact vacuoles are released from spheroplasts of Saccharomyces cerevisiae by means of a gentle mechanical disintegration method. They are purified by centrifugation in isotonic density gradients (flotation and subsequent sedimentation), and analyzed for their soluble amino acid content. The results indicate that about 60% of the total amino acid pool of spheroplasts is contained in the vacuoles. This may be an underestimate, as it presupposes no loss of amino acids from the vacuoles during the purification procedure. The amino acid concentration in the vecuoles is calculated to be approximately 5 times that in the cytoplasm if the total volumes of the two compartments are used for the calculation. The vacuolar amino acid pool is rich in basic amino acids, and in citrulline and glutamine, but contains a remarkably small amount of glutamate. Radioactive labeling experiments with spheroplasts indicate that the vacuolar amino acids are separated from the metabolically active pools located in the cytoplasm. This is particularly evident for the basic amino acids and glutamine; in contrast, the neutral amino acids and glutamate appear to exchange more rapidly between the cytoplasmic and the vacuolar compartments of the cells.     

A family of basic amino acid transporters of the vacuolar membrane from Saccharomyces cerevisiae

Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine. ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a vacuolar basic amino acid transporter 1 (VBA1)-deleted strain, whereas uptake of tyrosine or calcium was little affected. This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane. A defect in the uptake of histidine, lysine, or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCL069w (VBA3). These three VBA genes are closely related phylogenetically and constitute a new family of basic amino acid transporters in the yeast vacuole.     

Compartmentalization of metabolism of spatially-delimiting amino acid pools of yeast cells

The spatially different amino acid pools (i.e. cytoplasmic, vacuolar and mitochondrial) of yeast cells are metabolically compartmentalized. The accumulation of amino acids in these pools occurs at different rates; the highest rates are observed for glutamate and alanine. The former is predominantly accumulated in the cytoplasm, the latter--in the vacuoles. The renewal rates of the amino acid pools are also different. Each of them contains at least two subpools, readily convertible and relatively stable ones. The readily convertible subpools of the cytoplasmic and mitochondrial pools predominantly contain glutamate, aspartate, valine and alanine; that of the vacuolar pool--alanine. The bulk of the readily convertible alanine subpool (67%) is localized in the vacuoles, that of glutamate and aspartate (85 and 68%, respectively)--in the cytoplasm.     

Solute distribution between vacuole and cytosol of sugarcane suspension cells: Sucrose is not accumulated in the vacuole

The compartmentation of solutes in suspension cells of Saccharum sp. during different growth phases in batch culture was determined using CuCl₂ to permeabilize the plasma membrane of the cells. The efflux of cytosolic and vacuolar pools of sugars, cations and phosphate was monitored, and the efflux data for phosphate were compared and corrected using data from compartmentation analysis of phosphate as determined by ³¹P-nuclear magnetic resonance spectroscopy. The results show that sucrose is not accumulated in the vacuoles at any phase of the growth cycle. On the other hand, glucose and fructose are usually accumulated in the vacuole, except at the end of the cell-culture cycle when equal distribution of glucose and fructose between the cytosol and the vacuole is found. Both Na⁺ and Mg²⁺ are preferentially located in the vacuoles, but follow the same tendency as glucose and fructose with almost complete location in the vacuole in the early culture phases and increasing cytosolic concentration with increasing age of the cell culture. Potassium ions are always clearly accumulated in the cytosol at a concentration of about 80 mM; only about 20% of the cellular K⁺ is located inside the vacuole. Cytosolic phosphate is little changed during the cell cycle, whereas the vacuolar phosphate pool changes according to total cellular phosphate. In general there are two different modes of solute compartmentation in sugarcane cells. Some solutes, fructose, glucose, Mg²⁺ and Na⁺, show high vacuolar compartmentation when the total cellular content of the respective solute is low, whereas in the case of ample supply the cytosolic pools increase. For other solutes, phosphate and K⁺, the cytosolic concentration tends to be kept constant, and only excess solute is stored in the vacuole and remobilized under starvation conditions. The behaviour of sucrose is somewhat intermediate and it appears to equilibrate easily between cytosol and vacuole.Abbreviation NMR nuclear magnetic resonance The very cooperative help by Dr. J. Reiner with the ³¹P-NMR measurements and the technical assistance by D. Keis are gratefully acknowledged. This research was supported by the Deutsche Forschungsgemeinschaft and by Fonds der Chemischen Industrie.     

Growth of moderately thermophilic iron-oxidizing bacterium strain TI-1 in synthetic medium

The moderately thermophilic iron-oxidizing bacterium strain TI-1, which lacks enzyme systems involved in CO₂ fixation, grows at 45°C in Fe²⁺ medium supplemented with yeast extract to give a maximum cell growth of 1.0 × 10⁸ cells per ml, but does not grow in Fe²⁺ medium without yeast extract. To elucidate the physiology of the strain, a synthetic medium was developed. It was found that the best synthetic medium was Fe²⁺-6AA, containing Fe²⁺, salts, and the following six l-amino acids: alanine, aspartic acid, glutamic acid, arginine, serine, and histidine. In this medium, strain TI-1 showed a maximum cell growth of 10 × 10⁸ cells/ml. The six amino acids in the Fe²⁺-6AA medium were used not only as a carbon source but also as a source of nitrogen. Inorganic nitrogen sources, such as ammonium ion, hydrazine, hydroxylamine, nitrite, and nitrate, were not used as a sole source of nitrogen, but rather strongly inhibited the utilization of the six amino acids at 1 mM. In the Fe²⁺ (10 mM)-6AA medium supplemented with 21 mM Fe³⁺, reduction of Fe³⁺ to Fe²⁺ that was dependent on the added amino acids was observed, suggesting another role of the amino acids in the growth of strain TI-1. Washed, intact cells of strain TI-1 had the activity to reduce Fe³⁺ to Fe²⁺.     

The differential transport of amino acids into the phloem of Ricinus communis L. seedlings as shown by the analysis of sieve-tube sap

The cotyledons of castor bean (Ricinus communis L.) act as absorption organs for amino acids, which are supplied to the medium. The analysis of the sieve-tube sap, which exudes from the cut hypocotyl, demonstrated the ability of the cotyledons to load particular amino acids into the phloem and to reject the loading of others. The sieve-tube sap of cotyledons, which were embedded in the endosperm, contained 150 mM amino acids, with 50 mM glutamine as the major amino acid, and 10–15 mM each of valine, isoleucine, lysine and arginine. Removal of the endosperm led to a drastic decline in the amino-acid content of sieve-tube sap down to 16 mM. Addition of single amino acid species to the medium increased the amino acid concentration in the sieve-tube sap in specific manner: glutamine caused the largest increase (up to 140 mM in exudate), glutamate and alanine smaller increases (up to 60 mM), and arginine the smallest. In addition, the amino acid composition of the sieve-tube sap changed, for instance, glutamine or alanine readily appeared in the sieve-tube sap upon incubation in glutamine or alanine, respectively, whereas glutamate was hardly discernible even in the case of incubation with glutamate; arginine was loaded into the sieve tubes only reluctantly. In general, glutamine and alanine accumulated four- to tenfold in the sieve tubes. The uptake of amino acids and of sucrose into the sieve tubes was interdependent: the loading of sucrose strongly reduced the amino acid concentration in the sieve-tube exudate and loading of amino acids decreased the sucrose concentration. Comparison of the concentrations of various amino acids on their way from the endosperm via the cotyledon-endosperm interface, through the cotyledons and into the sieve tubes showed that glutamine, valine, isoleucine and lysine are accumulated on this pathway, whereas glutamate and arginine are more concentrated in the cotyledons than in the sieve tubes. Obviously the phloem-loading system has a transport specificity different from that of the amino acid uptake system of the cotyledon in general and it strongly discriminates between amino acids within the cotyledons.     

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 effects of high concentrations of sodium or calcium ions on the lipid composition and properties of Tetrahymena membranes

Tetrahymena pyriformis cells have been grown in media varying in NaCl concentration from 3.7 mM (normal medium) to 0.3 M and varying in CaCl2 from 0.2 mM (normal medium) to 0.1 M. Tetrahymena grown in 0.3 M NaCl showed relatively few alterations in phospholipid composition, with significant changes being found only in the cell surface membranes (pellicle), which incrased in phosphatidylethanolamine content from 39% (low Na+) to 48% (high Na+) of the total phospholipids. The small decrease in fatty acid unsaturation and increase in shorter chain fatty acids in pellicle phospholipids were not statistically significant. No significant changes in phospholipid head group composition or fatty acid distribution were observed in high Ca2+-grown cells. Complementary studies of membrane fluidity, as inferred from freeze-fracture electron microscopy analysis, indicated that membranes of high Na+-acclimated cells were similar to those of control cells, when each was measured in its respective medium. However, the outer alveolar membrane of the pellicle and the food vacuolar membrane were considerably less fluid in high-Ca2+ cells. The lower fluidity in vacuolar membranes may have been responsible for alterations in the cells' capacity to form food vacuoles.