Growth Inhibition by Amino Acids in Saccharomyces cerevisiae

AbstractAmino acids are essential metabolites but can also be toxic when present at high levels intracellularly. Substrate-induced down-regulation of amino acid transporters inSaccharomyces cerevisiaeis thought to be a mechanism to avoid this toxicity. It has been shown that unregulated uptake by the general amino acid permease Gap1 causes cells to become sensitive to amino acids. Here, we show that overexpression of eight other amino acid transporters (Agp1, Bap2, Can1, Dip5, Gnp1, Lyp1, Put4 or Tat2) also induces a growth defect when specific single amino acids are present at concentrations of 0.5–5 mM. We can now state that all proteinogenic amino acids, as well as the important metabolite ornithine, are growth inhibitory toS. cerevisiaewhen transported into the cell at high enough levels. Measurements of initial transport rates and cytosolic pH show that toxicity is due to amino acid accumulation and not to the influx of co-transported protons. The amino acid sensitivity phenotype is a useful tool that reports on thein vivoactivity of transporters and has allowed us to identify new transporter-specific substrates.

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Characterization of the Candida albicans Amino Acid Permease Family: Gap2 Is the Only General Amino Acid Permease and Gap4 Is an S-Adenosylmethionine (SAM) Transporter Required for SAM-Induced Morphogenesis

mSphere ◽

10.1128/msphere.00284-16 ◽

2016 ◽

Vol 1 (6) ◽

Cited By ~ 12

Author(s):

Lucie Kraidlova ◽

Sanne Schrevens ◽

Hélène Tournu ◽

Griet Van Zeebroeck ◽

Hana Sychrova ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Candida Albicans ◽

Amino Acid ◽

Virulence Factor ◽

Amino Acid Transporters ◽

Amino Acid Permease ◽

Content Type ◽

Important Virulence Factor ◽

General Amino Acid Permease

ABSTRACT Candida albicans is a commensal organism that can thrive in many niches in its human host. The environmental conditions at these different niches differ quite a bit, and this fungus must be able to sense these changes and adapt its metabolism to them. Apart from glucose and other sugars, the uptake of amino acids is very important. This is underscored by the fact that the C. albicans genome encodes 6 orthologues of the Saccharomyces. cerevisiae general amino acid permease Gap1 and many other amino acid transporters. In this work, we characterize these six permeases and we show that C. albicans Gap2 is the functional orthologue of ScGap1 and that C. albicans Gap4 is an orthologue of ScSam3, an S-adenosylmethionine (SAM) transporter. Furthermore, we show that Gap4 is required for SAM-induced morphogenesis, an important virulence factor of C. albicans. Amino acids are key sources of nitrogen for growth of Candida albicans. In order to detect and take up these amino acids from a broad range of different and changing nitrogen sources inside the host, this fungus must be able to adapt via its expression of genes for amino acid uptake and further metabolism. We analyzed six C. albicans putative general amino acid permeases based on their homology to the Saccharomyces cerevisiae Gap1 general amino acid permease. We generated single- and multiple-deletion strains and found that, based on growth assays and transcriptional or posttranscriptional regulation, Gap2 is the functional orthologue to ScGap1, with broad substrate specificity. Expression analysis showed that expression of all GAP genes is under control of the Csy1 amino acid sensor, which is different from the situation in S. cerevisiae, where the expression of ScGAP1 is not regulated by Ssy1. We show that Gap4 is the functional orthologue of ScSam3, the only S-adenosylmethionine (SAM) transporter in S. cerevisiae, and we report that Gap4 is required for SAM-induced morphogenesis. IMPORTANCE Candida albicans is a commensal organism that can thrive in many niches in its human host. The environmental conditions at these different niches differ quite a bit, and this fungus must be able to sense these changes and adapt its metabolism to them. Apart from glucose and other sugars, the uptake of amino acids is very important. This is underscored by the fact that the C. albicans genome encodes 6 orthologues of the Saccharomyces. cerevisiae general amino acid permease Gap1 and many other amino acid transporters. In this work, we characterize these six permeases and we show that C. albicans Gap2 is the functional orthologue of ScGap1 and that C. albicans Gap4 is an orthologue of ScSam3, an S-adenosylmethionine (SAM) transporter. Furthermore, we show that Gap4 is required for SAM-induced morphogenesis, an important virulence factor of C. albicans.

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The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.5010-5019.1993 ◽

1993 ◽

Vol 13 (8) ◽

pp. 5010-5019 ◽

Cited By ~ 1

Author(s):

J Heitman ◽

A Koller ◽

J Kunz ◽

R Henriquez ◽

A Schmidt ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Cyclosporin A ◽

Secretory Pathway ◽

Yeast Cells ◽

Amino Acid Transporters ◽

Yeast Saccharomyces Cerevisiae ◽

Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

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Rhizobium leguminosarum Has a Second General Amino Acid Permease with Unusually Broad Substrate Specificity and High Similarity to Branched-Chain Amino Acid Transporters (Bra/LIV) of the ABC Family

Journal of Bacteriology ◽

10.1128/jb.184.15.4071-4080.2002 ◽

2002 ◽

Vol 184 (15) ◽

pp. 4071-4080 ◽

Cited By ~ 76

Author(s):

A. H. F. Hosie ◽

D. Allaway ◽

C. S. Galloway ◽

H. A. Dunsby ◽

P. S. Poole

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Rhizobium Leguminosarum ◽

Binding Protein ◽

Amino Acid Transporters ◽

Acid Uptake ◽

Amino Acid Permease ◽

Branched Chain Amino Acid ◽

General Amino Acid Permease ◽

Branched Chain

ABSTRACT Amino acid uptake by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (BraRl). Characterization of the solute specificity of BraRl shows it to be the second general amino acid permease of R. leguminosarum. Although BraRl has high sequence identity to members of the family of hydrophobic amino acid transporters (HAAT), it transports a broad range of solutes, including acidic and basic polar amino acids (l-glutamate, l-arginine, and l-histidine), in addition to neutral amino acids (l-alanine and l-leucine). While amino and carboxyl groups are required for transport, solutes do not have to be α-amino acids. Consistent with this, BraRl is the first ABC transporter to be shown to transport γ-aminobutyric acid (GABA). All previously identified bacterial GABA transporters are secondary carriers of the amino acid-polyamine-organocation (APC) superfamily. Also, transport by BraRl does not appear to be stereospecific as d amino acids cause significant inhibition of uptake of l-glutamate and l-leucine. Unlike all other solutes tested, l-alanine uptake is not dependent on solute binding protein BraCRl. Therefore, a second, unidentified solute binding protein may interact with the BraDEFGRl membrane complex during l-alanine uptake. Overall, the data indicate that BraRl is a general amino acid permease of the HAAT family. Furthermore, BraRl has the broadest solute specificity of any characterized bacterial amino acid transporter.

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Amino Acid Signaling in Saccharomyces cerevisiae: a Permease-Like Sensor of External Amino Acids and F-Box Protein Grr1p Are Required for Transcriptional Induction of the AGP1 Gene, Which Encodes a Broad-Specificity Amino Acid Permease

Molecular and Cellular Biology ◽

10.1128/mcb.19.2.989 ◽

1999 ◽

Vol 19 (2) ◽

pp. 989-1001 ◽

Cited By ~ 180

Author(s):

Ismaïl Iraqui ◽

Stephan Vissers ◽

Florent Bernard ◽

Johan-Owen de Craene ◽

Eckhard Boles ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Glucose Sensors ◽

Amino Acid Permease ◽

Transporter Family ◽

F Box Protein ◽

Transcriptional Induction ◽

Amino Acid Signaling ◽

Broad Specificity

ABSTRACT The SSY1 gene of Saccharomyces cerevisiaeencodes a member of a large family of amino acid permeases. Compared to the 17 other proteins of this family, however, Ssy1p displays unusual structural features reminiscent of those distinguishing the Snf3p and Rgt2p glucose sensors from the other proteins of the sugar transporter family. We show here that SSY1 is required for transcriptional induction, in response to multiple amino acids, of theAGP1 gene encoding a low-affinity, broad-specificity amino acid permease. Total noninduction of the AGP1 gene in thessy1Δ mutant is not due to impaired incorporation of inducing amino acids. Conversely, AGP1 is strongly induced by tryptophan in a mutant strain largely deficient in tryptophan uptake, but it remains unexpressed in a mutant that accumulates high levels of tryptophan endogenously. Induction of AGP1requires Uga35p(Dal81p/DurLp), a transcription factor of the Cys6-Zn2 family previously shown to participate in several nitrogen induction pathways. Induction of AGP1by amino acids also requires Grr1p, the F-box protein of the SCFGrr1 ubiquitin-protein ligase complex also required for transduction of the glucose signal generated by the Snf3p and Rgt2p glucose sensors. Systematic analysis of amino acid permease genes showed that Ssy1p is involved in transcriptional induction of at least five genes in addition to AGP1. Our results show that the amino acid permease homologue Ssy1p is a sensor of external amino acids, coupling availability of amino acids to transcriptional events. The essential role of Grr1p in this amino acid signaling pathway lends further support to the hypothesis that this protein participates in integrating nutrient availability with the cell cycle.

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Different Ubiquitin Signals Act at the Golgi and Plasma Membrane to Direct GAP1 Trafficking

Molecular Biology of the Cell ◽

10.1091/mbc.e07-06-0627 ◽

2008 ◽

Vol 19 (7) ◽

pp. 2962-2972 ◽

Cited By ~ 39

Author(s):

April L. Risinger ◽

Chris A. Kaiser

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Plasma Membrane ◽

Amino Acid ◽

Protein Sorting ◽

High Capacity ◽

Amino Acid Permease ◽

Acid Addition ◽

Amino Acid Levels ◽

General Amino Acid Permease

The high capacity general amino acid permease, Gap1p, in Saccharomyces cerevisiae is distributed between the plasma membrane and internal compartments according to availability of amino acids. When internal amino acid levels are low, Gap1p is localized to the plasma membrane where it imports available amino acids from the medium. When sufficient amino acids are imported, Gap1p at the plasma membrane is endocytosed and newly synthesized Gap1p is delivered to the vacuole; both sorting steps require Gap1p ubiquitination. Although it has been suggested that identical trans-acting factors and Gap1p ubiquitin acceptor sites are involved in both processes, we define unique requirements for each of the ubiquitin-mediated sorting steps involved in delivery of Gap1p to the vacuole upon amino acid addition. Our finding that distinct ubiquitin-mediated sorting steps employ unique trans-acting factors, ubiquitination sites on Gap1p, and types of ubiquitination demonstrates a previously unrecognized level of specificity in ubiquitin-mediated protein sorting.

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Amino acids regulate the intracellular trafficking of the general amino acid permease of Saccharomyces cerevisiae

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.232591899 ◽

2002 ◽

Vol 99 (23) ◽

pp. 14837-14842 ◽

Cited By ~ 68

Author(s):

E. J. Chen ◽

C. A. Kaiser

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Intracellular Trafficking ◽

Amino Acid Permease ◽

General Amino Acid Permease

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The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.5010 ◽

1993 ◽

Vol 13 (8) ◽

pp. 5010-5019 ◽

Cited By ~ 65

Author(s):

J Heitman ◽

A Koller ◽

J Kunz ◽

R Henriquez ◽

A Schmidt ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Cyclosporin A ◽

Secretory Pathway ◽

Yeast Cells ◽

Amino Acid Transporters ◽

Yeast Saccharomyces Cerevisiae ◽

Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

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Changes in membrane proteins associated with inhibition of the general amino acid permease of yeast (Saccharomyces cerevisiae)

Biochemical Journal ◽

10.1042/bj1960531 ◽

1981 ◽

Vol 196 (2) ◽

pp. 531-536 ◽

Cited By ~ 10

Author(s):

J R Woodward ◽

H L Kornberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Membrane Proteins ◽

Membrane Protein ◽

Wild Type ◽

Amino Acid Permease ◽

Yeast Saccharomyces Cerevisiae ◽

General Amino Acid Permease ◽

Wild Type Yeast

The general amino acid permease (‘Gap’) system of the wild-type yeast (Saccharomyces cerevisiae) strain Y185 is inhibited by the uptake and accumulation of its substrate amino acids. Surprisingly, this inhibition persists even after ‘pools’ of amino acids, accumulated initially, have returned to normal sizes. Recovery from this inhibition depends on a supply of energy and involves the synthesis of a membrane protein component of the Gap system.

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Broad-Spectrum Amino Acid Transporters ClAAP3 and ClAAP6 Expressed in Watermelon Fruits

International Journal of Molecular Sciences ◽

10.3390/ijms20235855 ◽

2019 ◽

Vol 20 (23) ◽

pp. 5855 ◽

Cited By ~ 1

Author(s):

Tianran Shi ◽

Vijay Joshi ◽

Madhumita Joshi ◽

Stanislav Vitha ◽

Holly Gibbs ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Broad Spectrum ◽

De Novo ◽

Early Stage ◽

Expression Profiles ◽

Functional Characterization ◽

Gene Expression Profiles ◽

Amino Acid Transporters ◽

Amino Acid Permease

Watermelon fruit contains a high percentage of amino acid citrulline (Cit) and arginine (Arg). Cit and Arg accumulation in watermelon fruit are most likely mediated by both de novo synthesis from other amino acids within fruits and direct import from source tissues (leaves) through the phloem. The amino acid transporters involved in the import of Cit, Arg, and their precursors into developing fruits of watermelon have not been reported. In this study, we have compiled the list of putative amino acid transporters in watermelon and characterized transporters that are expressed in the early stage of fruit development. Using the yeast complementation study, we characterized ClAAP3 (Cla023187) and ClAAP6 (Cla023090) as functional amino acid transporters belonging to the family of amino acid permease (AAP) genes. The yeast growth and uptake assays of radiolabeled amino acid suggested that ClAAP3 and ClAAP6 can transport a broad spectrum of amino acids. Expression of translational fusion proteins with a GFP reporter in Nicotiana benthamiana leaves confirmed the ER- and plasma membrane-specific localization, suggesting the role of ClAAP proteins in the cellular import of amino acids. Based on the gene expression profiles and functional characterization, ClAAP3 and ClAAP6 are expected to play a major role in regulation of amino acid import into developing watermelon fruits.

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