Substrate specificities of active transport systems for amino acids in vacuolar-membrane vesicles of Saccharomyces cerevisiae. Evidence of seven independent proton/amino acid antiport systems.

Abstract The Ygr125w was previously identified as a vacuolar membrane protein by a proteomic analysis. We found that vacuolar levels of basic amino acids drastically decreased in ygr125wΔ cells. Since N- or C-terminally tagged Ygr125w was not functional, an expression plasmid of YGR125w with HA3-tag inserted in its N-terminal hydrophilic region was constructed. Introduction of this plasmid into ygr125w∆ cells restored the vacuolar levels of basic amino acids. We successfully detected the uptake activity of arginine by the vacuolar membrane vesicles depending on HA3-YGR125w expression. A conserved aspartate residue in the predicted first transmembrane helix (D223) was indispensable for the accumulation of basic amino acids. YGR125w has been recently reported as a gene involved in vacuolar storage of arginine; and it is designated as VSB1. Taken together, our findings indicate that Ygr125w/Vsb1 contributes to the uptake of arginine into vacuoles and vacuolar compartmentalization of basic amino acids.

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Nutrient regulation of oligopeptide transport in Saccharomyces cerevisiae

Microbiology ◽

10.1099/mic.0.29055-0 ◽

2006 ◽

Vol 152 (10) ◽

pp. 3133-3145 ◽

Cited By ~ 28

Author(s):

Amy M. Wiles ◽

Houjian Cai ◽

Fred Naider ◽

Jeffrey M. Becker

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Environmental Conditions ◽

Transport Systems ◽

Free Medium ◽

Small Peptides ◽

Functional Protein ◽

Nutrient Regulation ◽

Free Environment

Small peptides (2–5 amino acid residues) are transported into Saccharomyces cerevisiae via two transport systems: PTR (Peptide TRansport) for di-/tripeptides and OPT (OligoPeptide Transport) for oligopeptides of 4–5 amino acids in length. Although regulation of the PTR system has been studied in some detail, neither the regulation of the OPT family nor the environmental conditions under which family members are normally expressed have been well studied in S. cerevisiae. Using a lacZ reporter gene construct fused to 1 kb DNA from upstream of the genes OPT1 and OPT2, which encode the two S. cerevisiae oligopeptide transporters, the relative expression levels of these genes were measured in a variety of environmental conditions. Uptake assays were also conducted to measure functional protein levels at the plasma membrane. It was found that OPT1 was up-regulated in sulfur-free medium, and that Ptr3p and Ssy1p, proteins involved in regulating the di-/tripeptide transporter encoding gene PTR2 via amino acid sensing, were required for OPT1 expression in a sulfur-free environment. In contrast, as measured by response to toxic tetrapeptide and by real-time PCR, OPT1 was not regulated through Cup9p, which is a repressor for PTR2 expression, although Cup9p did repress OPT2 expression. In addition, all of the 20 naturally occurring amino acids, except the sulfur-containing amino acids methionine and cysteine, up-regulated OPT1, with the greatest change in expression observed when cells were grown in sulfur-free medium. These data demonstrate that regulation of the OPT system has both similarities and differences to regulation of the PTR system, allowing the yeast cell to adapt its utilization of small peptides to various environmental conditions.

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A PQ-loop protein Ypq2 is involved in the exchange of arginine and histidine across the vacuolar membrane of Saccharomyces cerevisiae

Scientific Reports ◽

10.1038/s41598-019-51531-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Miyuki Kawano-Kawada ◽

Kunio Manabe ◽

Haruka Ichimura ◽

Takumi Kimura ◽

Yuki Harada ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Kinetic Analysis ◽

Membrane Vesicles ◽

Vacuolar Membrane ◽

Coupled Systems ◽

Gene Encoding ◽

Proline Residue ◽

Uptake Activity ◽

Exchange Activity

Abstract In nutrient-rich conditions, basic amino acids are actively accumulated into the vacuoles by H+-coupled transporters in Saccharomyces cerevisiae. In addition to the H+-coupled systems, the existence of an exchanger for arginine and histidine was indicated by kinetic analysis using isolated vacuolar membrane vesicles; however, the gene(s) involved in the activity has not been identified. Here, we show that the uptake activity of arginine driven by an artificially imposed histidine gradient decreased significantly by the disruption of the gene encoding vacuolar PQ-loop protein Ypq2, but not by those of Ypq1 and Ypq3. The exchange activity was restored by the expression of YPQ2. Furthermore, the substitution of a conserved proline residue, Pro29, in Ypq2 greatly decreased the exchange activity. These results suggest that Ypq2 is responsible for the exchange activity of arginine and histidine across the vacuolar membrane, and the conserved proline residue in the PQ-loop motif is required for the activity.

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Mutations in Saccharomyces cerevisiae which confer resistance to several amino acid analogs

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2941-2949.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2941-2949

Author(s):

J H McCusker ◽

J E Haber

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Nitrogen Source ◽

Transport Systems ◽

Acid Uptake ◽

Acid Analog ◽

Amino Acid Analogs ◽

Amino Acid Analog ◽

Complementation Groups

Four new complementation groups of mutations which confer resistance to several amino acid analogs in Saccharomyces cerevisiae are described. These mutants were isolated on medium containing urea as the nitrogen source, in contrast to previous studies that had used medium containing proline. All four resistance to amino acid analog (raa) complementation groups appear to confer resistance by reducing amino acid analog and amino acid uptake. In some genetic backgrounds, raa leu2 and raa thr4 double mutants are inviable, even on rich medium. The raa4 mutation may affect multiple amino acid transport systems, since raa4 mutants are unable to use proline as a nitrogen source. raa4 is, however, unlinked to a previously described amino acid analog resistance and proline uptake mutant, aap1, or to the general amino acid permease mutant gap1. Both raa4 and gap1 prevent uptake of [3H]leucine in liquid cultures. The raa1, raa2, and raa3 mutants affect only a subset of the amino acid analogs and amino acids affected by raa4. The phenotypes of raa1, -2, and -3 mutants are readily observed on agar plates but are not seen in uptake and incorporation of amino acids measured in liquid media.

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AMINO ACID TRANSPORT SYSTEMS IN BRUSH-BORDER MEMBRANE VESICLES FROM LEPIDOPTERAN ENTEROCYTES

Journal of Experimental Biology ◽

10.1242/jeb.143.1.87 ◽

1989 ◽

Vol 143 (1) ◽

pp. 87-100

Author(s):

GIORGIO M. HANOZET ◽

BARBARA GIORDANA ◽

V. FRANCA SACCHI ◽

PAOLO PARENTI

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Glutamic Acid ◽

Brush Border ◽

Amino Acid Transport ◽

Brush Border Membrane ◽

Membrane Vesicles ◽

Transport Systems ◽

Acid Transport ◽

Luminal Membrane

The presence of different potassium-dependent amino acid transport systems in the luminal membrane of the larval midgut of Philosamia cynthia Drury (Saturnidae, Lepidoptera) was investigated by means of countertransport experiments performed with brush-border membrane vesicles. The vesicles were preloaded with 14 different unlabelled amino acids, whose ability to elicit an intravesicular accumulation over the equilibrium value of six labelled amino acids (L-alanine, L-leucine, L-phenylalanine, L-glutamic acid, L-lysine and L-histidine) was tested. For histidine, the results were compared with those obtained from inhibition experiments, in which the same 14 amino acids were used as inhibitors on the cis side of the brush-border membrane. The data demonstrate the presence in the lepidopteran luminal membrane of distinct transport pathways for lysine and glutamic acid. The transport of most neutral amino acids, with the exclusionof glycine and proline, seems to occur through a system that may be similar to the neutral brush-border system (NBB) found in mammalian intestinal membranes. This system is also able to handle histidine.

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Brush-border amino acid transport mechanisms in carnivorous eel intestine

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1989.257.3.r506 ◽

1989 ◽

Vol 257 (3) ◽

pp. R506-R510 ◽

Cited By ~ 1

Author(s):

C. Storelli ◽

S. Vilella ◽

M. P. Romano ◽

M. Maffia ◽

G. Cassano

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Brush Border ◽

Amino Acid Transport ◽

Membrane Vesicles ◽

Anguilla Anguilla ◽

Neutral Amino Acid ◽

Transport Systems ◽

European Eel ◽

Acid Transport

Brush-border membrane vesicles (BBMV) were prepared from European eel (Anguilla anguilla) intestinal epithelium by a magnesium-ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) precipitation technique. Amino acid transport by these purified vesicle preparations was investigated using either radiolabeled substrates or the voltage-sensitive fluorescent dye 3,3'-diethylthiadicarbocyanine iodide [DiSC2(5)]. All amino acids tested exhibited carrier-mediated, Na+-dependent and Na+-independent transfer processes plus diffusion. The only exceptions were glutamic acid and proline, which displayed Na+ dependency and diffusion but did not appear to be transported by Na+-independent agencies. Carrier-mediated transport kinetic constants (Kapp and Jmax) for several amino acids are reported. Cis-inhibition experiments suggested the presence of at least four distinct Na+-dependent transport systems in eel intestinal BBMV: 1) an anionic transport process for glutamic and aspartic acids; 2) a cationic mechanism for lysine and arginine; 3) a relatively specific neutral amino acid carrier for proline and alpha-(methylamino)isobutyric acid; and 4) a nonspecific neutral amino acid system for most other substrates of this group. This scheme for carnivorous fish intestine most closely approximates that reported for mammalian gut with minor dissimilarities that may relate to metabolic differences or specific dietary requirements of the two vertebrate groups.

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Some Observations On Platelet Amino Acid Transport Systems

10.1055/s-0038-1653244 ◽

1981 ◽

Author(s):

U T Yardimci ◽

A Özbilen ◽

O N Ulutin

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Active Transport ◽

Amino Acid Transport ◽

Acid Group ◽

Transport Systems ◽

Acid Transport ◽

Amino Acid Group ◽

Active Transport System ◽

Group Transport

We have studied the transport systems for amino acids in platelets. Na+/K+ dependent active transport systems were found to be responsible for the transport of amino acids through the platelet membrane (Km’s being at uM ranges). We have also isolated the binding proteins for amino acids from platelet membranes as the carriers involved in these active transport systems by cold osmotic shock procedure. Each amino acid besides being transported by a specific active transport system may be subject to transport by group amino acid transport systems.Group amino acid transport systems are classified by countertransport experiments as follows: Neutral amino acid group transport systems: IA: glycine, alanine, serine, threonine IB: valine, leucine, isoleucine, serine,threonine IC: cysteine, methionine, proline Basic amino acid group transport systems: lie: lysine IIB: histidine, arginine Acidic amino acid group transport systems: III A: Aspartic acid, glutamic acid Aromatic amino acid group transport systems: IVC: Phenylalanine,tyrosine, histidine, proline.

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Mutations in Saccharomyces cerevisiae which confer resistance to several amino acid analogs.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2941 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2941-2949 ◽

Cited By ~ 11

Author(s):

J H McCusker ◽

J E Haber

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Nitrogen Source ◽

Transport Systems ◽

Acid Uptake ◽

Acid Analog ◽

Amino Acid Analogs ◽

Amino Acid Analog ◽

Complementation Groups

Four new complementation groups of mutations which confer resistance to several amino acid analogs in Saccharomyces cerevisiae are described. These mutants were isolated on medium containing urea as the nitrogen source, in contrast to previous studies that had used medium containing proline. All four resistance to amino acid analog (raa) complementation groups appear to confer resistance by reducing amino acid analog and amino acid uptake. In some genetic backgrounds, raa leu2 and raa thr4 double mutants are inviable, even on rich medium. The raa4 mutation may affect multiple amino acid transport systems, since raa4 mutants are unable to use proline as a nitrogen source. raa4 is, however, unlinked to a previously described amino acid analog resistance and proline uptake mutant, aap1, or to the general amino acid permease mutant gap1. Both raa4 and gap1 prevent uptake of [3H]leucine in liquid cultures. The raa1, raa2, and raa3 mutants affect only a subset of the amino acid analogs and amino acids affected by raa4. The phenotypes of raa1, -2, and -3 mutants are readily observed on agar plates but are not seen in uptake and incorporation of amino acids measured in liquid media.

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SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2154 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2154-2164 ◽

Cited By ~ 50

Author(s):

D J DeMarini ◽

M Winey ◽

D Ursic ◽

F Webb ◽

M R Culbertson

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Gene Product ◽

Signal Sequence ◽

Null Alleles ◽

Nucleoside Triphosphate ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

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