Jaroslav Horák

l-Proline transport in Saccharomyces cerevisiae

Transport of L-proline into Saccharomyces cerevisiae K is mediated by two systems, one with a KT of 31 microM and Jmax of 40 nmol . s-1 . (g dry wt.)-1, the other with KT greater than 2.5 mM and Jmax of 150-165 nmol . s-1 . (g dry wt.)-1. The kinetic properties of the high-affinity system were studied in detail. It proved to be highly specific, the only potent competitive inhibitors being (i) L-proline and its analogs L-azetidine-2-carboxylic acid, sarcosine, D-proline and 3,4-dehydro-DL-proline, and (ii) L-alanine. The other amino acids tested behaved as noncompetitive inhibitors. The high-affinity system is active, has a sharp pH optimum at 5.8-5.9 and, in an Arrhenius plot, exhibits two inflection points at 15 degrees C and 20-21 degrees C. It is trans-inhibited by most amino acids (but probably only the natural substrates act in a trans-noncompetitive manner) and its activity depends to a considerable extent on growth conditions. In cells grown in a rich medium with yeast extract maximum activity is attained during the stationary phase, on a poor medium it is maximal during the early exponential phase. Some 50-60% of accumulated L-proline can leave cells in 90 min (and more if washing is done repeatedly), the efflux being insensitive to 0.5 mM 2,4-dinitrophenol and uranyl ions, the pH between 3 and 7.3, as well as to the presence of 10-100 mM unlabeled L-proline in the outside medium. Its rate and extent are increased by 1% D-glucose and by 10 micrograms nystatin per ml.

Anilinonaphthalene sulfonate fluorescence and amino acid transport in yeast

SummaryFluorescence of 1-anilinonaphthalene-8-sulfonate in yeast membranes appears to be caused predominantly by binding to lipids (ANSprotein∶ANSlipid≈1∶20) as indicated by the fluorescence lifetime, degree of polarization, and excitation spectra. It was insensitive to short-circuiting the membrane potential. Fluorescence intensity increased as cells (especially after pretreatment with energy donors such as glucose) were exposed to some amino acids, in particular, aspartic and glutamic acids. The character of fluorescence shifted to that of protein-bound ANS, suggesting an exposure of new protein sites accessible to the probe. This shift could be prevented by inhibitors of energy transduction as well as of transport. TheK1/2 of the shift was at 2.5mm aspartic acid.

Transport of L-lysine in the fission yeast Schizosaccharomyces pombe.

Systems of L-lysine transport in Schizosaccharomyces pombe are not constitutive, as at no phase of growth in a rich medium is lysine taken up. Transport activity appears only after preincubation of harvested cells with glucose or another suitable source of energy. If cycloheximide is added during this preincubation no transport systems are synthesized. After removal of glucose, the activity of the transport system decays with a half-time of 13 min. The transport of L-lysine into S. pombe cells from the stationary phase of growth preincubated for 60 min with 1% D-glucose is mediated by at least two systems, the high-affinity one with a Kt of 26 mumol/l and Jmax of 4.95 nmol/min per mg dry wt., the low-affinity one with a KT of 1.1 mmol/l and Jmax of 11.8 nmol/min per mg dry wt. The transport of lysine mediated by these two systems proceeds uphill. The high-affinity system has a pH optimum at 4.0-4.2, the accumulation ratio is highest at a cell density 2-5 mg dry wt. per ml and decreases with increasing lysine concentrations. Lysine accumulated by this system does not exit from cells. The only potent competitive inhibitors are L-arginine, L-histidine and D-lysine. The other amino acids tested do not behave as competitive inhibitors. Of the various metabolic inhibitors tested, the most potent were proton conductors and antimycin A.

Transport of l-glutamic acid in the fission yeast Schizosaccharomyces pombe

Transport of L-glutamic acid into the fission yeast Schizosaccharomyces pombe grown to the early stationary phase and preincubated for 60 min with 1% D-glucose is practically unidirectional and is mediated by a single uphill transport system with a KT of 170 microM and Jmax of 4.8 nmol min-1 (mg dry wt.)-1. The system proved to be rather non-specific since all the amino acids transported into the cells acted as potent competitive inhibitors. It has a pH optimum at 3.0-4.0, the accumulation ratio of L-glutamic acid is highest at a suspension density of 0.6-1.0 mg dry wt. per ml and decreases with increasing L-glutamic acid concentrations in the external medium. The system present in the cells after preincubation with D-glucose is unstable and its activity decays after washing the cells with water or after stopping the cytosolic proteinsynthesis with cycloheximide, with a half-time of 24 min in a reaction significantly retarded by phenylmethylsulfonyl fluoride, a serine proteinase inhibitor. The synthesis of the transport protein appears to be repressible by ammonium ions.

Temperature effects in amino acid transport by Saccharomyces cerevisiae

The maximum rate of transport of glycine, l -glutamic acid, l -arginine, and α -aminoisobutyric acid (AIB) in Saccharomyces cerevisiae is temperature-dependent with a break in the Arrhenius plot at 18–20°C, the apparent activation energies ranging from 0.8 to 39 kJ mol −1 above the break and from 85 to 120 kJ mol −1 below the break. With the exception of AIB, the half-saturation constants of amino acid uptake are not significantly affected by temperature. Competition between amino acids does not change the characteristic dependence but trans-inhibition by intracellular amino acids completely erases the transition temperature break. Temperature does not qualitatively alter the pH and cation effects on transport. On the other hand, utilization for amino acid transport of energy stored by preincubation with d -glucose shows a higher temperature dependence than the use of energy in depleted cells.

Critical findings on the activation cascade of yeast plasma membrane H+-ATPase

Strains of the yeast Saccharomyces cerevisiae, deficient in either of its two G-proteins, in the Snf3 and Rgt2 sensors, in the Gpr1 receptor and in various hexokinases were tested for their ability to start the activation cascade with a metabolizable monosaccharide that leads eventually to activation of plasma membrane H(+)-ATPase. The acidification rate after addition of glucose to glucose-grown cells and of galactose to galactose-grown ones, and the rate of ATP hydrolysis by purified plasma membranes in both types of cells were studied. It appears unequivocally that phosphorylation of the monosaccharide is essential for the activation; the role of the Gpa2 protein (possibly in combination with the Gpr1 receptor) is very probable while the two sensors appear to play somewhat ambiguous roles - in the absence of both the activation was actually higher than in the parent strain. The Gpa1 G-protein is not involved in acidification but may function in ATPase activity where, in addition to the phosphorylation step, other factors can play a role. There appear to be alternative pathways leading to the ultimate activation of the H(+)-ATPase, not necessarily involving G-proteins.

Thialysine-resistant mutants and uptake of lysine in Schizosaccharomyces pombe.

SummaryMutants defective in lysine transport were isolated and characterized. After UV-mutagenesis colonies resistant to thialysine, a toxic analogue of lysine, were isolated and L-lysine uptake into the mutant strains was analyzed. Among the thialysine-resistant strains a group of mutants was found, where the half-saturation constant, KT, of the high-affinity transport system for lysine was higher than in the wild-type, the high-affinity transport system for basic amino acids being specifically affected. This was confirmed by a complementation test in which all the thialysine-resistant strains with a higher KT for lysine uptake belonged to one complementation group. Kinetic and genetic analysis showed that our mutants were identical with can1-1 mutants, showing that a single high-affinity system for the transport of basic amino acids exists in S. pombe.

Phenylmethylsulfonyl fluoride protects l-lysine transport in Schizosaccharomyces pombe against inactivation by ammonium ions

Ammonium ions inactivate the basic amino acid transport system in Schizosaccharomyces pombe in an irreversible manner. The inactivation is accompanied by a 4-fold decrease of KT of L-lysine transport, leaving its Jmax unchanged; phenylmethylsulfonyl fluoride protects the system against inactivation. In contrast, two basic amino acid transport systems in a gap1 mutant of Saccharomyces cerevisiae are influenced by NH4+ ions in such a way that only the Jmax decreases while the KT of L-lysine transport is unchanged. Phenylmethylsulfonyl fluoride does not act here as a protective agent.

Effects of pH and of Temperature on Saturable Transport Processes

In their molecular nature, specific membrane transport processes resemble enzyme reactions taking place in a continuous phase: They involve binding of a “substrate” molecule to a specific receptor site, the translocation proper corresponding to the substrate → product conversion, and dissociation of the “product”, in the transport case simply release of the bound molecule or ion to another aqueous phase, separated from the starting aqueous phase by the membrane. The amount of evidence supporting the above sequence of events is vast and need not be reiterated here. Kinetically, all such transports are characterized by a half-saturation constant which is formally identical with the Michaelis constant of enzyme kinetics and is a similarly complicated function of various rate constants comprised in the mechanism, and by a maximum rate of transport, involving the total amount of carrier protein present in a given amount of cells and a combination of first-order rate constants, again depending on the complexity of the system under consideration.