The plasma membrane of Saccharomyces cerevisiae Molecular structure and asymmetry

The molecular structure of the plasma membrane of the haploid strain Saccharomyces cerevisiae X-2180 1A has been studied by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein and glycoprotein components have been identified and their apparent M_r determined. A glycoprotein showing an apparent M_r of 27 500 has been shown to be the main structural component. Treatment of the cells with cycloheximide prior to plasma membrane isolation resulted in a redistribution of the relative amounts of each protein band and a drastic reduction in the number of Schiff positive bands. It is postulated that treatment with this drug rids the plasma membrane of glycoprotein secretory components which are in the process of being secreted to the periplasmic space, thus allowing the study of the basic structural components of the organelle. The electrophoretic pattern of the internal membranes revealed close similarities with that of the plasma membrane and though two-dimensional electrophoresis might disclose greater differences, these similarities suggest a common origin for most of the components of both membranous systems. Finally, radioiodination techniques have been used in studying the asymmetric disposition of some of the components of the plasma membrane. At least five polypeptides were identified as located to the outer layer of the plasma membrane and two more glycopeptides were shown to span across the bilayer.     

Soluble and membrane-bound thiamine-binding proteins from Saccharomyces cerevisiae

Previous communications from this laboratory have indicated that there exists a thiamine-binding protein in the soluble fraction of Saccharomyces cerevisiae which may be implicated to participate in the transport system of thiamine in vivo.In the present paper it is demonstrated that both activities of the soluble thiamine-binding protein and thiamine transport in S. cerevisiae are greatest in the early-log phase of the growth and decline sharply with cell growth. The soluble thiamine-binding protein isolated from yeast cells by conventional methods containing osmotic shock treatment appeared to be a glycoprotein with a molecular weight of 140 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of the binding for thiamine was 29 nM which is about six fold lower than the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ (0.18 μM) of thiamine transport. The optimal pH for the binding was 5.5, and the binding was inhibited reversibly by 8 M urea but irreversibly by 8 M urea containing 1% 2-mercaptoethanol. Several thiamine derivatives and the analogs such as pyrithiamine and oxythiamine inhibited to similar extent both the binding of thiamine and transport in S. cerevisiae, whereas thiamine phosphates, 2-methyl-4-amino-5-hydroxymethylpyrimidine and $\text{O-}\text{benzoylthiamine}$ disulfide did not show similarities in the effect on the binding and transport in vivo. Furthermore, it was demonstrated by gel filtration of sonic extract from the cells that a thiamine transport mutant of S. cerevisiae (PT-R₂) contains the soluble binding protein in a comparable amounts to that in the parent strain, suggesting that another protein component is required for the actual translocation of thiamine in the yeast cell membrane. On the other hand, the membrane fraction prepared from S. cerevisiae showed a thiamine-binding activity with apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of 0.17μM at optimal pH 5.0 which is almost the same with the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for the thiamine transport system. The membrane-bound thiamine-binding activity was not only repressible by exogenous thiamine in the growth medium, but as well as thiamine transport it was markedly inhibited by both pyrithiamine and $\text{O-}\text{benzoylthiamine}$ disulfide. In addition, it was found that membrane fraction prepared frtom PT-R₂ has the thiamine-binding activity of only 3% of that from the parent strain of S. cerevisiae.These results strongly suggest that membrane-bound thiamine-binding protein may be directly involved in the transport of thiamine in S. cerevisiae.     

NMR analysis of a cell division cycle mutant of Saccharomyces cerevisiae

cdc 19.1 is a temperature-sensitive lesion in the genome of Saccharomyces cerevisiae. The phenotype of this mutant is a cell cycle specific arrest in G₁, which is expressed at 37°C. In the present study, ³¹P- and ¹³C-NMR spectroscopy were used to analyze the metabolism of the mutant at the permissive and restrictive temperatures. Our results confirm previous findings which have indicated that cdc 19.1 contains temperature-sensitive pyruvate kinase activity. In contrast to previous findings, however, the present investigation demonstrates that restriction of pyruvate kinase activity in vivo takes as long as 24 h to be fully expressed. In addition, analysis by NMR has allowed us to assess the metabolic consequences of pyruvate kinase restriction which may contribute to the arrest of cell growth in the early G₁ phase of the cell division cycle.     

l-Proline transport in Saccharomyces cerevisiae

Transport of l-proline into Saccharomyces cerevisiae K is mediated by two systems, one with a K_T of 31 μM and J_max of 40 nmol · s^?1 · (g dry wt.)^?1, the other with K_T > 2.5 mM and J_max 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°C and 20–21°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, to 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 μg nystatin per ml.     

Study of the interaction of Saccharomyces cerevisiae with glucose by particle microelectrophoresis

Saccharomyces cerevisiae NCYC 239 in the presence of glucose at temperatures under 303 K shows a time-dependent lowering of electrophoreric mobility $\text{υ}$. At temperatures above 303 K, this time-dependent change in $\text{υ}$ is in the direction of increased mobilities. Cells suspended in buffer indicate a surface pK_a of less than 4, whereas for cells suspended in buffered glucose it is impossible to derive a surface pK_a. A kinetic study of the interaction of S. cerevisiae with glucose as a function of temperature allows calculation of an activation energy of 140 kJ·mol^?1 for the combined processes of (i) uptake of glucose onto the cell wall, (ii) transfer through the cell wall and membrane, and (iii) the establishment of a steady glucose flux through the wall and membrane.     

Solubilization and reconstitution of the glucose transport system from Saccharomyces cerevisiae

The glucose transport system from Saccharomyces cerevisiae was solubilized from isolated plasma membranes by the nonionic detergent, octylglucoside. The transport system was reconstituted into proteoliposomes with removal of detergent from the extract by dialysis, followed by the addition of asolectin liposomes to the dialyzed proteins with a freeze-thaw and brief bath-sonication step. The reconstituted proteoliposomes exhibit specific carrier-mediated facilitated diffusion of d-glucose, including stimulated equilibrium exchange and influx counterflow. Furthermore, the reconstituted facilitated diffusion system shows substrate specificities similar to those of the intact cell d-glucose transport system.     

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.     

The isolation of Saccharomyces cerevisiae nuclear membranes with nuclease and high-salt treatment

Saccharomyces cerevisiae nuclear membranes were prepared from isolated nuclei by digesting chromatin with deoxyribonuclease and ribonuclease, washing of residual nuclei with 0.5 M MgCl₂, and discontinuous gradient centrifugation in buffered Ficoll solutions. Electron microscopic examination of the preparations showed single membrane and double membrane vesicles and membrane sheets. Pores or residual pores were often visible. In double membrane profiles the two unit membranes were often separated by the remains of the perinuclear cistern. The nuclear membrane fragments contained 58% protein, 23.8% phospholipid, 6% sterols, 7.1% neutral acylglycerols, 4.8% RNA, and 0.3% DNA. The phospholipid content of the membrane preparations was influenced by a phospholipase activity with acidic pH optimum.     

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.     

Turnover of protein components of the plasma membrane of Saccharomyces cerevisiae

The peptide composition of plasma membrane in Saccharomyces cerevisiae cells growing at different temperatures between 18 and 38°C was studied using SDS-polyacrylamide gel electrophoresis. Stability of the proteins, both qualitative and quantitative, was observed at the tested temperatures. Treatment for 2 h with cycloheximide decreased by about 50% the amount of a 80 kDa membrane peptide at 18, 23, 28 and 33°C, with no other apparent effects. At 38°C the 80 kDa peptide was not affected by the presence of the drug. Addition of tunicamycin to cultures at concentrations partially inhibitory to growth caused a large accumulation of the 80 kDa peptide in the plasma membrane, which cycloheximide did not modify. Pulse-chase experiments indicated a low rate of turnover of total plasma membranes in cells growing at 18 and 28°C. In contrast, at 38°C about 50% of the radioactivity in plasma membranes disappeared after a 2 h chase. The 80 kDa protein band was the only one with significant differential decay.     

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 mmol 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 $\text{K}{}^{\mathrm{+}}\text{>}\text{Rb}{}^{\mathrm{+}}\text{>>}\text{Li}{}^{\mathrm{+}}\text{～-}\text{Cs}{}^{\mathrm{+}}\text{～-}\text{Na}{}^{\mathrm{+}}$. 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.     

Stimulation of dehydrogenase synthesis by cadmium in a cadmium-resistant strain of Saccharomyces cerevisiae

The activity of dehydrogenase in Saccharomyces cerevisiae was estimated by reduction of 2,3,5-triphenyltetrazolium chloride. By the adaptation of yeast to cadmium, the high activity of dehydrogenase was observed. Furthermore, the activity of dehydrogenase in Cd-resistant cells was increased by growing in medium containing CdSO₄. However, the activity of dehydrogenase was inhibited by the addition of CdSO₄ to the reaction mixture. The activity of dehydrogenase in Cd-sensitive cells was increased slightly by incubation with low concentrations of CdSO₄.High activity of dehydrogenase in Cd-resistant cells was completely negated by the addition of cycloheximide to the incubation medium. The increase of dehydrogenase activity is due partly to de novo synthesis of protein.     

Effects of phenothiazines on inhibition of plasma membrane ATPase and hyperpolarization of cell membranes in the yeast Saccharomyces cerevisiae

The transmembranal potential, in Saccharomyces cerevisiae, has been calculated from the distribution of the lipophilic cation tetraphenylphosphonium (TPP⁺) between the intracellular and extracellular water. Trifluoperazine at concentrations of 10 to 50 μM, caused a substantial increase in the membrane potential (negative inside). This increase was observed only in the presence of a metabolic substrate and was eliminated by the addition of the protonophores 2,4-dinitrophenol and sodium azide, removal of glucose, replacement of glucose by the nonmetabolizable analog 3-O-methyl glucose, or by the addition of 100mM KCl. An increase in ⁴⁵CaCl₂ accumulation from solutions of low concentrations (1 μM) was observed under all conditions where membrane potential was increased. Proton ejection activity was monitored by measurements of the rates of the decrease in the pH of unbuffered cell suspensions in the presence of glucose. Trifluoperazine inhibited the changes in medium pH; this inhibition was not the result of an increase in the permeability of cell membranes to protons since in the absence of glucose, trifluoperazine did not cause a change in the rate of pH change generated by proton influx. The activity of plasma membrane ATPase was measured in crude membrane preparations in the presence of sodium azide to inhibit mitochondrial ATPase. Trifluoperazine strongly inhibited the activity of the plasma membrane ATPase. The effect of phenothiazines on transport and on membrane potential reported in this study and in the previous one (Eilam, Y. (1983) Biochim. Biophys. Acta 733, 242–248) were observed only in the presence of a metabolic substrate. The possibility that energy is required for the uptake of phenothiazines into the cells was eliminated by results showing energy-independent uptake of [³H]chlorpromazine. The results strongly suggest that phenothiazines activate energy-dependent K⁺-extrusion pumps, which lead to increased membrane potential. Increased influx of calcium seems to be energized by membrane potential, and therefore stimulated under all conditions where membrane potential is increased. The analog which does not bind to calmodulin, trifluoperazine sulfoxide, had no effect on the cells, but the involvement of calmodulin in the processes altered by trifluoperazine cannot as yet, be determined.     

Studies on compartmentation of S-adenosyl-L-methionine in Saccharomyces cerevisiae and isolated rat hapatocytes

The existence of metabolically distinct pools of $\text{S-}\text{adenosyl-L-methionine}$ in Saccharomyces cerevisiae and isolated rat hepatocytes was investigated. Utilizing a relatively long labeling period with [methyl-¹⁴C]methionine, a metabolically ‘stable’ pool was labeled. A subsequent short labeling with [methyl-³H]methionine selectively labeled a putative metabolically ‘labile’ pool. The existence of these distinguishable pools was ascertained by following the ³H and ¹⁴C label disappearance in $\text{S-}\text{adenosyl-L-methionine}$ during the chase-period in label-free media containing cycloleycine to prevent futher synthesis of $\text{S-}\text{adenosyl-L-methionine}$. In both yeast and hepatocytes, the ${}^3\text{H}{}^{14}\text{C}$ ratio in $\text{S-}\text{adenosyl-L-methionine}$ decreased sharply. The individual ³H and ¹⁴C decrease in $\text{S-}\text{adenosyl-L-methionine}$ showed $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values of 3 and 8 min for yeast and 4 and 18 min for hepatocytes. The results strongly indicate that at least two metabolically distinct $\text{S-}\text{adenosyl-L-methionine}$ pools actually do exist in both systems. Subcellular fractionation revealed that the ‘labile’ pool exist in the cytosol for both yeast and hepatocytes while the ‘stable’ pool exists in the vacuolar and the mitochondrial fraction for the yeast and hepatocytes respectively. The $\text{S-}\text{adenosyl-L-methionine}$ pools were also studied in normal yeast under anaerobic chase condition and petite mutant yeast. Sharply contrasting with aerobically chased normal yeast, both showed closely parallel ³H and ¹⁴C decreases in $\text{S-}\text{adenosyl-L-methionine}$.     

A petite mitochondrial DNA segment arising in exceptionally high frequency in a mit− mutant of Saccharomyces cerevisiae

In cultures of the mit^? mutant strain Mb12 of Saccharomyces cerevisiae (carrying a mutation in the oli2 gene), 70% of the cells are petite mutants. More than 80% of the petites from Mb12 contain a particular mtDNA segment, denoted BB5, that is 880 bp long and carries a single MboI site. Thus, in cultures of Mb12, about 56% of the cells are petites containing the defective BB5 mtDNA genome, and only 30% are mit^? cells containing parental Mb12 mtDNA. The BB5 mtDNA segment is also found in petites arising from the wild-type strain J69-1B (from which Mb12 was derived), but in this case mtDNA from only five out of 24 petites produced an 880 bp band after MboI digestion. Since J69-1B cultures carry a petite frequency of about 5%, approximately 1% of cells in J69-1B cultures contain the BB5 mtDNA segment. The difference between Mb12 and J69-1B cultures is reflected in the MboI digestion patterns of the respective mtDNAs. While Mb12 mtDNA contains a grossly superstoicheiometric 880 bp MboI fragment, the corresponding fragment in J69-1B mtDNA cannot be seen on stained gels, but can be readily visualized in Southern blots hybridized to a ³²P-labelled DNA probe obtained from the 880 bp MboI fragment. The BB5 mtDNA segment was shown to contain the oril sequence (one of several very similar sequences in wild-type mtDNA thought to act as origins of replication of mtDNA) which confers the genetic property of very high suppressiveness on petites carrying this mtDNA. The efficient replication of BB5 mtDNA may contribute to its abundance in Mb12 cultures. Nevertheless, other factors must operate to influence the abundance of the BB5 mtDNA segment in cultures of different strains, the most important of which is likely to be the rate of excision of this mtDNA segment from the parental mtDNA genome.     

Barotropic and thermotropic bilayer phase behavior of positional isomers of unsaturated mixed-chain phosphatidylcholines

The bilayer phase transitions of six kinds of mixed-chain phosphatidylcholines (PCs) with an unsaturated acyl chain in the sn-1 or sn-2 position, 1-oleoyl-2-stearoyl- (OSPC), 1-stearoyl-2-oleoyl- (SOPC), 1-oleoyl-2-palmitoyl- (OPPC), 1-palmitoyl-2-oleoyl- (POPC), 1-oleoyl-2-myristoyl- (OMPC) and 1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light transmittance measurements. Bilayer membranes of SOPC, POPC and MOPC with an unsaturated acyl chain in the sn-2 position exhibited only one phase transition, which was identified as the main transition between the lamellar gel (L_β) and liquid crystalline (L_α) phases. On the other hand, the bilayer membranes of OSPC, OPPC and OMPC with an unsaturated acyl chain in the sn-1 position exhibited not only the main transition but also a transition from the lamellar crystal (L_c) to the L_β (or L_α) phase. The stability of their gel phases was markedly affected by pressure and chain length of the saturated acyl chain in the sn-2 position. Considering the effective chain lengths of unsaturated mixed-chain PCs, the difference in the effective chain length between the sn-1 and sn-2 acyl chains was proven to be closely related to the temperature difference of the main transition. That is, a mismatch of the effective chain length promotes a temperature difference of the main transition between the positional isomers. Anomalously small volume changes of the L_c/L_α transition for the OPPC and OMPC bilayers were found despite their large enthalpy changes. This behavior is attributable to the existence of a cis double bond and to significant inequivalence between the sn-1 and sn-2 acyl chains, which brings about a small volume change for chain melting due to loose chain packing, corresponding to a large partial molar volume, even in the L_c phase. Further, the bilayer behavior of unsaturated mixed-chain PCs containing an unsaturated acyl chain in the sn-1 or sn-2 position was well explained by the chemical-potential diagram of a lipid in each phase.     

Differential scanning calorimetry of milk fat globule membranes

Differential scanning calorimetry was employed as an aid in examining the structure of the bovine milk fat globule membrane. At least six major endotherms are observed between 10 and 90°C, corresponding to order-disorder transitions of discrete structural domains of the membrane. These endothermic transitions occur at 16, 28, 43, 58, 68, and 75°C. The transitions occurring between 10 and 50°C were reversible, suggesting the involvement of lipid. However, the high temperature transitions were irreversible. The calorimetric C transition, centered at 43°C, was shown to involve neutral lipid, since the endotherm was reversible, insensitive to proteolysis, and similar to the endotherm of the isolated neutral lipid fraction of the milk fat globule membrane. The glycolipid and phospholipid fractions of the milk fat globule membrane yielded endotherms outside of the temperature range of the C transition. Another endotherm, the D transition (58°C), was found to involve the denaturation of the major membrane coat protein, butyrophilin (band 12). Evidence for this assignment included the following observations: (i) the nearly selective proteolysis of butyrophilin resulted in the complete removal of the D transition, (ii) the butyrophilin-enriched, Triton X-100-insoluble pellet of milk fat globule membrane yielded a relatively normal D transition, and (iii) the irreversible, disulfide-stabilized aggregation of butyrophilin occurred in the membrane solely at the temperature of the D transition. Furthermore, no other prominent milk fat globule membrane polypeptide formed these non-native disulfide crossbridges during the D transition. The sources of the other major endotherms of the milk fat globule membrane have not yet been assigned.     

The physical state of quick-frozen membranes and lipids

Lipid bilayers and biomembranes produce nearly identical calorimeter scans regardless of whether they are slowly cooled under near-equilibrium conditions or rapidly frozen at rates used in freeze-fracture electron microscopy. Except for the melting of ice at 273 K, for both cooling regimens no significant thermal events occur from 100 K to the usual gel to liquid crystal transition. The gel to liquid crystal transition itself is somewhat altered by rapid cooling when bilayers contain mixed lipid species. Combined with X-ray diffraction studies, the results indicate that quickly frozen bilayers are crystalline, but that the crystalline domains are quite small or otherwise disordered. In contrast to the behavior of lipids in bilayers, hexagonal-phase calcium cardiolipid easily forms a glass upon cooling.     

Transport and metabolic effects of α-aminoisobutyric acid in saccharomyces cerevisiae

α-Aminoisobutyric acid is actively transported into yeast cells by the general amino acid transport system. The system exhibits a K_m for α-aminoisobutyric acid of 270 μM, a V_max of 24 nmol/min per mg cells (dry weight), and a pH optimum of 4.1–4.3. α-Aminoisobutyric acid is also transported by a minor system(s) with a V_max of 1.7 nmol/min per mg cells. Transport occurs against a concentration gradient with the concentration ratio reaching over 1000:1 (in/out). The α-aminoisobutyric acid is not significantly metabolized or incorporated into protein after an 18 h incubation. α-Aminoisobutyric acid inhibits cell growth when a poor nitrogen source such as proline is provided but not with good nitrogen sources such as NH₄⁺. During nitrogen starvation α-aminoisobutric acid strongly inhibits the synthesis of the nitrogen catabolite repression sensitive enzyme, asparaginase II. Studies with a mutant yeast strain (GDH-CR) suggest that α-aminoisobutyric acid inhibition of asparaginase II synthesis occurs because α-aminoisobutyric acid is an effective inhibitor of protein synthesis in nitrogen starved cells.     

Functional characterization of the Saccharomyces cerevisiae ABC-transporter Yor1p overexpressed in plasma membranes

Yor1p, a Saccharomyces cerevisiae plasma membrane ABC-transporter, is associated to oligomycin resistance and to rhodamine B transport. Here, by using the overexpressing strain Superyor [A. Decottignies, A.M. Grant, J.W. Nichols, H. de Wet, D.B. McIntosh, A. Goffeau, ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p, J. Biol. Chem. 273 (1998) 12612-12622], we show that Yor1p also confers resistance to rhodamine 6G and to doxorubicin. In addition, Yor1p protects cells, although weakly, against tetracycline, verapamil, eosin Y and ethidium bromide. The basal ATPase activity of the overexpressed form of Yor1p was studied in membrane preparations. This activity is quenched upon addition of micromolar amounts of vanadate. V_max and K_m values of ∼ 0.8 s^− 1 and 50 ± 8 μM are measured. Mutations of essential residues in the nucleotide binding domain 2 reduces the activity to that measured with a Δyor1 strain. ATP hydrolysis is strongly inhibited by the addition of potential substrates of the transporter. Covalent reaction of 8-azido-[α-³²P]ATP with Yor1p is not sensitive to the presence of excess oligomycin. Thus, competition of the drug with ATP binding is unlikely. Finally, we inspect possible hypotheses accounting for substrate inhibition, rather than stimulation, of ATP hydrolysis by the membrane preparation.