Milan Höfer

Uphill transport of sugars in the yeast Rhodotorula gracilis

Abstract The lipid-forming yeast Rhodotorula gracilis was found to transport both metabolizable and non-metabolizable sugars against a concentration gradient. The process appears to require metabolic energy but operates to a limited extent even anaerobically when no gas exchange and substrate utilization are detectable in the cells. The accumulated sugar is present intracellularly in an osmotically active state and is readily exchangeable for external sugar. The transport is pH-dependent with an optimum near pH 5 but it is Na + - and K + -independent. A carrier system appears to be involved which transports 1 sugar molecule at a time and possesses different effective affinities for substrates at the two sides of the membrane. Of all the sugars tested only d -glucose does not penetrate the cell anaerobically although it prevents the transport of other sugars.

Metabolism of the obligatory aerobic yeast Rhodotorula gracilis IV. Induction of an enzyme necessary for D-xylose catabolism

Abstract 1. The metabolic fate of D -xylose taken up by Rhodotorula gracilis cells has been investigated with the following results: 2. 1. D -Xylose added to glucose-grown cells stimulates the cell respiration. However, the sugar taken up is not immediately broken down, more than 90% of it was recovered after a 30-min incubation as free intracellular D -xylose. 3. 2. The stimulated cell respiration is supported exclusively from an endogenous source of substrate. 4. 3. Only prolonged incubation of cells with D -xylose leads to exponentially growing ability to catabolize the pentose. This adaptation is completed in about 4 h. 5. 4. During the adaptation an enzyme activity appears, which was found in both the supernatant and the sediment of xylose-grown cell-free extract, but not in those of glucose-grown cells. 6. 5. This enzyme activity corresponds to that of D -xylose isomerase (EC5.3.1.5) because: (a) the enzyme requires for its activity neither NADH nor NADPH; (b) xylose-grown cells which readily catabolized D -xylose fail to break down added xylitol even if (c) xylitol is accumulated in the cells. 7. 6. Application of 3 mM actidione inhibited the enzyme induction without profoundly affecting the cell metabolism, i.e. we are dealing here with a de novo enzyme synthesis.

Proton pumps of the plasmalemma of the yeast Rhodotorula gracilis Their coupling to fluxes of potassium and other ions

Abstract (1) Intact cells of the obligatory aerobic yeast Rhodotorula gracilis (glutinis) generate a difference of the electrochemical proton potential ( Δμ H + ) across the plasmalemma. In the range from pH 4.0 to 7.0 its value remains close to 12 kJ/mol. At pH 4.0 it is composed of the pH difference (inside alkaline) alone, at pH 7.0 of the membrane potential alone. (2) Both components of Δμ H + are generated by an active process, as shown by their rapid dissipation under anaerobic conditions. (3) In order to find out by which type of mechanism Δμ H + is generated the effect of a number of inhibitors of transport-ATPases (among them ouabain, triphenyltin chloride, quercetin, oligomycin, venturicidin, dicyclohexylcarbodiimide, Dio-9) were tested both on the generation of the membrane potential and on the extrusion of protons either in the absence or the presence of potassium ions. We found that all three processes were inhibited by Dio-9 and dicyclohexylcarbodiimide, which are specific for H+-ATPases. Triphenyltin chloride inhibited the K+/H+-exchange without having any effect either on the extrusion of H+ alone or on the membrane potential. (4) Dicyclohexylcarbodiimide and Dio-9, but not triphenyltin chloride inhibited at pH 4.0 the active transport of sugars. This class of substrates has been shown earlier to be transported by an electrogenic H+ symport driven by Δμ H + across the cell membrane. (5) Neither the rate of respiration nor the intracellular level of ATP were significantly decreased by any of these inhibitors (except for venturicidin). (6) We conclude that in Rhodotorula gracilis the difference of the electrochemical potential of H+ is created by an electrogenic proton pump, presumably in ATPase. The extrusion of protons in exchange against potassium is catalyzed by a different energy-dependent but electroneutral system. This conclusion is based on the observation that the H+/K+ exchange does not work under conditions where the membrane potential is large, and vice versa.

Mobile membrane carrier for monosaccharide transport inRhodotorula gracilis

SummaryEvidence for a mobile membrane carrier mediating the uphill monosaccharide transport in the yeastRhodotorula gracilis is based on two types of observations: (1) Countertransport was found with14C-labelledd-xylose,l-xylose,l-rhamnose and withl-rhamnose in a cell suspension preincubated with unlabelledd-xylose. This finding indicates, moreover, that both the hexoses and the pentose share the same membrane carrier. (2) The mobility of occupied carrier molecules is higher than that of free carrier molecules. This conclusion has been drawn from: (a) comparison of the initial rates of uptake of a labelled sugar into cells preincubated in the absence and in the presence of unlabelled sugar; (b) comparison on the half-saturation constant of transport with the dissociation constant of the sugar-carrier complex; and (c) comparison of the initial rates of efflux of a labelled sugar into sugar-free and sugar-containing medium.

Measurement of plasma membrane potentials of yeast cells with glass microelectrodes

Glass microelectrodes were used to measure the electrical potential difference (Δψ) across plasma membrane of the yeast Pichia humboldtii. The cells were captured in the neck of a glass microfunnel and impaled with a glass microelectrode. The measurements were reproducible and stable for several minutes. The highest Δψ values were obtained in cells metabolizing glucose at pH 6. Δψ in cells deenergized by uncouplers or in dead cells was reduced to about one third of the maximal value. This residual Δψ probably represented Donnan potential. Δψ also was reduced by increasing concentrations of K+ in the medium. Other monovalent cations were distinctly less effective: Li+ ⪡ Na+ < K+, and Ca2+ was without effect. These experiments prove the applicability of the electrophysiological technique on yeast cells and thus open the way for direct determination of the electrical component of the plasma membrane electrochemical proton gradient.

Metabolism of the obligatory aerobic yeast Rhodotorula gracilis

The transients in metabolite concentrations of the yeast Rhodotorula gracilis have been analyzed following the addition of D-glucose and D-xylose. In addition, the effect of varied metabolite concentrations on pyruvate kinase (important for the switch: glycolysis vs. gluconeogenesis) were tested. These results when seen together with the already known enzyme equipment of the yeast, lead to the following conclusions:SummaryThe transients in metabolite concentrations of the yeast Rhodotorula gracilis have been analyzed following the addition of D-glucose and D-xylose. In addition, the effect of varied metabolite concentrations on pyruvate kinase (important for the switch: glycolysis vs. gluconeogenesis) were tested. These results when seen together with the already known enzyme equipment of the yeast, lead to the following conclusions:n 1.The pentose phosphate cycle is the main pathway of D-glucose metabolism; glucose molecules are broken down not only oxidatively, but also through the reversed transaldolase and transketolase reactions, eventually forming xylulose-5-P.2.Xylulose-5-P is split to a C2-unit and glyceraldehyde-3-P. The former compound (presumably glycollate) is oxidized to glyoxylate and further metabolized in the glyoxylate shunt of the tricarboxylic acid cycle, the latter compound is transformed to acetyl-CoA through the glycolytic reactions.3.Addition of D-xylose stimulates endogenous metabolism. It is postulated that under these conditions fragments of an endogenous substrate (predominantly cell lipids) are transformed to glucose-6-P in reactions combined from the lower part of the glycolytic pathway and the non-oxidative part of the pentose phosphate shunt.4.Crossover plots indicate the loci of glycolytic regulation between triosephosphates and phosphoglyceric acids, between phosphoglyceric acids and pyruvate (or PEP) as well as between PEP (or pyruvate) and dicarboxylic acids of the TCA cycle (malate).5.A scheme of metabolic pathways in the yeast is postulated and discussed. For comparison, the metabolite concentrations in Saccharomyces carlsbergensis, a yeast with complete glycolysis, were also analyzed.

Protoplasten der Hefe Rhodotorula gracilis@@@Protoplasts from the yeast Rhodotorula gracilis: II. Physiologische und Transporteigenschaften@@@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).ZusammenfassungDie 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.