Characterization of amino acid pools in the vacuolar compartment of Saccharomyces cerevisiae

Intact vacuoles are released from spheroplasts of Saccharomyces cerevisiae by means of a gentle mechanical disintegration method. They are purified by centrifugation in isotonic density gradients (flotation and subsequent sedimentation), and analyzed for their soluble amino acid content. The results indicate that about 60% of the total amino acid pool of spheroplasts is contained in the vacuoles. This may be an underestimate, as it presupposes no loss of amino acids from the vacuoles during the purification procedure. The amino acid concentration in the vecuoles is calculated to be approximately 5 times that in the cytoplasm if the total volumes of the two compartments are used for the calculation. The vacuolar amino acid pool is rich in basic amino acids, and in citrulline and glutamine, but contains a remarkably small amount of glutamate. Radioactive labeling experiments with spheroplasts indicate that the vacuolar amino acids are separated from the metabolically active pools located in the cytoplasm. This is particularly evident for the basic amino acids and glutamine; in contrast, the neutral amino acids and glutamate appear to exchange more rapidly between the cytoplasmic and the vacuolar compartments of the cells.     

Novel families of vacuolar amino acid transporters

Amino acids are compartmentalized in the vacuoles of microorganisms and plants. In Saccharomyces cerevisiae, basic amino acids accumulate preferentially into vacuoles but acidic amino acids are almost excluded from them. This indicates that selective machineries operate at the vacuolar membrane. The members of the amino acid/auxin permease family and the major facilitator superfamily involved in the vacuolar compartmentalization of amino acids have been recently identified in studies using S. cerevisiae. Homologous genes for these transporters are also found in plant and mammalian genomes. The physiological significance in response to nitrogen starvation can now be discussed.     

Dynamic aspects of vacuolar and cytosolic amino acid pools of Saccharomyces cerevisiae.

By using the Cu2+ method (Y. Ohsumi, K. Kitamoto, and Y. Anraku, J. Bacteriol. 170:2676-2682, 1988) for differential extraction of the vacuolar and cytosolic amino acid pools from yeast cells, the amino acid compositions of the two pools extracted from Saccharomyces cerevisiae cells, grown in synthetic medium supplemented with various amino acids, were determined. Histidine and lysine in the medium expanded the vacuolar pool extremely. Glutamate also accumulated in the cells, but mainly in the cytosol. The composition of amino acids in the cytosolic pool was fairly constant, in contrast to that in the vacuolar pool. Cells grown in synthetic medium supplemented with 10 mM arginine accumulated arginine in the vacuoles at a concentration of about 430 mM. This large arginine pool was metabolically active and was effectively utilized during nitrogen starvation. Arginine efflux from the vacuoles was coupled with K+ influx, with an arginine/K+ exchange ratio of 1, as judged by the initial rate. The vacuolar arginine pool was exchangeable with lysine added to the medium and was decreased by treatment of the cells with the mating pheromone, alpha-factor.     

Molecular study on the role of vacuolar transporters in glycyrrhetinic acid production in engineered Saccharomyces cerevisiae

Glycyrrhetinic acid (GA) is one of the major bioactive components of the leguminous plant, Glycyrrhiza spp. (Chinese licorice). Owing to GA's complicated chemical structure, its production by chemical synthesis is challenging and requires other efficient strategies such as microbial synthesis. Earlier investigations employed numerous approaches to improve GA yield by refining the synthetic pathway and improving the metabolic flux. Nevertheless, the metabolic role of transporters in GA biosynthesis in microbial cell factories has not been studied so far. In this study, we investigated the role of yeast ATP binding cassette (ABC) vacuolar transporters in GA production. Molecular docking of GA and its precursors, β-Amyrin and 11-oxo-β-amyrin, was performed with five vacuolar ABC transporters (Bpt1p, Vmr1p, Ybt1p, Ycf1p and Nft1p). Based on docking scores, two top scoring transporters were selected (Bpt1p and Vmr1p) to investigate transporters' functions on GA production via overexpression and knockout experiments in one GA-producing yeast strain (GA166). Results revealed that GA and its precursors exhibited the highest predicted binding affinity towards BPT1 (ΔG = ?10.9, ?10.6, ?10.9 kcal/mol for GA, β-amyrin and 11-oxo-β-amyrin, respectively). Experimental results showed that the overexpression of BPT1 and VMR1 restored the intracellular as well as extracellular GA production level under limited nutritional conditions, whereas knockout of BPT1 resulted in a total loss of GA production. These results suggest that the activity of BPT1 is required for GA production in engineered Saccharomyces cerevisiae.     

Biogenesis of vacuolar membrane glycoproteins of yeast Saccharomyces cerevisiae

To investigate the biogenesis of the yeast vacuole, we have sought novel marker proteins localized to the vacuolar membrane. Glycoproteins were prepared from vacuolar membrane vesicles by concanavalin A-Sepharose column chromatography and used to raise monoclonal antibodies. The antibodies obtained recognize several vacuolar proteins that have N-linked oligosaccharide chains. A set of the antibodies reacts with a vacuolar glycoprotein with a major molecular species of 72 kDa (vgp72), which appears to associate peripherally with the vacuolar membrane. The biosynthesis of vgp72 has been examined in detail by pulse-chase experiments and by analyses using various secretory mutants (sec18, sec7, and sec1) and a vacuolar protease mutant (pep4). vgp72 first appears in the endoplasmic reticulum as a 74-kDa species and is quickly modified in the Golgi apparatus to two distinct species: a 79-kDa form, and a heterogeneously glycosylated form (90-150 kDa). Subsequently, both species are proteolytically processed in the vacuole giving rise to a 72-kDa species as well as heavily glycosylated form. Thus, the biogenesis of vgp72 utilizes the early part of the secretory pathway as is the case of vacuolar soluble enzymes. A unique feature is that two species that are different in the extent of glycosylation appear to follow the same destination to the vacuolar membrane.     

Phylogenetic classification of transporters and other membrane proteins from Saccharomyces cerevisiae

On the basis of functional and phylogenetic criteria, we have identified a total of 229 subfamilies and 111 singletons predicted to carry out transport or other membrane functions in Saccharomyces cerevisiae. We have extended the Transporter Classification (TC) and created a Membrane Classification (MC) for non-transporter membrane proteins. Using the preliminary phylogenetic digits X, Y, Z (for new families, subfamilies, and clusters, respectively), we allocated a five-digit number to 850 proteins predicted to contain more than two transmembrane domains. Compared with a previous TC of the yeast genome, we classified an additional set of 538 membrane proteins (transporters and non-transporters) and identified 111 novel phylogenetic subfamilies. Electronic Publication     

Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae

Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1? mutation. Uptake activity was inhibited by the V-ATPase inhibitor: concanamycin A and a protonophore. Isoleucine uptake was inhibited by various neutral amino acids and histidine, but not by γ-aminobutyric acid, glutamate, and aspartate. V-ATPase-dependent acidification of the vesicles was declined by the addition of isoleucine or histidine, depending upon Avt1p. Taken together with the data of the amino acid contents of vacuolar fractions in cells, the results suggested that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids.     

A family of yeast proteins mediating bidirectional vacuolar amino acid transport

Seven genes in Saccharomyces cerevisiae are predicted to code for membrane-spanning proteins (designated AVT1-7) that are related to the neuronal gamma-aminobutyric acid-glycine vesicular transporters. We have now demonstrated that four of these proteins mediate amino acid transport in vacuoles. One protein, AVT1, is required for the vacuolar uptake of large neutral amino acids including tyrosine, glutamine, asparagine, isoleucine, and leucine. Three proteins, AVT3, AVT4, and AVT6, are involved in amino acid efflux from the vacuole and, as such, are the first to be shown directly to transport compounds from the lumen of an acidic intracellular organelle. This function is consistent with the role of the vacuole in protein degradation, whereby accumulated amino acids are exported to the cytosol. Protein AVT6 is responsible for the efflux of aspartate and glutamate, an activity that would account for their exclusion from vacuoles in vivo. Transport by AVT1 and AVT6 requires ATP for function and is abolished in the presence of nigericin, indicating that the same pH gradient can drive amino acid transport in opposing directions. Efflux of tyrosine and other large neutral amino acids by the two closely related proteins, AVT3 and AVT4, is similar in terms of substrate specificity to transport system h described in mammalian lysosomes and melanosomes. These findings suggest that yeast AVT transporter function has been conserved to control amino acid flux in vacuolar-like organelles.     

Ypq3p-dependent histidine uptake by the vacuolar membrane vesicles of Saccharomyces cerevisiae

The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA³ was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate.     

Proton potential-dependent polyamine transport by vacuolar membrane vesicles of Saccharomyces cerevisiae.

Vacuolar membrane vesicles of Saccharomyces cerevisiae accumulated spermine and spermidine in the presence of ATP, not in the presence of ADP. Spermine and spermidine transport at pH 7.4 showed saturation kinetics with Km values of 0.2 mM and 0.7 mM, respectively. Spermine uptake was competitively inhibited by spermidine and putrescine, but was not affected by seven amino acids, substrates of active transport systems of vacuolar membrane. Spermine transport was inhibited by the H(+)-ATPase-specific inhibitors bafilomycin A1 and N,N'-dicyclohexylcarbodiimide, but not by vanadate. It was also sensitive to Cu2+ or Zn2+ ions, inhibitors of vacuolar H(+)-ATPase. Both 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847) and nigericin blocked completely the spermine uptake, but valinomycin did not. [14C]Spermine accumulated in the vesicles was exchangeable with unlabeled spermine and spermidine. However, it was released by a protonophore only in the presence of a counterion such as Ca2+. These results indicate that a polyamine-specific transport system depending on a proton potential functions in the vacuolar membrane of this organism.     

Localization of the thermosensitive X-prolyl dipeptidyl aminopeptidase in the vacuolar membrane of Saccharomyces cerevisiae

Most of the X-prolyl dipeptidyl aminopeptidase activity of Saccharomyces cerevisiae was found to be associated with purified vacuolar membranes (specific activity approx. 75-times higher than in the protoplast lysate). The tonoplast-bound enzyme is thermosensitive. Another heat-resistant enzyme was found in the protoplast lysate. The tonoplast-bound thermosensitive enzyme shows an apparent Km of 0.06 mM against L-alanyl-L-prolyl-p-nitroanilide while the heat-resistant enzyme shows an apparent Km of 0.4 mM against the same substrate.     

Zinc status and vacuolar zinc transporters control alkaline phosphatase accumulation and activity in Saccharomyces cerevisiae

Little is known about how metalloproteins in the secretory pathway obtain their metal ion cofactors. We used the Pho8 alkaline phosphatase of the yeast Saccharomyces cerevisiae to probe this process in vivo . We found that both Pho8 activity and protein accumulation are zinc-dependent and decrease in zinc-limited cells. Low Pho8 accumulation was the result of degradation by vacuolar proteases. Surprisingly, the protective effect of zinc on Pho8 stability was not solely due to Zn²⁺ binding to the active-site ligands suggesting that the Pho8 protein is targeted for degradation in zinc-limited cells by another mechanism. Pho8 appears to be a rare example of a metalloprotein whose stability is regulated by its metal cofactor independently of active-site binding. We also assessed which zinc transporters are responsible for supplying zinc to Pho8. We found that the Zrc1 and Cot1 vacuolar zinc transporters play the major role while the Msc2/Zrg17 zinc transporter complex active in the endoplasmic reticulum is not involved. These results demonstrate that the vacuolar zinc transporters, previously implicated in metal detoxification, also deliver zinc to certain metalloproteins within intracellular compartments. These data suggest that Pho8 receives its metal cofactor in the vacuole rather than in earlier compartments of the secretory pathway.     

The ABC transporters of Saccharomyces cerevisiae

The ABC transporters (ATP Binding Cassette) compose one of the bigest protein family with the great medical, industrial and economical impact. They are found in all organism from bacteria to man. ABC proteins are responsible for resistance of microorganism to antibiotics and fungicides and multidrug resistance of cancer cells. Mutations in ABC transporters genes cause seriuos deseases like cystic fibrosis, adrenoleucodystrophy or ataxia. Transport catalized by ABC proteins is charged with energy from the ATP hydrolysis. The ABC superfamily contains transporters, canals, receptors. Analysis of the Saccharomyces cerevisiae genome allowed to distinguish 30 potential ABC proteins which are classified into 6 subfamilies. The structural and functional similarity of the yeast and human ABC proteins allowes to use the S. cerevisiae as a model organism for ABC transporters characterisation. In this work the present state of knowleadge on yeast S. cerevisiae ABC proteins was summarised.     

Purification and amino acid sequence of mating factor from Saccharomyces cerevisiae.

Mating factor is a peptide excreted into the culture fluid by alpha-mating type cells of Saccharomyces cerevisiae X-2180 1B. The purification of the mating factor was carried out by ion exchange chromatography on phosphocellulose and Amberlite IRC 50 columns, followed by gel filtration on a Sephadex LH 20 column. The factor thus prepared was a peptide composed of Lys1, His1, Trp2, Gln2, Pro2, Gly1, Met1, Leu2 and Tyr1, and was able to induce morphological changes on alpha-mating type cells at a concentration of 5 pg/ml. The amino acid sequence of the mating factor was determined by the manual Edman degradation method using intact mating factor and its thermolytic peptides. The C-terminal amino acid residue was determined by digesting the factor with carboxypeptidase A. The complete amino acid sequence of the mating factor was established to be as follows: Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr.