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

2019 ◽  
Vol 9 (1) ◽  
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.

Ygr125w/Vsb1-dependent accumulation of basic amino acids into vacuoles of Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Miyuki Kawano-Kawada ◽  
Haruka Ichimura ◽  
Shota Ohnishi ◽  
Yusuke Yamamoto ◽  
Yumi Kawasaki ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Transmembrane Helix ◽  
Membrane Vesicles ◽  
Vacuolar Membrane ◽  
Expression Plasmid ◽  
Basic Amino Acids ◽  
Uptake Activity ◽  
Hydrophilic Region ◽  
Aspartate Residue

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.

Identification and isolation of the gene encoding the small subunit of ribonucleotide reductase from Saccharomyces cerevisiae: DNA damage-inducible gene required for mitotic viability

1987 ◽  
Vol 7 (8) ◽  
pp. 2783-2793
Author(s):  
S J Elledge ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Dna Damage ◽  
Ribonucleotide Reductase ◽  
Small Subunit ◽  
Cross Reaction ◽  
Expression Library ◽  
Gene Encoding ◽  
Ribonucleotide Reductases ◽  
Colony Color

Ribonucleotide reductase catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. The gene encoding the small subunit of ribonucleotide reductase was isolated from a Saccharomyces cerevisiae genomic DNA expression library in lambda gt11 by a fortuitous cross-reaction with anti-RecA antibodies. The cross-reaction was due to an identity between the last four amino acids of each protein. The gene has been named RNR2 and is centromere linked on chromosome X. The nucleotide sequence was determined, and the deduced amino acid sequence, 399 amino acids, shows extensive homology with other eucaryotic ribonucleotide reductases. Transplason mutagenesis was used to disrupt the RNR2 gene. A novel assay using colony color sectoring was developed to demonstrate visually that RNR2 is essential for mitotic viability. RNR2 encodes a 1.5-kilobase mRNA whose levels increase 18-fold after treatment with the DNA-damaging agent 4-nitroquinoline 1-oxide. CDC8 was also found to be inducible by DNA damage, but POL1 and URA3 were not inducible by 4-nitroquinoline 1-oxide. The expression of these genes defines a new mode of regulation for enzymes involved in DNA biosynthesis and sharpens our picture of the events leading to DNA repair in eucaryotic cells.

scholarly journals ZDS1 and ZDS2, genes whose products may regulate Cdc42p in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (10) ◽  
pp. 5264-5275 ◽  
Author(s):  
E Bi ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Intracellular Signaling ◽  
Double Mutant ◽  
Genome Project ◽  
Yeast Genome ◽  
Glutathione S Transferase ◽  
Gene Encoding ◽  
Biological Phenomena ◽  
Wide Range

A genetic screen for GTPase-activating proteins (GAPs) or other negative regulators of the Rac/Rho family GTPase Cdc42p in Saccharomyces cerevisiae identified ZDS1, a gene encoding a protein of 915 amino acids. Sequence from the yeast genome project identified a homolog, ZDS2, whose predicted product of 942 amino acids is 38% identical in sequence to Zds1p. Zds1p and Zds2p have no detectable homology to known Rho-GAPs or to other known proteins. However, by several assays, it appears that overexpression of either Zds1p or Zds2p decreases the level of Cdc42p activity. Deletion analysis also suggests that Zds1p and Zds2p are at least partially overlapping in function. Deletion of ZDS2 produced no obvious phenotype, and deletion of ZDS1 produced no obvious phenotype other than a mild effect on cell shape. However, the zds1 zds2 double mutant grew slowly with an apparent mitotic delay and produced elongated cells and buds with other evidence of abnormal morphogenesis. A glutathione S-transferase-Zds1p fusion protein that fully complemented the double mutant localized to presumptive bud sites and the tips of small buds. The similarity of this localization to that of Cdc42p suggests that Zds1p may interact directly with Cdc42p. As ZDS1 and ZDS2 have recently been identified also by numerous other groups studying a wide range of biological phenomena, the roles of Cdc42p in intracellular signaling may be more diverse than has previously been appreciated.

scholarly journals AnkB, a Periplasmic Ankyrin-Like Protein inPseudomonas aeruginosa, Is Required for Optimal Catalase B (KatB) Activity and Resistance to Hydrogen Peroxide

2000 ◽  
Vol 182 (16) ◽  
pp. 4545-4556 ◽  
Author(s):  
Michael L. Howell ◽  
Eyad Alsabbagh ◽  
Ju-Fang Ma ◽  
Urs A. Ochsner ◽  
Martin G. Klotz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hydrogen Peroxide ◽  
Cytoplasmic Membrane ◽  
Membrane Vesicles ◽  
Gene Encoding ◽  
Rnase Protection ◽  
C Terminus ◽  
Hydrophobic Amino Acids ◽  
Increased Sensitivity ◽  
Membrane Spanning

ABSTRACT In this study, we have cloned the ankB gene, encoding an ankyrin-like protein in Pseudomonas aeruginosa. TheankB gene is composed of 549 bp encoding a protein of 183 amino acids that possesses four 33-amino-acid ankyrin repeats that are a hallmark of erythrocyte and brain ankyrins. The location ofankB is 57 bp downstream of katB, encoding a hydrogen peroxide-inducible catalase, KatB. Monomeric AnkB is a 19.4-kDa protein with a pI of 5.5 that possesses 22 primarily hydrophobic amino acids at residues 3 to 25, predicting an inner-membrane-spanning motif with the N terminus in the cytoplasm and the C terminus in the periplasm. Such an orientation in the cytoplasmic membrane and, ultimately, periplasmic space was confirmed using AnkB-BlaM and AnkB-PhoA protein fusions. Circular dichroism analysis of recombinant AnkB minus its signal peptide revealed a secondary structure that is ∼65% α-helical. RNase protection and KatB- and AnkB-LacZ translational fusion analyses indicated that katBand ankB are part of a small operon whose transcription is induced dramatically by H2O2, and controlled by the global transactivator OxyR. Interestingly, unlike the spherical nature of ankyrin-deficient erythrocytes, the cellular morphology of anankB mutant was identical to that of wild-type bacteria, yet the mutant produced more membrane vesicles. The mutant also exhibited a fourfold reduction in KatB activity and increased sensitivity to H2O2, phenotypes that could be complemented in trans by a plasmid constitutively expressing ankB. Our results suggest that AnkB may form an antioxidant scaffolding with KatB in the periplasm at the cytoplasmic membrane, thus providing a protective lattice work for optimal H2O2 detoxification.

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

2016 ◽  
Vol 80 (6) ◽  
pp. 1125-1130 ◽  
Author(s):  
Kunio Manabe ◽  
Miyuki Kawano-Kawada ◽  
Koichi Ikeda ◽  
Takayuki Sekito ◽  
Yoshimi Kakinuma
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Vesicles ◽  
Vacuolar Membrane

scholarly journals Proton Gradient-Driven Nickel Uptake by Vacuolar Membrane Vesicles of Saccharomyces cerevisiae

1998 ◽  
Vol 180 (7) ◽  
pp. 1962-1964 ◽  
Author(s):  
Ken Nishimura ◽  
Kazuei Igarashi ◽  
Yoshimi Kakinuma
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Parent Strain ◽  
Membrane Vesicles ◽  
Vacuolar Membrane ◽  
Ph Gradient ◽  
Dependent Manner ◽  
Nickel Ion ◽  
Nickel Uptake ◽  
Highly Sensitive ◽  
Negative Mutant

ABSTRACT A vacuolar H+-ATPase-negative mutant ofSaccharomyces cerevisiae was highly sensitive to nickel ion. Accumulation of nickel ion in the cells of this mutant of less than 60% of the value for the parent strain arrested growth, suggesting a role for this ATPase in sequestering nickel ion into vacuoles. An artificially imposed pH gradient (interior acid) induced transient nickel ion uptake by vacuolar membrane vesicles, which was inhibited by collapse of the pH difference but not of the membrane potential. Nickel ion transport into vacuoles in a pH gradient-dependent manner is thus important for its detoxification in yeast.

scholarly journals Evidence for a low-affinity, high-capacity uniport for amino acids in Bombyx mori larval midgut

1998 ◽  
Vol 274 (5) ◽  
pp. R1372-R1375 ◽  
Author(s):  
M. G. Leonardi ◽  
M. Casartelli ◽  
P. Parenti ◽  
B. Giordana
Keyword(s):  
Amino Acids ◽  
Kinetic Analysis ◽  
Bombyx Mori ◽  
Electrical Potential ◽  
High Capacity ◽  
Membrane Vesicles ◽  
Electrical Potential Difference ◽  
Larval Midgut ◽  
Posterior Midgut ◽  
Transmembrane Electrical Potential

We investigated the kinetics of leucine influx as a funtion of external substrate concentration between 0.03 and 16 mM in brush-border membrane vesicles (BBMV) prepared from the middle region of Bombyx mori larval midgut. A detailed kinetic analysis of leucine uptake led to the identification, in parallel with the K+-dependent symporter for neutral amino acids, of a K+-independent, low-affinity, high-capacity system. The parameter values of the Michaelis constant (7.12 mM) and maximal rate of transport (4.48 nmol ⋅ 7 s−1 ⋅ mg protein−1) were not influenced by an external alkaline pH nor by a transmembrane electrical potential difference. The uniporter is poorly specific, as it displayed the following rank of preference: Leu, His, Val, Ile, Phe, Ser > Lys, Arg, Gln > Pro, 2-amino-2-norbornane-carboxylic acid, Ala, Gly. The kinetic analysis performed in BBMV prepared from the posterior midgut portion indicates that the low-affinity, high-capacity uniporter is present along the entire length of the silkworm larval midgut with similar expression and functional properties.

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