L. Říhová

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.

Derepression of the cell cycle by starvation is involved in the induction of tobacco pollen embryogenesis

SummaryMicrospectrophotometry following Feulgen staining and autoradiography following (3H)-thymidine labelling were used to study cell-cycle events during pollen development in tobacco (Nicotiana tabacum L.). During normal gametophytic pollen development in the anther and in vitro the generative nucleus passes through the S phase to the G2 phase soon after microspore mitosis, while the vegetative nucleus remains arrested in G1 (=G0). During embryogenie induction by an in vitro starvation treatment of immature pollen ongoing DNA replication in the generative nucleus is completed and followed by DNA replication in the vegetative cell in a large fraction of the pollen grains. Addition of the DNA replication inhibitor hydroxyurea to the starvation medium postpones S phase entry until the pollen is transferred to a rich medium and does not affect embryo formation. These results demonstrate that one of the crucial events of embryogenic induction is the derepression of the G1 arrest in the cell cycle of the vegetative cell.

Developmental changes in gene expression during pollen differentiation and maturation inNicotiana tabacum L

Total and polysome-bound ribosomes and the uptake and incorporation of3H-uridine and14C-leucine were examined in dividing microspores and in pollen grains isolated from anthers of 6 different developmental stages. Direct evidence was obtained that the formation of cytoplasm of the vegetative cell following microspore division is related to a rapid activation of RNA and protein synthesis and of ribosomes in differentiating pollen. Total ribosomes associated with gametophytic programme rose about 10times and the process of differentiation was accompanied by a rapid increase in uptake capacity of pollen grains for both uridine and leucine. Pollen development after cytoplasm synthesis and starch deposition continued by pollen maturation, which was characterized by a decline in RNA synthesis, dissociation of polysomes and by a further rise of transport activity of pollen grain wall for exogenous substrates, indicating probable pollen adaptation for utilization of metabolites from the degenerating tapetal cytoplasm.

Uptake of amino acids by actidione-treated yeast cells. I. Specificity of carriers.

Uptake of glyeine,l-cysteine,l-leucine,l-methionine,l-aspartic acid andl-lysine was investigated in resting cells ofSaccharomyces cerevisiae treated with 0.3mm actidione for blocking protein synthesis. The amino acids were taken up against substantial concentration gradients (up to nearly 1,000∶1 for μm l-cysteine and glycine). They were present in the free form inside the cells. Their unidirectional transmembrane fluxes were under a negative feedback control by the intracellular concentration of the amino acid involved. The amino acids tested apparently employed more than one transport agéncies for their membrane passage, the half-saturation constants being 6.2–7.7×10−4m for glycine, 2.5×10−4m forl-cysteine, 6×10−5 and 4×10−4m forl-lysine, 3×10−5 and 6×10−4m forl-methionine, 7–18×10−5 and 1.6×10−3m forl-aspartic acid and 6×10−5 and 2×10−3m forl-leucine. The specificities of the transport systems are overlapping but there emerges a wide-affinity transport system for glycine, alanine, leucine, methionine, serine, cysteine, phenylalanine, aspartic acid, asparagine, glutamic acid and tryptophan (and possibly for other amino acids), and more specific systems for each of the following: glycine, lysine, methionine, histidine, arginine, and aspartic and glutamic acids. Proline had the peculiar effect of stimulating the transport of all the amino acids tested. The amino acids apparently interacted in the uptake not only by competition for the binding site but also by allotopic inhibition (e.g.l-cysteine) and possibly stimulation (l-proline). The initial rate of uptake of amino acids and their steady-state level of distribution were characterized by identical activation energies: 7.5 kcal/mole forl-lysine, 6.9 kcal/mole forl-aspartic acid, and 13.2 kcal/mole for glycine.

Energy requirement for amino acid uptake inSaccharomyces cerevisiae

The uptake of glycine and α-aminoisobutyric acid by baker’s yeast was substantially increased by preincubation withd-glucose,d-fructose, sucrose, and maltose, but much less with ethanol or acetate. The increments in uptake are in rough agreement with the intracellular amount of acid-non-extractable high-energy phosphate (probably polyphosphate). The energy for amino acid transport is thus provided predominantly by the nonmitochondrial catabolic processes.

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.

RNA Synthesis and Ribosome Status in Pollen Tube Growth of Nicotiana tabacum L.; Effects of External pH

Summary RNA synthesis during the development of tobacco pollen is at its maximum shortly after microspore division and then decreases steadily, being only about 3 % in mature germinating pollen at its highest rate, and almost absent in pollen tubes after 2 days culture. The decrease in cultured mature pollen is not modified by keeping the external pH near the optimum for pollen tube growth and extending the duration of growth to almost 4 days. Inhibition of transcription by -y-rays, cordycepin, or a-amanitin does not inhibit growth, and growth inhibition by actinomycin D is independent of whether the drug is applied at the beginning of culture or when RNA synthesis is practically absent. RNA content in pollen tubes exhibits little variation during 4 days culture at optimal pH. A slowing down of growth is associated with a disassembly of polyribosomes and with a decrease in extractability of the total ribosomes. The process is enhanced in a non-buffered culture medium as a result of external pH acidification by pollen tubes. It can be concluded that the growth of tobacco pollen tubes does not require de novo synthesis of RNA, and that the dependence of tube growth on exogenous pH is associated with the importance of proton elimination from the cytosol for the stability in the cell distribution and function of ribosomes.

DEGRADATION AND TURNOVER OF BACTERIAL CELL WALL MUCOPEPTIDES IN GROWING BACTERIA.

The mucopeptide layer of the cell wall ofBacillus megaterium is broken down into separate components during growth of the cells. The released diaminopimelic acid is partly decarboxylated to lysine, which is incorporated in the proteins and partly used for cell wall resynthesis. The smaller portion of the degraded mucopeptide is released into the medium in the form of non-utilized fragments. The rate of the mucopeptide turnover is a function of the rate of growth of the culture. About 15–20% of the rigid layer of the cell wall is degraded during on cell division. The sensitivity ofBacillus megaterium to lysozyme and the rate of its conversion to protoplasts is also proportionate to the rate of growth of the culture. There is no measurable mucopeptide turnover in non-growing cells, either in the stationary phase of the culture or in starvation in nitrogen-free medium. The resistance of the cell wall to lysozyme also increases during the stationary phase. The rigid component of the cell wall is probably also broken down during growth ofBacillus cereus andEscherichia coli cultures.AbstractСлой мукопептидов в клеточной оболочке Bacillus megaterium в течение роста клеток расщепляется на отдельные составные части. Освобождающаяся диаминопимеловая кислота частично декарбоксируется на лизин, который включается в белки. Меньшая часть расщепленного мукопептида выделяется в среду в форме неиспользованных осколков. Скорость «turnover» мукопептидов зависит от скорости роста культуры. В течение одного клеточного деления деградируется около 15–20% плотного слоя клеточной оболочки. Чувствительность Bacillus megaterium к лизоциму и скорость его превращения в протопласты также пропорциональна скорости роста культуры. В нерастущих клетках—как в стационарной фазе культуры, так в при ее голодании в безазотной среде—«turnover” мукопептидов не осуществляется. В то же время в стационарной фазе повышается устойчивость клеточных оболочек к лизоциму. Расщепление плотного компонента клеточной оболочки имеет место и в ходе размножения культур Bacillus cereus и Escherichia coli.

Amino Acid Uptake and Protein Synthesis in Cultured Tobacco Pollen

Summary The uptake of amino acids and their incorporation into proteins by tobacco pollen tubes in suspension culture was studied as a function of pollen tube growth. Leucine transport over a 0.01–0.5 mM concentration range occurs against the concentration gradient via an energy-dependent saturable system with an apparent K m × 10 -4 M and maximum velocity 1.25 fmol min -1 per pollen tube in a 4 h culture. A part of the absorbed leucine is released into the medium, the efflux being about 6 % of influx in 10 min preloaded pollen tubes. The rate of uptake per pollen tube increases proportionally to the duration of growth up to at least 8 h and decreases after 12 h of culture. In the period between 6 and 12 h, the first callose plug is formed nearly in 90 % of pollen tubes, separating the about 0.9 mm apical part. The uptake is linear for 30 min and in 2 h an equilibrium between the endogenous and exogenous amino acid pools is achieved in 1 mg · ml -1 of casein hydrolysate. The rate of protein synthesis, as estimated from the proportion of incorporated to free amino acids absorbed, does not decrease and the amount of incorporated exogenous amino acids increases continuously for at least 18 h of culture, provided that pollen tube growth is not depressed. A comparison of the results from different periods of labelling with 14 C-leucine suggest that the free leucine pool is compartmentalized. The protein precursor poll appears to be small and rapidly fed from exogenous source.

Biochemical and Cytological Changes in Young Tobacco Pollen during in vitro Starvation in Relation to Pollen Embryogenesis

The potentiality of microspores or young pollen grains to deviate from the normal gametophytic development to embryo-genic pathway and sporophyte formation raises the possibility to use the pollen system as an unique tool to study fundamental questions of cytodifferentiation and developmental biology. From this aspect, isolated pollen culture presents a number of advantages over anther culture as it eliminates uncontrolled effects of anther wall and enables directly affect pollen grains by changes of culture conditions.