Table 1.
Klbat1Δ mutants are impaired in VIL biosynthesis and catabolism.
Figure 1.
Growth phenotype of single and double mutants complemented with plasmids harboring BAT1, BAT2 or KlBAT1.
Wild type, bat1Δ, bat2Δ and bat1Δ bat2Δ strains were grown on ammonium-glucose (A) or VIL-glucose (B). Values are shown relative to growth rate of the wild type strain (0.20 h−1 and 0.13 h−1 on ammonium-glucose and VIL glucose respectively) and represent the mean of three independent experiments ± S. D. Cells were complemented with a centromeric plasmid (CEN) harboring BAT1 (CEN BAT1), BAT2 (CEN BAT2) or the K. lactis orthologous gene KlBAT1 whose expression was driven by its own promoter (CEN PKlBAT1-KlBAT1) or by BAT1 (CEN PBAT1-KlBAT1) or BAT2 (CEN PBAT2-KlBAT1) promoters.
Figure 2.
Bat1 is mitochondrially located, while Bat2 is cytoplasmic.
Fluorescence images showing the subcellular localization of the paralogous proteins Bat1 and Bat2. Samples were taken from exponentially grown cultures of tagged mutants grown on glucose-ammonium (A, B) or on glucose-VIL (C, D). Mitochondrial localization of Bat1, the signal of the Bat1-yECitrine fusion co-localizes with mitotracker signal (A, C). Cytoplasmic localization of the Bat2-yECitrine fusion (B, D).
Table 2.
In S. cerevisiae bat1Δ mutant is impaired in VIL biosynthesis, while a bat2Δ mutant is mainly impaired in VIL catabolism.
Figure 3.
Saccharomyces cerevisiae BAT1 and KlBAT1 expression is repressed by VIL.
Northern analysis was carried out on total RNA obtained from K. lactis 155 (wild type) and CLA34 (Klbat1Δ) strains (A), and S. cerevisiae strain CLA1-2 (wild type B, C). Strains were grown on 2% glucose with either valine (V) (150 mg/l), leucine (L) (100 mg/l), isoleucine (I) (30 mg/l), γ-aminobutiric acid (GABA 7 mM ), γ-aminobutiric acid+VIL (GABA VIL),VIL (valine+isoleucine+leucine), NH4 (40 mM NH4 SO2), NH4 VIL (40mM NH4 SO2+VIL), glutamine (GLN 7mM), glutamine+VIL (GLN VIL), as nitrogen sources. Filters were sequentially probed with the BAT1, BAT2, KlBAT1- specific PCR products described in experimental procedures and a BamH1-HindIII 1500 bp ACT1 DNA or an SCR 400bp PCR fragment as loading controls. Numbers indicate relative expression as compared to WT grown on ammonium-glucose. Four biological replicates were carried out, representative results are shown.
Figure 4.
S. cerevisiae BAT1 and BAT2 display divergent expression profile.
Northern analysis was carried out on total RNA obtained from S. cerevisiae strains CLA1-2 (wild type), CLA31 (bat1Δ BAT2) and CLA32 (BAT1 bat2Δ). Strains were grown on 2% glucose with either 40mM NH4 SO2, VIL (150 mg/l, 100 mg/l or 30 mg/l of valine (V), leucine (L) or isoleucine (I) respectively or NH4 SO2+VIL as nitrogen sources. Filters were sequentially probed with a 1500 bp BAT1 fragment, a 1450 bp BAT2 and a BamH1-HindIII 1500 bp ACT1 DNA fragment as loading control. Numbers indicate relative expression as compared to: Lane 1 the WT grown on glucose VIL, Lane 2 WT grown on glucose NH4. Four biological replicates were performed, and representative results are shown.
Figure 5.
Branched chain aminotransferase activity.
S. cerevisiae wild type, bat1Δ, bat2Δ and K. lactis wt (KlWT) strains were grown on glucose (A) or ethanol (B) as carbon source and ammonium or VIL as nitrogen sources. Aminotransferase activity was determined in cell free extracts as indicated in Materials and methods using α-ketoisovalerate (α-KIV), α-ketoisocaproate (α-KIC) or α-ketomethylvalerate (α-KMV) as substrates. Transaminase activity is reported as nmol mg/l min−1. Values are presented as mean from at least three measurements ± S. D.
Table 3.
Growth phenotypes of single and double bat1Δ and bat2Δ mutants.
Figure 6.
BAT1 and KlBAT1 expression is repressed under respiratory conditions.
Northern analysis was carried out with total RNA samples obtained from S. cerevisiae WT, bat1Δ BAT2 and BAT1 bat2Δ and K. lactis WT cultures grown on ammonium-glucose or ammonium-ethanol to mid exponential growth phase. Filters were sequentially probed with the BAT1, BAT2, KlBAT1- specific PCR products described in experimental procedures and a BamH1-HindIII 1500 bp ACT1 DNA or an SCR1 400bp PCR fragment as loading controls. Numbers indicate relative expression as compared to the WT strains grown on ammonium-glucose. Four biological replicates were performed, and representative results are shown.
Table 4.
Strains used in this work.