O-acetylserine and O-acetylhomoserine sulfhydrylase of yeast; studies with methionine auxotrophs.

The nutritional requirements of three yeast mutants, previously shown to possess low O-acetyl-L-serine (OAS) and O-acetyl-L-homoserine (OAH) sulfhydrylase activities, were reinvestigated. It was thus found that one mutant (strain No. 16), previously identified as a homocysteine auxotroph, is in fact a double mutant requiring both cysteine and OAH. In agreement with the previous assignment, the other two strains (strains No. 13 and 17) were shown to be true cysteine auxotrophs. These results can best be explained by assuming the cystathionine pathway to be the main route of homocysteine synthesis in this organism. It was further found that extracts of the three mutants contain genetically modified OAS-OAH sulfhydrylases with much reduced catalytic activities. Modified sulfhydrylase was partially purified from strain No. 16 by the same procedure as for the wild-type enzyme. Both OAS and OAH sulfhydrylase activities of the mutant enzyme were copurified and behaved identically on polyacrylamide gel electrophoresis. The enzymatic and physicochemical properties of the purified mutant enzyme were shown to be very similar to those of the wild-type enzyme, except that the catalytic activities of the former were only 3-5% of those of the latter, and that the ratio of OAH sulfhydrylase to OAS sulfhydrylase activity was somewhat lower in the former than in the latter.     

Extracellular trehalose utilization by Saccharomyces cerevisiae

Two haploid strains of Saccharomyces cerevisiae viz. MATα and MATa were grown in glucose and trehalose medium and growth patterns were compared. Both strains show similar growth, except for an extended lag phase in trehalose grown cells. In both trehalose grown strains increase in activities of both extracellular trehalase activities and simultaneous decrease in extracellular trehalose level was seen. This coincided with a sharp increase in extracellular glucose level and beginning of log phase of growth. Alcohol production was also observed. Secreted trehalase activity was detected, in addition to periplasmic activity. It appeared that extracellular trehalose was hydrolyzed into glucose by extracellular trehalase activity. This glucose was utilized by the cells for growth. The alcohol formation was due to the fermentation of glucose. Addition of extracellular trehalase caused reduction in the lag phase when grown in trehalose medium, supporting our hypothesis of extracellular utilization of trehalose.     

The flavoproteome of the yeast Saccharomyces cerevisiae

Genome analysis of the yeast Saccharomyces cerevisiae identified 68 genes encoding flavin-dependent proteins (1.1% of protein encoding genes) to which 47 distinct biochemical functions were assigned. The majority of flavoproteins operate in mitochondria where they participate in redox processes revolving around the transfer of electrons to the electron transport chain. In addition, we found that flavoenzymes play a central role in various aspects of iron metabolism, such as iron uptake, the biogenesis of iron–sulfur clusters and insertion of the heme cofactor into apocytochromes. Another important group of flavoenzymes is directly (Dus1-4p and Mto1p) or indirectly (Tyw1p) involved in reactions leading to tRNA-modifications. Despite the wealth of genetic information available for S. cerevisiae, we were surprised that many flavoproteins are poorly characterized biochemically. For example, the role of the yeast flavodoxins Pst2p, Rfs1p and Ycp4p with regard to their electron donor and acceptor is presently unknown. Similarly, the function of the heterodimeric Aim45p/Cir1p, which is homologous to the electron-transferring flavoproteins of higher eukaryotes, in electron transfer processes occurring in the mitochondrial matrix remains to be elucidated. This lack of information extends to the five membrane proteins involved in riboflavin or FAD transport as well as FMN and FAD homeostasis within the yeast cell. Nevertheless, several yeast flavoproteins, were identified as convenient model systems both in terms of their mechanism of action as well as structurally to improve our understanding of diseases caused by dysfunctional human flavoprotein orthologs.     

Control of methionine synthesis by lysine in Lemna minor

When Lemna minor was cultured in the presence of 0.25 mM l-lysine, the concentration of free methionine and formyl and methyl tetrahydrofolate (THFA) were decreased. l-lysine, l-homoserine, l-threonine and l-methionine at concentrations up to 8 mM did not affect N¹⁰-formyl THFA synthetase (E.C. 6.3.4.3) and N⁵,N¹⁰-methylene THFA reductase (E.C. 1.1.1.68). In contrast, serine hydroxymethyltransferase (E.C. 2.1.2.1) activity was inhibited by lysine. This inhibition gave a sigmoidal curve when plotted for a range of l-lysine or THFA concentrations. Exogenous lysine also reduced the incorporation of glycine [¹⁴C] and serine [3-¹⁴C] into free and protein methionine. Lysine, which is known to control synthesis of homocysteine in L. minor, may also regulate production of C-1 units for methionine synthesis by inhibition of serine hydroxymethyltransferase.     

Pathways of glutathione degradation in the yeast Saccharomyces cerevisiae

The degradation of glutathione (GSH) in the yeast Saccharomyces cerevisiae appears to be mediated only by γ-glutamyltranspeptidase and cysteinylglycine dipeptidase. Other enzymes of the γ-glutamyl cycle, γ-glutamyl cyclotransferase and 5-oxo-l-prolinase, are not present in the yeast. In vivo transpeptidation was shown in the presence of a high intracellular level of γ-glutamyltranspeptidase, but only when the de-repressing nitrogen source was a suitable acceptor of the transferase reaction. In contrast, when the de-repressing source was not an acceptor of the transferase reaction (e.g. urea), only glutamate was detected. Intracellular GSH is virtually inert when the level of γ-glutamyltranspeptidase is low. Possible roles for in vivo transpeptidation are discussed.     

Mapping of the mitochondrial 16S ribosomal RNA gene and its expression in the cytoplasmic petite mutants ofSaccharomyces cerevisiae

The 16S ribosomal RNA gene of yeast mitochondria was titrated in various cytoplasmic petite mutants by DNA-RNA hybridization. The gene was located close to the prolyl transfer RNA gene. The properties of the rho^? strains suggest that the gene order would be: - P_I - 16S - prolyl tRNA - valyl tRNA - (tRNAs) - R_I - R_III -; the 23S ribosomal gene is far from the 16S one. Several petite mutants were found which have retained, in addition to many transfer RNA genes, both of the 23S and 16S ribosomal RNA genes. The two genes seem to be transcribed in these mutants.     

Control of high osmolarity signalling in the yeast Saccharomyces cerevisiae

Signal transduction pathways control cellular responses to extrinsic and intrinsic signals. The yeast HOG (High Osmolarity Glycerol) response pathway mediates cellular adaptation to hyperosmotic stress. Pathway architecture as well as the flow of signal have been studied to a very high degree of detail. Recently, the yeast HOG pathway has become a popular model to analyse systems level properties of signal transduction. Those studies addressed, using experimentation and modelling, the role of basal signalling, robustness against perturbation, as well as adaptation and feedback control. These recent findings provide exciting insight into the higher control levels of signalling through this MAPK system of potential general importance.     

Derepression of the high-affinity phosphate uptake in the yeast Saccharomyces cerevisiae

Phosphate starvation derepresses a high-affinity phosphate uptake system in Saccharomyces cerevisiae strain A294, while in the same time the low-affinity phosphate uptake system disappears. The protein synthesis inhibitor cycloheximide prevents the derepression, but has no effect as soon as the high-affinity system is fully derepressed. Two other protein synthesis inhibitors, lomofungin and 8-hydroxyquinoline, were found to interfere also with the low-affinity system and with Rb⁺ uptake. After incubation of the yeast cells in the presence of phosphate the high-affinity system is not derepressed, but the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the low-affinity system has decreased for about 35%. Phosphate supplement after derepression causes the high-affinity system to disappear to a certain extent while in the meantime the low-affinity system reappears. The results are compared with those found in the yeast Candida tropicalis for phosphate uptake.     

Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae

In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127-6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1? deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1? mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cell. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ‘Crabtree effect’, was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate hydrogenase was subject to glucose inactivation.     

Induction and complementation of lysine auxotrophs in Saccharomyces

Four chemical agents, EMS EMS: Ethyl methanesulfonate; MNNG: N-methyl-N\t'-nitro-N\t'-nitrosoguanidine; NA: Nitrous acid; ICR-170: 2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl) aminopropylamino] acridine 2 HCl; UV: Ultra violet radiation. , MNNG, NA, ICR-170, as well as UV were used to induce mutations in the wild-type haploid strain X2180-1B (α) of Saccharomyces. A total of 2053 (EMS, 427; MNNG, 444; NA, 469; ICR-170, 456; UV, 257) lysine-requiring mutant clones were isolated from many independent treatments and by nystatin enrichment technique. Mutants were classified into various functional groups on the basis of complementation analysis with 14 tester strains (lys 1 to lys 15 except lys 3). Of the clones analyzed, the number of isolates unable to complement with a given tester strain ranged from 2 for lys 5 to 918 for lys 4. Three of the mutually complementing lysine loci (lys 1, lys 2, and lys 4) accounted together for over 85% of the mutant clones whereas lys 6, lys 7, lys 8, and lys 14 had less than 10 noncomplementing isolates each. Mutants for lys 4 were most frequent with all of the mutagens tested except with NA in which case the mutants for lys 2 were most frequent. A total of 56 isolates failed to complement with lys 10, lys 11, and lys 12. Similarly, 47 isolates failed to complement with lys 9 and lys 13 simultaneously. Only 44 isolates complemented with all of the tester strains used.     

Mitotic recombination induced by chemical and physical agents in the yeast Saccharomyces cerevisiae

The treatment of diploid cultures of yeast with ultraviolet light (UV), γ-rays, nitrous acid (NA) and ethyl methane sulphonate (EMS) results in increases in cell death, mitotic gene conversion and crossing-over. Acridine orange (AO) treatment, in contrast, was effective only in increasing the frequency of gene conversion. The individual mutagens were effective in the order UV > NA > γ-rays > AO > EMS. Prior treatment of yeast cultures in starvation medium produced a significant reduction in the yield of induced gene conversion.The results have been interpreted on the basis of a general model of mitotic gene conversion which involves the post-replication repair of induced lesions involving de novo DNA synthesis without genetic exchange. In contrast mitotic crossing-over appears to involve the action of a repair system independent from excision or post-replication repair which involves genetic exchange between homologous chromosomes.     

Water structure in vitro and within Saccharomyces cerevisiae yeast cells under conditions of heat shock

The OH stretch mode from water and organic hydroxyl groups have strong infrared absorption, the position of the band going to lower frequency with increased H-bonding. This band was used to study water in trehalose and glycerol solutions and in genetically modified yeast cells containing varying amounts of trehalose. Concentration-dependent changes in water structure induced by trehalose and glycerol in solution were detected, consistent with an increase of lower-energy H-bonds and interactions at the expense of higher-energy interactions. This result suggests that these molecules disrupt the water H-bond network in such a way as to strengthen molecule–water interactions while perturbing water–water interactions. The molecule-induced changes in the water H-bond network seen in solution do not translate to observable differences in yeast cells that are trehalose-deficient and trehalose-rich. Although comparison of yeast with low and high trehalose showed no observable effect on intracellular water structure, the structure of water in cells is different from that in bulk water. Cellular water exhibits a larger preference for lower-energy H-bonds or interactions over higher-energy interactions relative to that shown in bulk water. This effect is likely the result of the high concentration of biological molecules present in the cell. The ability of water to interact directly with polar groups on biological molecules may cause the preference seen for lower-energy interactions.     

Lap3 is a selective target of autophagy in yeast, Saccharomyces cerevisiae

Autophagy is a primarily non-selective degradation system of cytoplasmic constituents in lysosomes/vacuoles during starvation. In yeast, autophagy is also involved in the selective transport of Ape1, a vacuolar hydrolase, as a biosynthetic route. Ald6, a soluble cytoplasmic enzyme, is preferentially eliminated from cytoplasm via autophagy. However, little is known about the mechanisms of Ald6 targeting to autophagosomes. Here, we show that Lap3, a soluble cytosolic cysteine protease, is spatially associated with Ape1 and selectively transported to the vacuole during nitrogen starvation. The rate of Lap3 transport is much higher than that of Ald6 and is similar to that of Ape1. Moreover, ATG11 and ATG19, essential factors for Ape1 transport, are important for Lap3 transport. Most Lap3 is degraded within a couple of hours in the vacuole in contrast to Ape1; therefore, we conclude that the machinery required for Ape1 biosynthesis is used for selective degradation of Lap3.