Figure 1.
Mpc3 is a highly regulated member of the yeast MPC gene family.
A, Amino acid sequence comparison of the yeast Mpc mitochondrial pyruvate carriers. Mpc2 and Mpc3 are 83% identical in the N-terminal 106 amino acids. Mpc1 is 28% identical to Mpc2 or Mpc3 in the N-terminal 116 amino acids. Putative transmembrane domains were identified using the TMpred tool at ch.EMBnet.org. B, Mpc3 abundance is activated by salt stress and diauxic shift. Yeast strains expressing the indicated TAP-tagged Mpc proteins from the endogenous promoters were used. Cells were subjected to osmotic stress (0.4 M NaCl) or shifted from glucose to glycerol medium for the indicated times. Mpc proteins were visualized by anti-PAP immunoblotting and compared to total protein loading. C, the relative distribution of Mpc proteins changes during adaptation to salt stress or non fermentative growth conditions. The individual Mpc protein levels were quantified as in (B) using cells continuously growing in normal medium (YPD), high salt medium (0.4 M NaCl) or glycerol medium (YPGlyc). The relative abundance of each Mpc protein was calculated under each growth condition. Data are shown as mean +/− standard deviation from three independent experiments. Mpc3 protein levels are significantly higher (p<0.05) as compared to Mpc1 or Mpc2 on glycerol medium according to the Students t-test.
Figure 2.
Transcriptional regulation of MPC genes in response to salt stress and diauxic shift.
Yeast wild type (BY4741, wt) and hog1 mutant strains were treated for 10 min with 0.4 M NaCl (A) or shifted from YPD to YPGlyc medium for 30 min (B). The expression levels were determined by RT-PCR and normalized for the ACT1 messenger. Data are shown as mean from two independent experiments; error bars represent standard deviation. MPC3 transcript levels are significantly lower in the hog1 mutant vs. wild type after NaCl induction (p = 0.001) or diauxic shift (p = 0.0003) according to the Students t-test.
Figure 3.
Differential requirement of Mpc proteins for amino acid biosynthesis and respiratory growth.
The growth of the wild type (BY4741, wt) and the indicated mutant strains was monitored in SD medium with (A) or without valine (B), with valine in the presence of 0.4 M NaCl (C) and in SGlyc medium with valine (D). Data presented are mean values from three biological replicates. The standard deviation is 2–5% for (A), 2–4% for (B) and (C), and 0.5–1% for (D). (E) Plate assay of yeast wild type and the indicated mpc mutant strains upon fermentative (SD) or respiratory (SLac and SGlyc) growth conditions.
Figure 4.
The effect of Mpc overexpression on respiratory growth and oxidative stress tolerance.
Individual Mpc proteins were overexpressed as dsRed fusions in yeast wild type cells. A. All three Mpc-dsRed fusion proteins localize exclusively to the mitochondria. Yeast cells were cotransformed with the respective Mpc overexpressing constructs and the mitochondrial GFP expressing pVT100 plasmid. Representative pictures are shown for GFP, dsRed and the overlay obtained by confocal fluorescence microscopy. B. The overexpression of Mpc1 or Mpc2 inhibits respiratory growth. The growth of yeast wild type cells overexpressing the indicated Mpc proteins was assayed on glucose (SD) or glycerol (SGlyc) containing minimal medium and compared to the respective empty vector control. C. Overexpression of Mpc3 leads to oxidative stress tolerance. The growth of yeast strains described in (B) was continuously monitored in normal medium (SD) and in the presence of 2 mM hydrogen peroxide or 25 µM menadione. Data presented are mean values from three biological replicates. The standard deviation is 1–3% for SD, 1–2% for hydrogen peroxide and 2–10% for menadione growth.
Figure 5.
Effects of gain and loss of Mpc function on the oxygen consumption rate.
The rate of oxygen consumption was measured in intact yeast cells corresponding to the indicated overexpression (A) or deletion strains (B). The control strain in the upper panel is wild type BY4741 with the empty overexpression vector and untransformed wild type in the lower panel. Data represent mean values +/− SD from at least three independent measurements. Mpc1 and Mpc2 overexpressing strains have significantly reduced oxygen consumption rates (p = 0.02), while Mpc3 overexpressing cells significantly increase the oxygen consumption rate (p = 0.05) according to the Students t-test (A). Oxygen consumption is significantly reduced (p = 0.05) in the mpc1 and mpc3 mutants as compared to wt according to the Students t-test (B).
Figure 6.
The function of Mpc proteins in ROS balance and survival in stationary phase.
A–B. The yeast strains overexpressing individual Mpc proteins described in Fig. 4 were grown in normal SD medium and subjected to a brief oxidative stress caused by hydrogen peroxide (A) or menadione (B). ROS was measured by the oxidation of dichlorodihydrofluorescein as described in “Experimental Procedures”. Fluorescence of the wild type before stress was arbitrarily set to 1. ROS levels were determined for three independent cultures in duplicate. Data presented are mean values +/− SD. Mpc1 and Mpc2 overexpressing strains have significantly increased ROS levels (p = 0.02 for panel A; p = 0.05 for panel B) as compared to wt according to the Students t-test (B). C. The survival in stationary phase in YPD medium of the indicated yeast strains was determined by plate assays after the indicated time.
Figure 7.
Mpc3 interacts preferentially with Mpc1 and other Mpc3 subunits.
Coprecipitation experiments were performed from mitochondrial extracts from yeast cells expressing Mpc3-HA in the presence or not of the indicated Mpc-TAP protein. Immunoblots are shown for the Mpc-TAP and Mpc3-HA proteins in the input and for Mpc3-HA after copurification of the respective TAP-tagged Mpc proteins (IP).
Figure 8.
The inducible Crc1 function is important to sustain respiratory growth together with Mpc1 and Mpc3.
A. Growth of the indicated yeast strains was assayed on synthetic agar medium containing glucose (SD) or glycerol (SGlyc) as the energy source. Leucine and valine were supplemented to the plates to exclude growth effects caused by diminished mitochondrial amino acid biosynthesis. B. CRC1 expression is highly activated by osmostress and during the diauxic shift. Yeast wild type (BY4741, wt) and hog1 mutant strains were treated for 20 min with 0.4 M NaCl (upper panel) or shifted from YPD to YPGlyc medium for 30 min (lower panel). The expression levels were determined by RT-PCR and normalized for the ACT1 messenger. Data are shown as mean from two independent experiments; error bars represent SD. CRC1 transcript levels are significantly lower in the hog1 mutant vs. wild type after NaCl induction (p = 0.003) or diauxic shift (p = 0.05) according to the Students t-test.