A. Kotyk

[Cell-membrane transport].

The cell membrane forms a boundary that is essential for life. This boundary supports life by controlling the movement of molecules and substances across it. Some molecules and substances are transported across the cell membrane by processes that require the expenditure of energy. However, many molecules cross the cell membrane by passive processes that do not require an expenditure of energy. In this laboratory we will demonstrate two passive processes, diffusion and osmosis.

Properties of the sugar carrier in baker's yeast

Incubation ofSaccharomyces cerevisiae cells withd-galactose induced the formation of galactose-utilizing enzymes, among them a monosaccharide carrier, apparently synthesized as a proteinde novo. The synthesis of the carrier preceded that of galactokinase by as much as 2 h. The inducible carrier shows a preference for monosaccharides with an axial hydroxyl group at carbon 4 of theC1 chair conformation or at carbon 2 of the1C chair conformation. Through its mediation, some sugars normally poorly transported (d-galactose,d-fucose,l-xylose,l-arabinose) can enter into the entire cell water, occupying then one more kinetic (and morphological ?) compartment than before induction. Some other monosaccharides, readily transported even by a constitutive carrier system (e.g.l-sorbose,d-xylose,d-arabinose) share the newly synthesized carrier.

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.

Factors governing substrate-induced generation and extrusion of protons in the yeast Saccharomyces cerevisiae

Experiments with respiration deficient (rho-), ADP/ATP transport deficient (op1) and double (op1 rho-) mutants, with glycolytic and tricarboxylic acid cycle substrates showed that the substrate-induced acidification of yeast suspensions is closely associated with glycolysis. The glucose/proton stoichiometry is 2.5 : 1 to 4 : 1 depending on glucose concentration. The kinetics of the process are complex, the acidification curve having a very fast initial component and two slower exponential components. The first component suggests an initial proton efflux from endogenous sources, triggered by exogenous substrates. The acidification process exhibits two Km values at about 1 and 15 mM D-glucose, indicating two distinct saturable pathways of proton extrusion. The total extent of acidification and thus the final pHout reaches a saturation value with increasing glucose concentration and suspension density. Both the total extent and the rate of acidification are subject to control by extracellular pH which reflects the tendency of the cells to build a fixed [H+]out/[H+]in ratio. When the control is lifted, both quantities are considerably increased. A crucial role in the substrate-induced acidification is thus played by active membrane processes and their control mechanisms.

Transport of α-aminoisobutyric acid in Saccharomyces cerevisiae: Feedback control

Abstract The uptake of α-aminoisobutyric acid in bakers yeast proceeds at the expense of metabolic energy and does not reach a steady-state level if energy and substrate are provided. The uptake shows two components, one with a K m of 5.4 mM and a V of II μmoles/g dry wt per min, the other with a K m of 0.15 mM and a V of 0.5 μmole/g dry wt per min. α-Aminoisobutyric acid does not leave the cells under any conditions, except after treatment with nystatin. The uptake is trans-inhibited by a number of different amino acids, including α-aminoisobutyric acid itself, in a non-competitive manner, the K i for α-aminoisobutyric acid vs α-aminoisobutyric acid uptake being 27 mM for the major component. A model involving two forms of carrier and strictly unidirectional fluxes is described, suggesting a feedback control by the intracellular amino acid at the key step of uptake.

Processes involved in the creation of buffering capacity and in substrate-induced proton extrusion in the yeast Saccharomyces cerevisiae

The high pH-maintaining capacity of yeast suspension after glucose-induced acidification, measured as its ability to neutralize added alkali, was found to be due mainly to actively extruded acidity (H+). The buffering action of passively excreted metabolites (CO2, organic acids) and cell surface polyelectrolytes contributed only 15--40% to the overall pH-maintaining capacity which was 10 mmol NaOH/l per pH unit between pH 3 and 4 and 3.5 nmol NaOH/l per pH unit between pH 4 and 7. The buffering capacity of yeast cell-free extract was still higher (up to 4.5-times) than that of glucose-supplied cell suspension; addition of glucose to the extract thus produced considerable titratable acidity but negligible net acidity. The glucose-induced acidification of yeast suspension was stimulated by univalent cations in the sequence K+ greater than Rb+ much greater than Li+ congruent to Cs+ congruent to Na+. The processes participating in the acidification and probably also in the creation of extracellular buffering capacity include excretion of CO2 and organic acids, net extrusion of H+ and K+ (in K+-free media; in K+-containing media this is preceded by an initial rapid K+ uptake), and movements of some anions (phosphate, chlorides). The overall process appears to be electrically silent.

Uptake of trehalose by Saccharomyces cerevisiae.

Trehalose, a storage sugar of bakers yeast, is known not to be metabolized when added to a cell suspension in water or a growth medium and to support growth only after a lag of about 10 h. However, it was transported into cells by at least two transport systems, the uptake being active, with a pH optimum at 5.5. There was no stoicheiometry with the shift of protons into cells observed at high trehalose concentrations. Trehalose remained intact in cells and was not appreciably lost to a trehalose-free medium. The uptake systems were present directly after growth on glucose, then decayed with a half-life of about 25 min but could be reactivated by aerobic incubation with trehalose, maltose, alpha-methyl-D-glucoside, glucose or ethanol. The uptake systems thus induced were different as revealed by competition experiments. At least one of the systems for trehalose uptake showed cooperative kinetics. Comparative anaysis with other disaccharides indicated the existence in Saccharomyces cerevisiae, after induction with trehalose, of at least four systems for the uptake of alpha-methyl-D-glucoside, four systems for maltose, together with the two for trehalose, variously shared by the sugars, the total of alpha-glucoside-transporting systems being five.

Membrane potential in yeast cells measured by direct and indirect methods

The membrane potential, delta psi, of various yeasts estimated from the distribution of tetraphenylphosphonium cations ranged from -50 to -120 mV, depending on species, incubation conditions and technique of measurement. Values obtained directly with a microelectrode in Endomyces magnusii were consistently lower than those determined indirectly.

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.

Intracellular pH distribution and transmembrane pH profile of yeast cells

The pH-dependent fluorescence excitation of fluorescein located intracellularly and in the vicinity of cells of the yeast Saccharomyces cerevisiae and Endomyces magnusii was used to obtain local pH values at a linear resolution 0.2 micron. Cells suspended in water or in a diluted (5 mM) acidic buffer had a relatively alkaline interior (about 7.0-7.5) with pH decreasing gradually toward the periphery and further out through the cell wall to the value of the bulk solution. In slightly alkaline weak buffers the cells also showed an alkaline center and a slightly acidic ring-shaped area, but the peripheral region close to the membrane was again alkaline with pH increasing toward the bulk solution. The heterogeneity of intracellular pH was reduced or nearly abolished in starved or antimycin-treated cell. Suspension of cells in strong (200 mM) buffer resulted within 15-20 min in a nearly homogeneous pH pattern throughout the cell, attaining pH values of 5.5-7.5, depending on the pH of the buffer. Addition of glucose with concomitant pH decrease of the extracellular medium did not change appreciably the intracellular pattern for 20-30 min, except with diethylstilbestrol (inhibitor of proton-extruding ATPase) when the cell became more acidic. It appears that the delta pH measurements between the cell as a whole and the bulk solution (as are used for the calculation of the electrochemical potential of protons in proton-driven transports) are not substantiated, the probable pH difference across the plasma membrane being substantially smaller than previously supposed.