Effects of the physiological state of five yeast species on H+-ATPase-related processes

Effects of starvation and glucose preincubation on membrane potential, ATPase-mediated acidification and glutamic acid transport were studied in yeast speciesSaccharomyces cerevisiae, Schizosaccharomyces pombe, Dipodascus magnusii, Lodderomyces elongisporus andRhodotorula gracilis. Themembrane potential was highest after preincubation with glucose in all species butL. elongisporus andR. gracilis. In all cases the membranes were depolarized in the presence of 20 mmol/L KCl and hyperpolarized with 50 μmol/L diethylstilbestrol (DES). Theextracellular acidification caused by addition of glucose was highest after preincubation with glucose in all cases except inR. gracilis where there was none. In all cases except inR. gracilis addition of KCl caused a marked increase in the acidification rate. Addition of DES with glucose caused a large decrease in rate inS. cerevisiae but had much less effect on the other species.Transport of glutamic acid was clearly increased after pretreatment with glucose inS. cerevisiae, S. pombe andD. magnusii (mainly due to enhanced synthesis of the carrier) but actually decreased inR. gracilis andL. elongisporus. Addition of DES had an inhibitory effect in all species but much more pronounced inS. cerevisiae andS. pombe than in others. In general, both the acidification and the transport of glutamate were enhanced after preincubation with glucose but much more so in the semianaerobic species, such asS. cerevisiae, than in the strict aerobes (R. gracilis) where the effect was occasionally negative. There was no relationship between the ATPase-mediated acidification and the membrane potential.     

Protoplasten der Hefe Rhodotorula gracilis

Zusammenfassung Die Protoplasten der obligat aeroben Hefe Rhodotorula gracilis wurden hinsichtlich ihrer charakteristischen physiologischen und Transporteigenschaften mit intakten Zellen verglichen. Folgende Ergebnisse wurden gewonnen: 1. Endogene und durch d-Glucose stimulierte Atmung entsprach den Werten von intakten Hefezellen. 2. d-Glucose wurde von Protoplasten aus dem Medium aufgenommen und abgebaut. 3. Die Aufnahme von d-Xylose führte zu vielfacher Akkumulation der Pentose im Zellinnern. Nach 50 min wurde ein für den Xyloseabbau induziertes System wirksam. 4. Bei Zugabe im Gemisch wurde die Aufnahme von d-Xylose durch d-Glucose unterbunden. 5. Akkumulierte d-Xylose wurde bei Zugabe von d-Glucose im Austauschtransport durch den mobilen Träger aus der Zelle heraus befördert. 6. Der Zuckertransport, gemessen an der d-Xyloseaufnahme, war streng stoffwechselenergieabhängig und wurde durch Entkoppler vollständig gehemmt.Diese Ergebnisse zeigen, daß die Stoffwechsel- und Transportfunktionen der intakten Hefezellen in ihren Protoplasten vollstädig erhalten bleiben. Die Anwendung von R. gracilis-Protoplasten zur Klärung spezieller Fragestellungen ergab: 1. Der Transport von d-Trehalose erfolgte nach extracellulärer Spaltung des Disaccharides durch Aufnahme der entstandenen Glucose. 2. Densitometrische Messungen an Protoplastensuspensionen zeigten sich geeignet zur kontinuierlichen Aufzeichnung von Zuckeraufnahmevorgängen.

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Protoplasts from the yeast Rhodotorula gracilis II. Physiological and transport properties

The protoplasts of the obligatory aerobic yeast Rhodotorula gracilis (5/Fres/Harrison) were compared with the intact yeast cells with respect to the identity of their physiological and transport properties. It was found: 1. The rates of endogenous and glucose-stimulated respiration of protoplasts were similar to those of the whole cells. 2. d-glucose was taken up from the medium with constant velocity; no free glucose could be detected inside the protoplasts. 3. The uptake of d-xylose led to manifold accumulation of the pentose intracellularly. Within 50 min incubation an enzyme system for the degradation of d-xylose became effective. 4. In a mixture of d-xylose and d-glucose the latter blocked the uptake of the pentose. 5. d-xylose once accumulated was exchanged by the mobile membrane carrier for d-glucose after its addition to the protoplast suspension. 6. Addition of NaN₃ or CCCP resulted in an inhibition of d-xylose uptake. The transport process is tightly coupled to cell metabolism.It is concluded that the metabolic and transport functions of R. gracilis protoplasts equal those of the intact yeast cells. The application of the protoplasts to study some special transport problems revealed: 1. In the course of d-trehalose uptake the disaccharide was cleaved to glucose, which was actually transported across the cell membrane. 2. Densitometry of protoplasts suspensions was found suitable for the continuous recording of sugar uptake processes. This observation is of special importance for further investigations of the oscillations in sugar transport observed earlier (Heller and Höfer, 1973).

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Herrn Professor Dr. Maximilian Steiner zum 70. Geburtstag gewidmet.     

Uptake ofd-Glucosamine bySaccharomyces cerevisiae

d-glucosamine does not serve as a metabolic substrate inSaccharomyces cerevisiae although it stimulates by 15% endogenous respiration. It is taken up by a system or systems shared withd-glucose,d-fructose andd-xylose but apparently not fully with 2-deoxy-d-glucose. Its half-saturation constant is 38±14 mmol/L, in agreement with its inhibitor constant versusd-glucose andd-xylose uptake. Its maximum rate is 69±17 μmol per g dry mass per min. The transport is thermodynamically passive butd-glucosamine distribution follows the membrane potential, reaching ratios of 80∶1 at pH 7.5 and about 1∶1 at pH 4.0. These rations decrease with increasingd-glucosamine concentration as well as with increasing suspension density, and are affected by metabolic inhibitors.     

Effects of pH on the growth rate, motility and photosynthesis inEuglena gracilis

The influence of pH 3–10 on the growth, motility and photosynthesis inEuglena gracilis was demonstrated during a 7-d cultivation. The cells did not survive at pH<4 and >8, highest growth rate being detected at pH 7. Motility followed a similar patterns as growth rate. Photosynthetic response curves were shown to be of the same type over the whole pH range. High respiration was characteristic for cells grown at pH 5 and 6, the lowest one at 7. At high and also at low pH more active respiration was found which can be considered as a protective response on proton stress. Respiration was not completely inhibited with potassium cyanide. Photosynthesis was the most effective at pH 6; lower and higher pH decreased photosynthetic efficiency. pH affected more the growth rate than the photosynthesis.     

Water-soluble vitamins in cells and spent culture supernatants of Poteriochromonas stipitata,Euglena gracilis,and Tetrahymena thermophila

Vitamins B₆ and B₁₂, biotin, folates, riboflavin, nicotinate, pantothenate, biopterin, and vitamin C (l-ascorbate) were assayed in Poteriochromonas stipitata, Euglena gracilis, and Tetrahymena thermophila cells grown in defined media and in spent culture supernatants. P. stipitata and E. gracilis synthesized, stored and excreted folates (mainly as 5-methyltetrahydrofolate), B₆, riboflavin, pantothenate, nicotinate, biopterin, and ascorbate. E. gracilis synthesized and stored biotin. T. thermophila did not synthesize the above vitamins except for B₁₂, biopterin, and ascorbate; it excreted biopterin and stored B₁₂ and ascorbate. Thiamin was left of consideration because all 3 organisms are thiamin auxotrophs. Possible ecological implications of these findings are considered.     

tight coupling of monosaccharide transport and metabolism inRhodotorula gracilis

A variety of agents have been applied to separate the uphill, metabolically linked transport of monosaccharides from facilitated diffusion inRhodotorula gracilis. The substances used were iodoacetamide, 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, sodium azide, sodium fluoride and heavy-metal cations, including Cd²⁺, Hg²⁺, Cu²⁺, UO²⁺ ₂, La³⁺ and Th⁴⁺. No agent examined led to such separation. There was always either a complete inhibition of both cell metabolism (or uncoupling of oxidative phosphorylation) and the transport mechanism or the transport of sugars uphill persisted (when the metabolism was only partly inhibited). It is concluded that the monosaccharide transporting system inRhodotorula gracilis is tightly coupled to cell metabolism and cannot operate without sufficient energy supply even in cases when no uphill movement is involved.     

The polypeptide 310-helix as a template and a spacer

Summary X-ray diffraction analyses have provided detailed structural information on the 3₁₀-helices of (i) pBrBz-d-(Me)Phe-(Aib)₂-d-(Me)Phe-Aib-OtBu and Ac-(Aib)₂-l-Lys(Bz)-(Aib)₂-l-Lys(Bz)-(Aib)₂-NHMe as suitable templates for molecular recognition studies, and (ii) pBrBz-TOAC-(l-Ala)₂-TOAC-l-Ala-NHtBu as an appropriate spacer for an ESR study of side chain to side chain interactions. In addition, in Ac-TOAC-(Aib)₂-l-Trp-Aib-OMe, forming a 3₁₀-helix, the TOAC residue plays the role of an effective quencher of the fluorescence of the tryptophan residue located one turn apart.     

Short-term effects of fire frequency on vegetation composition and biomass in mixed prairie in south-western Manitoba

The net effects of one, two, and three spring burns in consecutive yearson the aboveground biomass, species composition, and soil variables wereassessed in two different mixed-grass prairie sites in south-western Manitoba.Precipitation in the first year was greater than the 30-year average but lowerduring the next three years. The first site (Area 6) was characterized byBouteloua gracilis, Stipa spartea,Selaginella densa and lichens, while the second, somewhatdrier site (Area 10) was dominated by B. gracilis andCarex spp. Each burn treatment was applied to 15 plots inarandom block design. Vegetation and soil data collected following the thirdburnare presented. In general, the effects of repeated burning were more pronouncedin the drier Area 10, where litter was significantly reduced with eachadditional burn. In Area 6, litter was significantly reduced only after threeyears of burning. In both sites, the cumulative effect of fire had nosignificant effect on total standing crop, even after three consecutive yearsofburning. In the drier Area 10, however, the biomass of several componentsshifted such that B. gracilis biomass increasedsignificantly and forb biomass decreased significantly after three burns. Plantcover was also affected more in the drier Area 10. The cover of B.gracilis, the dominant C₄ grass, significantly increasedand Carex spp. cover decreased after two burns in thedriersite. In Area 6, the most significant effect of fire was a reduction inSelaginella and lichen cover. Bare ground increased inbothsites as the number of burns increased. There was little change in soilnutrients with burning, but soil moisture somewhat decreased and surfacetemperature significantly increased after three burns.     

The influence of ATP on sugar uptake mediated by the constitutive glucose carrier of Saccharomyces cerevisiae

The glucose carrier of Saccharomyces cerevisiae transports the phosphorylatable sugars glucose, mannose, fructose and 2-deoxy-D-glucose (2-dGlc) and the non-phosphorylatable sugar 6-deoxy-D-glucose (6-dGlc). Reduction of the ATP concentration by, for example, incubating cells with antimycin A, results in a decrease in uptake of 2-dGlc and fructose. These uptake velocities can be increased again by raising the ATP level. These results establish a role of ATP in sugar transport. Transport of glucose and mannose is less affected by changes in the ATP concentration than 2-dGlc and fructose uptake, while the 6-dGlc transport is independent of the amount of ATP in the cells. Also, reduction of the kinase activity by incubation with xylose diminished transport of 2-dGlc and fructose, while the uptake of glucose and mannose remained unchanged. It is discussed that these results are due to transport-associated phosphorylation with ATP as substrate and the hexokinases and the glucokinase as phosphorylating enzymes.     

Characterization of 2-deoxyglucose and 6-deoxyglucose transport in Kluyveromyces marxianus: evidence for two different transport mechanisms

Transport of 2-deoxy-d-glucose (2-dGlc) and 6-deoxy-d-glucose (6-dGlc) is studied in Kluyveromyces marxianus, grown under different conditions. It is shown that early stationary phase cells contain only one glucose transporter, with low affinity for 6-dGlc and high affinity for 2-dGlc. This transporter is recognized by glucose and fructose. In late stationary phase cells, two transport systems are operative for 6-dGlc, one with a high and one with a low affinity. The high-affinity system appears to be a glucose-galactose carrier, catalyzing uphill transport, energized by coupling sugar transport to translocation of protons. Induction (or derepression) of the high-affinity 6-dGlc transport seems to be coupled, in an as yet unknown way, to citrate consumption and a strong alkalinization of the medium during growth. It is concluded that glucose transport in K. marxianus can proceed by at least two mechanisms: a glucose-fructose carrier, probably having phosphotransferase characteristics, and a derepressible glucose/galactose-proton symporter.     

Field measurements of photosynthesis,water-use efficiency,and growth inAgropyron smithii (C3) andBouteloua gracilis (C4) in the Colorado shortgrass steppe

Summary Field measurements of gas exchange and growth were conducted on a C₃ grass,Agropyron smithii, and a C₄ grass,Bouteloua gracilis, in order to further establish the adaptive significance of the C₄ pathway under natural conditions. Maximum rates of leaf area expansion in tillers and maximum seasonal photosynthesis rates of both species occurred during the cool, early summer month of June. The occurrence of maximum growth and photosynthesis inB. gracilis during this cool period was apparently related to its occupation of warm microenvironments next to the ground surface. As temperatures increased during the midsummer, photosynthesis rates decreased to 47% and 55% of the seasonal maximum inB. gracilis andA. smithii, respectively. Water-use efficiencies in both species were similar or slightly higher forB. gracilis during June, the period of maximum growth. By mid-July, however, leaves of the C₃ grass,A. smithii, exhibited water-use efficiencies approximately half as high asB. gracilis. These differences in water-use efficiency were the result of differences in stomatal conductance, rather than differences in daily CO₂ uptake rates which were similar in both species. The results demonstrate that in certain environments there are no offset periods of growth and maximum photosynthesis during the growing season in these C₃ and C₄ species. The greater amounts of daily water use inA. smithii during the midsummer might contribute to its much greater abundance in lowland sites in the shortgrass steppe. The C₄ grass,B. gracilis, occurs in dry upland sites in addition to the more mesic lowland sites.     

Evidence for a specific fructose carrier inRhodotorula glutinis based on kinetic studies with a mutant defective in glucose transport

The mutant R₃₃ of the obligatory aerobic yeastRhodotorula glutinis exhibited a defect ind-glucose uptake. Detailed kinetic studies ofd-glucose andd-fructose transport in wild-type and mutant strains provided evidence for the existence in the plasma membrane of a carrier specific for fructose. The transport ofd-fructose in the mutant exhibited saturation kinetics up to 1 mmol/Ld-fructose; at higher concentrations the rate ofd-fructose uptake decreased. In the wild-type strain biphasicd-fructose uptake kinetics were observed; the low-affinity component was not found in the mutant, but the high-affinity transport system persisted. During the exponential phase of growth (ond-glucose) the high-affinityd-fructose system was repressed in the wild-type strain. Mutual competition betweend-fructose andd-glucose as well as the pH dependence of transport of the two hexoses further supported the following conclusion: In the wild-type strain,d-fructose is taken up both by the specific fructose carrier (K _T=0.22 mmol/L) and the glucose carrier (K _T=9.13 mmol/L). The former does not translocated-glucose, the latter is damaged by the mutation. Finally H⁺ co-transport and plasma membrane depolarization induced by the onset ofd-fructose transport indicated that the fructose carrier is an H⁺ symporter.     

Effect of pH on inhibition of cyanide-resistant respiration by hydroxamic acids inRhodotorula glutinis

The extent of inhibition of cyanide-insensitive respiration inRhodotorula glutinis by salicylhydroxamic (SHAM) and bezhydroxamic acid depends on the pH of the cell suspension; maximum inhibition occurs at pH 4–6, at higher pH values the inhibitory capacity Tapidly drops. The acidity of the medium affects primarily the degree of dissociation of hydroxamic acids which, as weak acids, pass through the yeast cell membrane only as neutral molecules (Kotyk 1963; de la Peánaet al. 1982). In a medium more alkaline than the cell interior the fraction of the acids distributed into the cell decreases and a full inhibition requires an increased concentration of the acid in the medium. Cyanide-resistant respiration was also found inSaccharomyces cerevisiae; even here medium alkalinization eliminates the inhibitory effect of hydroxamic acids but, in contrast toR. glutinis, the cell membrane is permeable also to 2-iodobenzhydroxamie acid. Comparison of the effect of pH on the action of SHAM on cyanide-resistant respiration inR. glutinis, on the concentration of dissociated and undissociated form of SHAM in the intracelluler fluid and on its chelating ability refutes the notion that chelation of nonheme iron forms the basis of inhibiton of the alternative oxidase. The authors thank Dr. Karel Janáček, DrSc., for valuable comments concerning the evaluation of experimental results.     

Evidence for an inducible glucose transport system in Kluyveromyces lactis

To find the cause of delayed glucose oxidation in succinate-grown Kluyveromyces lactis, glucose transport was studied in glucose- and in succinate-grown cells. The initial rate of 2-deoxyglucose (2-dGlc) accumulation, as well as the appearance of 2-deoxyglucose 6-phosphate, was higher in the glucose-grown cells. In both cell types, 2-dGlc was apparently transported in the free form to be phosphorylated intracellularly . In glucose-grown cells the level of free 2-dGlc in the pool was always less than the external concentration. Exchange transport in starved, poisoned cells loaded with unlabeled 2-dGlc was 140-fold greater in glucose- than in succinate-grown cells, probably because of the presence of an inducible transport component. The development of the increased rate of transport in a succinate-grown uracil-requiring auxotroph after transfer to glucose depends on the presence of uracil.     

Effect of hydrogen peroxide on sugar transport inSchizosaccharomyces pombe. Absence of membrane lipid peroxidation

Stationary unaerated cells ofS. pombe containing endogenous substrates but not energized by any exogenous ones take up 2-deoxy-d-glucose, 6-deoxy-d-glucose,d-xylose andd-arabinose actively over diffusion equilibrium. The active uptake is inhibited by 20–100 mmol/L H₂O₂ which causes an increase inK _T but has no effect onJ _max. This “competitive inhibition” indicates that H₂O₂ affects directly the sugar binding sites of the transporters. The ATP-binding site of the plasma membrane H⁺-ATPase is also affected by 100 mmol/L H₂O₂; theK _T decreases 7-fold,J _max about 2.5-fold. These effects are not likely to be mediated by membrane lipid peroxidation which appears to be lacking inS. pombe, and this lack may be one of the reasons for the high resistance of this yeast to H₂O₂. Because of thisS. pombe represents a suitable system for studying direct effects of oxidants on membrane proteins.     

Pollen dimorphism in threeTeucrium species (Lamiaceae)

In the Iberian Peninsula and Balearic Islands pollen dimorphism was found inTeucrium fruticans L.,T. pseudochamaepitys L., andT. rotundifolium Schreber. Generally, this dimorphism shows two sizes of pollen grains, the smaller more or less collapsed. The percentage of pollen viability is calculated. Differences in size between viable and nonviable pollen grains are similar, being about 40% inT. fruticans andT. pseudochamaepitys, and about 26% inT. rotundifolium. With regard to pollen viability, the percentage of male sterility is higher inT. fruticans, in which from male sterile (ms) plants, 100% nonviable pollen was obtained from every flower observed. InT. pseudochamaepitys andT. rotundifolium with rare exceptions, the percentage of nonviable pollen does not seem to be significant.     

Internal oxygen transport inRumex species and its significance for respiration under hypoxic conditions

Rumex thyrsiflorus, Rumex crispus andRumex maritimus show a differential flood-tolerance in the river ecosystem in the Netherlands.R. thyrsiflorus occurs at high-elevated habitats and is flood-intolerant, the other two species occur at lower-elevated habitats and are flood-tolerant. We compared their respiratory activity under aerobic and anaerobic conditions in the root environment and quantified the internal gas transport. The results indicate that aerial oxygen can be used for root respiration in both aerobically and anaerobically grown plants. The amount of oxygen used via internal aeration increased with decreasing oxygen concentration in the root environment. Aerobically grown plants ofR. maritimus andR. crispus already showed a high internal aeration, but there was a significant increase in internal oxygen transport in anaerobic plants, where new, aerenchymatous roots had formed. This indicates the functional significance of new root formation for respiration in these species upon hypoxia. After two weeks of anaerobiosis, more than 50% of the total respiration of the roots of young plants ofR. maritumus and 40% of roots of young plants ofR. crispus was due to internal aeration at low oxygen concentrations in the root environment. InR. maritimus both young and old plants performed in this way, inR. crispus only young plants, whileR. thyrsiflorus showed some internal aeration, but this was hardly detectable. These differences can be explained on the basis of a different morphology and concomitant diffusive resistance of both root and shoot system. In experiments with different submergence levels of the shoot, the amount of internal aeration was positively correlated to the total leaf area protruding above the water surface inR. maritimus. This indicates a functional significance of the petiole and leaf elongation response upon total submergence of this species.     

Facultative parasites and host respiration. I. Healthy tissue adjacent to the site of infection

Summary The respiration, as measured by oxygen uptake, was higher for healthy tissue adjacent to soft rots of apple (var. Bramley) caused byPenicillium expansum Link andThom. andBotrytis cinerea Fr. and of potato tuber (var. Arran Banner) caused byErwinia aroideae Townsend, than tissue at a more distant site. The respiration of tissue adjacent to a physiological rot caused by bruising was not affected. It was concluded that there is diffusion of a substance(s) from the site of infection which causes an increase in the rate of respiration of the adjacent tissue.This work formed part of a Ph. D. thesis submitted to the University of London, July 1961.     

A reconsideration of the genusRabdosiella (Lamiaceae,Nepetoideae, Ocimeae)

The two generaPlectranthus andIsodon are compared and found to be very dissimilar.Isodon ist considered to be misplaced inOcimeae subtribePlectranthinae and apparently is more closely related to subtribeHyptidinae. The disjunct genusRabdosiella is compared to these two genera and regarded to be polyphyletic. The AfricanR. calycina (Benth.)Codd is returned toPlectranthus and calledP. calycinus Benth., while the AsianR. ternifolia (D. Don)Codd is placed inIsodon sect.Pyramidium and calledI. ternifolius (D. Don)Kudo.     

Evidence for the role of intracellular glutamine level in the regulation of glutamine uptake in the cyanobacteriumAnabaena 7120

The role of intracellular glutamine concentration in the regulation of¹⁴C-glutamine uptake was studied in a diazotrophic cyanobacteriumAnabaena 7120. The uptake pattern was found to be biphasic, consisting of a rapid first phase lasting up to 60 s followed by a slower second phase. Azaserine, which could not inhibit in vitro and in vivo glutamine synthetase (GS) activity effectively, inhibited the¹⁴C-glutamine uptake. Glutamine uptake was also not significantly affected when glutamate, methylglutamate, aspartate, arginine, lysine, hydroxylysine, ornithine, and GS inhibitor,L-methionine-DL-sulfoximine (MSX) were simultaneously available during uptake assay, suggesting that glutamine uptake takes place via a general amino acid permease which does not, however, transport basic and acidic amino acids. The azaserine-treated cells had increased and decreased levels of glutamine and glutamate, respectively, suggesting that the increased intracellular glutamine level is responsible for the inhibition of¹⁴C-glutamine uptake and provides evidence here for the role of an intracellular glutamine pool in the regulation of¹⁴C-glutamine uptake inAnabaena 7120.