A Novel High-Affinity Sucrose Transporter Is Required for Virulence of the Plant Pathogen Ustilago maydis methods
Aim. Evidence-backed execution summary for A Novel High-Affinity Sucrose Transporter Is Required for Virulence of the Plant Pathogen Ustilago maydis methods from A Novel High-Affinity Sucrose Transporter Is Required for Virulence of the Plant Pathogen Ustilago maydis.
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Srt1 Is an Energy-Dependent, Sucrose-Specific Transporter of the Fungal Plasma Membrane
reagent used in the protocol.
- Use
- To functionally characterize Srt1, the gene was expressed in the monosaccharide transport-deficient S. cerevisiae strain EBY.VW4000, and uptake was analyzed with radiolabeled putative substrates ( d -glucose, d -fructose, d -ribose, d -xylose, d -galactose, mannitol, sorbitol, xylitol, myo -inositol). As Srt1...
Srt1 Is an Energy-Dependent, Sucrose-Specific Transporter of the Fungal Plasma Membrane
reagent used in the protocol.
- Use
- (A) Transport is activated in the presence of the metabolizable carbon source glucose (Glc). (B) The pH optimum for sucrose uptake by Srt1 is in the acidic pH range. (C) Sucrose uptake is sensitive to the protonophore CCCP, but not to the SH-group inhibitor PCMBS. w/o, without. (D) The plateau of sucrose accumulatio...
Srt1 Is an Energy-Dependent, Sucrose-Specific Transporter of the Fungal Plasma Membrane
reagent used in the protocol.
- Use
- These activities of plant sucrose transporters can be inhibited very specifically by the SH-group inhibitor p -chloro-mercuribenzene sulfonate (PCMBS) that does not affect plant hexose transporters. In fact, the specificity of this inhibitor is so high that sucrose fluxes and phloem loading can be inhibited by PCMB...
Materials and Methods
reagent used in the protocol.
- Use
- Escherichia coli strain TOP10 (Invitrogen) was used for cloning purposes. For plant infections, U. maydis cells were grown at 28°C in YEPSL. For RNA extraction, U. maydis was grown in glutamine minimal medium, which is based an the minimal medium described by Holliday with 30 mM l -glutamine as nitrogen source...
Materials and Methods
reagent used in the protocol.
- Use
- SG200Δ srt:: srt1 - GFP was generated by fusing the ORF for eGFP to the 3′-end of the srt1 ORF deleting the srt1 stop codon. Primer pairs used to generate the flanks for homologous recombination were 2374_LB1Pf ( 5′-CGG GTC TCC CTT TCC TTC TTT TGC-3′ ) and 2374_LB2Pf ( 5′-GTT GGC CGC GT...
Materials and Methods
reagent used in the protocol.
- Use
- SG200Δ srt - srt1:: ip: The Δ srt1 deletion strain was complemented with the srt1 gene under the control of its native promoter (about 2.5 kb of upstream sequence) three times independently by homologous recombination of pSRT1-GW into the ip-locus. pSRT1-GW constructs were cloned according to the Gatewa...
DNA and RNA Procedures
reagent used in the protocol.
- Use
- Molecular methods followed described protocols. DNA isolation from U. maydis and transformation procedures were performed as described. Homologous integration of constructs was verified by gel blot analyses. Transformation of S. cerevisiae followed the protocol given in. Total RNA from U. maydis cells grown in ax...
Transport Studies with Radiolabeled Substrates
reagent used in the protocol.
- Use
- S. cerevisiae cells were grown to an absorbance at 600 nm (A 600 nm ) of 1.0, harvested, washed twice with water, and resuspended in buffer to an A 600 nm of 10.0. If not otherwise indicated, uptake experiments were performed in 50 mM Na-phosphate buffer (pH 5.0) with an initial substrate concentration of 1 mM 14 C-...
Srt1 Enables U. maydis to Feed on Apoplastic Sucrose without Extracellular Hydrolysis
Under growth chamber conditions, an U. maydis mutant that had its srt1 gene replaced by an srt1 promoter/ AtSUC9 cDNA fusion showed wild-type virulence ( ). With a K M -sucrose of 0.5 mM, AtSUC9 has a lower substrate affinity than Srt1, but still one of the lowest K M -sucrose values determined for plant sucrose tr...
- Use
- Under growth chamber conditions, an U. maydis mutant that had its srt1 gene replaced by an srt1 promoter/ AtSUC9 cDNA fusion showed wild-type virulence ( ). With a K M -sucrose of 0.5 mM, AtSUC9 has a lower substrate affinity than Srt1, but still one of the lowest K M -sucrose values determined for plant sucrose tr...
Materials and Methods
SG200Δ srt - srt1:: ip: The Δ srt1 deletion strain was complemented with the srt1 gene under the control of its native promoter (about 2.5 kb of upstream sequence) three times independently by homologous recombination of pSRT1-GW into the ip-locus. pSRT1-GW constructs were cloned according to the Gatewa...
- Use
- SG200Δ srt - srt1:: ip: The Δ srt1 deletion strain was complemented with the srt1 gene under the control of its native promoter (about 2.5 kb of upstream sequence) three times independently by homologous recombination of pSRT1-GW into the ip-locus. pSRT1-GW constructs were cloned according to the Gatewa...
DNA and RNA Procedures
Molecular methods followed described protocols. DNA isolation from U. maydis and transformation procedures were performed as described. Homologous integration of constructs was verified by gel blot analyses. Transformation of S. cerevisiae followed the protocol given in. Total RNA from U. maydis cells grown in ax...
- Use
- Molecular methods followed described protocols. DNA isolation from U. maydis and transformation procedures were performed as described. Homologous integration of constructs was verified by gel blot analyses. Transformation of S. cerevisiae followed the protocol given in. Total RNA from U. maydis cells grown in ax...
Light and Epifluorescence Microscopy
Light microscopic analyses were performed using a Zeiss Axioplan 2 microscope. Photomicrographs were obtained with an Axiocam HrM camera, and the images were processed with Axiovision (Zeiss) and Photoshop (Adobe). Chlorazole Black E staining of fungal cells in planta was performed as described. GFP signals of Srt1...
- Use
- Light microscopic analyses were performed using a Zeiss Axioplan 2 microscope. Photomicrographs were obtained with an Axiocam HrM camera, and the images were processed with Axiovision (Zeiss) and Photoshop (Adobe). Chlorazole Black E staining of fungal cells in planta was performed as described. GFP signals of Srt1...
Confocal Microscopy
Subcellular localization of the Srt1::GFP fusion protein in S. cerevisiae was determined by confocal microscopy (Leica TCS SPII; Leica Microsystems) and processed with the Leica Confocal Software 2.5 (Leica Microsystems). Emitted fluorescence was monitored at detection wavelengths longer than 510 nm.
- Use
- Subcellular localization of the Srt1::GFP fusion protein in S. cerevisiae was determined by confocal microscopy (Leica TCS SPII; Leica Microsystems) and processed with the Leica Confocal Software 2.5 (Leica Microsystems). Emitted fluorescence was monitored at detection wavelengths longer than 510 nm.
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Srt1 Is an Energy-Dependent, Sucrose-Specific Transporter of the Fungal Plasma Membrane
(A) Uptake of 14 C-sucrose by srt1 -expressing (closed circles) and control cells (open circles). (B) Competition analysis (0.1 mM 14 C-sucrose) with different potential substrates added at 100-fold molar excess. w/o, without. (C) Michaelis-Menten kinetics of sucrose uptake rates (pH 5.0) indicate a K M of 26±4.3 µM (standard error [SE]). Error bars represent SE ( n = 3).
The Arabidopsis Sucrose Transporter AtSUC9 Can Functionally Replace Srt1
To validate that sucrose uptake is the primary function of Srt1 during biotrophic growth, we tested whether another transporter with a well-characterized sucrose uptake activity can functionally replace Srt1. We selected the sucrose transporter AtSUC9 from Arabidopsis thaliana. This plant transporter is plasma membrane localized, transports sucrose and maltose, and is sensitive to CCCP and PCMBS. Moreover, AtSUC9 has a K M -sucrose of 0.5 mM, which is quite low for a plant sucrose transporter but still 20-fold higher than the K M -sucrose of Srt1 ( ). In strain SG200Δ srt1:: AtSUC9, the AtSUC9 cDNA was inserted into the srt1 locus.
Srt1 Enables U. maydis to Feed on Apoplastic Sucrose without Extracellular Hydrolysis
Under growth chamber conditions, an U. maydis mutant that had its srt1 gene replaced by an srt1 promoter/ AtSUC9 cDNA fusion showed wild-type virulence ( ). With a K M -sucrose of 0.5 mM, AtSUC9 has a lower substrate affinity than Srt1, but still one of the lowest K M -sucrose values determined for plant sucrose transporters. In contrast, the K M -sucrose of ZmSUT1, the sucrose transporter responsible for phloem loading in maize and, thus, the competing transporter at the U. maydis /maize interface, varies from 3.7 mM at pH 5.6 to 12.4 mM at pH 6.5. These different K M values may explain the successful replacement of Srt1 by AtSUC9. Nevertheless, it could well be that SG200Δ srt1:: AtSUC9 would show reduced virulence in the field, where growth conditions are more competitive.
Materials and Methods
Escherichia coli strain TOP10 (Invitrogen) was used for cloning purposes. For plant infections, U. maydis cells were grown at 28°C in YEPSL. For RNA extraction, U. maydis was grown in glutamine minimal medium, which is based an the minimal medium described by Holliday with 30 mM l -glutamine as nitrogen source. Plant infections with U. maydis were performed as described. The U. maydis strain used in this study is SG200, a haploid, solopathogenic strain that can infect maize plants without a mating partner. S. cerevisiae strains used for analyses of Srt1 were EBY.VW4000 ( MATa; leu2-3,112; ura3-52; trp1-289; his3-Δ1; MAL2-8c; SUC2; Δhxt1-17; Δgal2; Δstl1; Δagt1; Δmph2; Δmph3), SEY2102 ( MATα; ura3-52; leu2-3,112; his4-519; suc2-Δ9; gal2), D458-1B ( MATα; leu2; itr1; ino1), and DBY2617 ( MATa; his4-539; lys2-801; ura3-52; suc2-43...
Transport Studies with Radiolabeled Substrates
S. cerevisiae cells were grown to an absorbance at 600 nm (A 600 nm ) of 1.0, harvested, washed twice with water, and resuspended in buffer to an A 600 nm of 10.0. If not otherwise indicated, uptake experiments were performed in 50 mM Na-phosphate buffer (pH 5.0) with an initial substrate concentration of 1 mM 14 C-labeled sucrose (or another 14 C-labeled or 3 H-labeled substrate). Cells were shaken in a rotary shaker at 29°C, and transport tests were started by adding labeled substrate. Samples were withdrawn at given intervals, filtered on nitrocellulose filters (0.8-µm pore size), and washed with an excess of distilled H 2 O. Incorporation of radioactivity was determined by scintillation counting. Competition analyses were performed with 0.1 mM 14 C-sucrose in the presence of 10 mM competitor (100-fold excess). For analyses of the energy dependence of sucrose transport, d...
Transport Studies with Radiolabeled Substrates
For influx/efflux analyses in the plateau of sucrose accumulation ( ), identical amounts of S. cerevisiae cells were incubated in two flasks with either 100 µM 14 C-labeled sucrose or with unlabeled sucrose, and sucrose uptake was determined in the flask with the labeled substrate. When the plateau was reached (after 35 min), the cells were quickly pelleted and washed in Na-phosphate buffer (pH. 5.0). Cells from the unlabeled flask were then resuspended to the initial volume with 100 µM 14 C-sucrose, cells from the labeled flask with 100 µM unlabeled sucrose, and uptake experiments were continued.
Quantitative Real-Time PCR Analysis
To analyze srt1 expression on different carbon sources, SG200 was grown in glutamine minimal media supplemented with the indicated amount of the respective carbon source to an optical density at 600 nm (OD 600 ) of 1.0 for 6 h. Precultures were grown overnight in glutamine minimal medium containing 1% of glucose. RNA samples were frozen in liquid nitrogen for two independently conducted replicates.
Supporting Information
14 C-maltose is not a substrate for Srt1. Uptake of 14 C-maltose (closed circles) was determined in parallel with the uptake of 14 C-sucrose (open circles) in the same srt1 -expressing S. cerevisiae cells that had been used to determine transport in. The extracellular pH was 5.0, substrate concentration was 1 mM. Although 14 C-maltose transport was analyzed for much longer than the transport of 14 C-sucrose (see also ), no significant import of 14 C-maltose into srt1 -expressing cells could be observed. Error bars represent standard error ( n = 3).
Measurement outputs
What raw and processed outputs should exist?
(A) Uptake of 14 C-sucrose by srt1 -expressing (closed circles) and control cells (open circles). (B) Competition analysis (0.1 mM 14 C-sucrose) with different potential substra...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Demonstrates that infections with SG200Δ srt1:: AtSUC9 are indistinguishable from wild-type infections with respect to tumor formation and frequency. Thus, the virulence o...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Expression of srt1 in an S. cerevisiae strain (DBY2617) that possesses a cytoplasmic but no secreted invertase enabled this strain not only to import 14 C-sucrose, but also to g...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Additional analyses of the subcellular localization in S. cerevisiae with a functional Srt1::GFP fusion protein demonstrated that, as expected from the transport assays ( and )...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
(A) Uptake of 14 C-sucrose by srt1 -expressing (closed circles) and control cells (open circles).
from paperScoring or quantification
Quantify the primary readouts for this experiment: (A) Uptake of 14 C-sucrose by srt1 -expressing (closed circles) and control cells (open circles). (B) Competition analysis (0.1 mM 14 C-sucrose) with different potential substra...; Demonstrates that infections with SG200Δ srt1:: AtSUC9 are indistinguishable from wild-type infections with respect to tumor formation and frequency. Thus, the virulence o...; Expression of srt1 in an S. cerevisiae strain (DBY2617) that possesses a cytoplasmic but no secreted invertase enabled this strain not only to import 14 C-sucrose, but also to g...; Additional analyses of the subcellular localization in S. cerevisiae with a functional Srt1::GFP fusion protein demonstrated that, as expected from the transport assays ( and )....
from paperNormalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
inferred from protocolStatistical comparison
(A) Uptake of 14 C-sucrose by srt1 -expressing (closed circles) and control cells (open circles). (B) Competition analysis (0.1 mM 14 C-sucrose) with different potential substra...; (A) Transport is activated in the presence of the metabolizable carbon source glucose (Glc). (B) The pH optimum for sucrose uptake by Srt1 is in the acidic pH range. (C) Sucrose...; 14 C-maltose is not a substrate for Srt1. Uptake of 14 C-maltose (closed circles) was determined in parallel with the uptake of 14 C-sucrose (open circles) in the same srt1 -exp...
from paperReporting output
Report representative outputs alongside summary comparisons for (A) Uptake of 14 C-sucrose by srt1 -expressing (closed circles) and control cells (open circles). (B) Competition analysis (0.1 mM 14 C-sucrose) with different potential substra..., Demonstrates that infections with SG200Δ srt1:: AtSUC9 are indistinguishable from wild-type infections with respect to tumor formation and frequency. Thus, the virulence o..., Expression of srt1 in an S. cerevisiae strain (DBY2617) that possesses a cytoplasmic but no secreted invertase enabled this strain not only to import 14 C-sucrose, but also to g..., Additional analyses of the subcellular localization in S. cerevisiae with a functional Srt1::GFP fusion protein demonstrated that, as expected from the transport assays ( and )....
inferred from protocolStructured statistical methods
(A) Uptake of 14 C-sucrose by srt1 -expressing (closed circles) and control cells (open circles). (B) Competition analysis (0.1 mM 14 C-sucrose) with different potential substra...; (A) Transport is activated in the presence of the metabolizable carbon source glucose (Glc). (B) The pH optimum for sucrose uptake by Srt1 is in the acidic pH range. (C) Sucrose...; 14 C-maltose is not a substrate for Srt1. Uptake of 14 C-maltose (closed circles) was determined in parallel with the uptake of 14 C-sucrose (open circles) in the same srt1 -exp...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
(A) Uptake of 14 C-sucrose by srt1 -expressing (closed circles) and control cells (open circles). (B) Competition analysis (0.1 mM 14 C-sucrose) with different potential substrates added at 100-fold molar excess. w/o, without. (C) Michaelis-Menten kinetics of sucrose uptake rates (pH 5.0) indicate a K M of 26±4.3 µM (standard error [SE]). Error bars represent SE ( n = 3).
To validate that sucrose uptake is the primary function of Srt1 during biotrophic growth, we tested whether another transporter with a well-characterized sucrose uptake activity can functionally replace Srt1. We selected the sucrose transporter AtSUC9 from Arabidopsis thaliana. This plant transporter is plasma membrane localized, transports sucrose and maltose, and is sensitive to CCCP and PCMBS. Moreover, AtSUC9 has a K M -sucrose of 0.5 mM, which is quite low for a plant sucrose transporter but still 20-fold higher than the K M -sucrose of Srt1 ( ). In strain SG200Δ srt1:: AtSUC9, the AtSUC9 cDNA was inserted into the srt1 locus.
Under growth chamber conditions, an U. maydis mutant that had its srt1 gene replaced by an srt1 promoter/ AtSUC9 cDNA fusion showed wild-type virulence ( ). With a K M -sucrose of 0.5 mM, AtSUC9 has a lower substrate affinity than Srt1, but still one of the lowest K M -sucrose values determined for plant sucrose transporters. In contrast, the K M -sucrose of ZmSUT1, the sucrose transporter responsible for phloem loading in maize and, thus, the competing transporter at the U. maydis /maize interface, varies from 3.7 mM at pH 5.6 to 12.4 mM at pH 6.5. These different K M values may explain the successful replacement of Srt1 by AtSUC9. Nevertheless, it could well be that SG200Δ srt1:: AtSUC9 would show reduced virulence in the field, where growth conditions are more competitive.
Escherichia coli strain TOP10 (Invitrogen) was used for cloning purposes. For plant infections, U. maydis cells were grown at 28°C in YEPSL. For RNA extraction, U. maydis was grown in glutamine minimal medium, which is based an the minimal medium described by Holliday with 30 mM l -glutamine as nitrogen source. Plant infections with U. maydis were performed as described. The U. maydis strain used in this study is SG200, a haploid, solopathogenic strain that can infect maize plants without a mating partner. S. cerevisiae strains used for analyses of Srt1 were EBY.VW4000 ( MATa; leu2-3,112; ura3-52; trp1-289; his3-Δ1; MAL2-8c; SUC2; Δhxt1-17; Δgal2; Δstl1; Δagt1; Δmph2; Δmph3), SEY2102 ( MATα; ura3-52; leu2-3,112; his4-519; suc2-Δ9; gal2), D458-1B ( MATα; leu2; itr1; ino1), and DBY2617 ( MATa; his4-539; lys2-801; ura3-52; suc2-438). Cells were grown in minimal medium (0.67% yeast nitrogen base without amino acids plus required amino acids depending on the strain) containing 2% maltose (EBY.VW4000) or glucose (all other strains) at 29°C.
S. cerevisiae cells were grown to an absorbance at 600 nm (A 600 nm ) of 1.0, harvested, washed twice with water, and resuspended in buffer to an A 600 nm of 10.0. If not otherwise indicated, uptake experiments were performed in 50 mM Na-phosphate buffer (pH 5.0) with an initial substrate concentration of 1 mM 14 C-labeled sucrose (or another 14 C-labeled or 3 H-labeled substrate). Cells were shaken in a rotary shaker at 29°C, and transport tests were started by adding labeled substrate. Samples were withdrawn at given intervals, filtered on nitrocellulose filters (0.8-µm pore size), and washed with an excess of distilled H 2 O. Incorporation of radioactivity was determined by scintillation counting. Competition analyses were performed with 0.1 mM 14 C-sucrose in the presence of 10 mM competitor (100-fold excess). For analyses of the energy dependence of sucrose transport, d -glucose was added to the yeast cells 2 min before the start of the experiment to a final concentration of 10 mM. For inhibitor analyses, CCCP (carbonylcyanide m -chlorophenylhydrazone) or PCMBS ( p -chloromercuribencene sulfonate) were used at final concentrations of 50 µM.
For influx/efflux analyses in the plateau of sucrose accumulation ( ), identical amounts of S. cerevisiae cells were incubated in two flasks with either 100 µM 14 C-labeled sucrose or with unlabeled sucrose, and sucrose uptake was determined in the flask with the labeled substrate. When the plateau was reached (after 35 min), the cells were quickly pelleted and washed in Na-phosphate buffer (pH. 5.0). Cells from the unlabeled flask were then resuspended to the initial volume with 100 µM 14 C-sucrose, cells from the labeled flask with 100 µM unlabeled sucrose, and uptake experiments were continued.
To analyze srt1 expression on different carbon sources, SG200 was grown in glutamine minimal media supplemented with the indicated amount of the respective carbon source to an optical density at 600 nm (OD 600 ) of 1.0 for 6 h. Precultures were grown overnight in glutamine minimal medium containing 1% of glucose. RNA samples were frozen in liquid nitrogen for two independently conducted replicates.
14 C-maltose is not a substrate for Srt1. Uptake of 14 C-maltose (closed circles) was determined in parallel with the uptake of 14 C-sucrose (open circles) in the same srt1 -expressing S. cerevisiae cells that had been used to determine transport in. The extracellular pH was 5.0, substrate concentration was 1 mM. Although 14 C-maltose transport was analyzed for much longer than the transport of 14 C-sucrose (see also ), no significant import of 14 C-maltose into srt1 -expressing cells could be observed. Error bars represent standard error ( n = 3).
Machine-readable layer
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"name": "The Arabidopsis Sucrose Transporter AtSUC9 Can Functionally Replace Srt1",
"text": "To validate that sucrose uptake is the primary function of Srt1 during biotrophic growth, we tested whether another transporter with a well-characterized sucrose uptake activity can functionally replace Srt1. We selected the sucrose transporter AtSUC9 from Arabidopsis thaliana. This plant transporter is plasma membrane localized, transports sucrose and maltose, and is sensitive to CCCP and PCMBS. Moreover, AtSUC9 has a K M -sucrose of 0.5 mM, which is quite low for a plant sucrose transporter but still 20-fold higher than the K M -sucrose of Srt1 ( ). In strain SG200Δ srt1:: AtSUC9, the AtSUC9 cDNA was inserted into the srt1 locus."
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"text": "Escherichia coli strain TOP10 (Invitrogen) was used for cloning purposes. For plant infections, U. maydis cells were grown at 28°C in YEPSL. For RNA extraction, U. maydis was grown in glutamine minimal medium, which is based an the minimal medium described by Holliday with 30 mM l -glutamine as nitrogen source. Plant infections with U. maydis were performed as described. The U. maydis strain used in this study is SG200, a haploid, solopathogenic strain that can infect maize plants without a mating partner. S. cerevisiae strains used for analyses of Srt1 were EBY.VW4000 ( MATa; leu2-3,112; ura3-52; trp1-289; his3-Δ1; MAL2-8c; SUC2; Δhxt1-17; Δgal2; Δstl1; Δagt1; Δmph2; Δmph3), SEY2102 ( MATα; ura3-52; leu2-3,112; his4-519; suc2-Δ9; gal2), D458-1B ( MATα; leu2; itr1; ino1), and DBY2617 ( MATa; his4-539; lys2-801; ura3-52; suc2-43..."
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"text": "To analyze srt1 expression on different carbon sources, SG200 was grown in glutamine minimal media supplemented with the indicated amount of the respective carbon source to an optical density at 600 nm (OD 600 ) of 1.0 for 6 h. Precultures were grown overnight in glutamine minimal medium containing 1% of glucose. RNA samples were frozen in liquid nitrogen for two independently conducted replicates."
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"text": "14 C-maltose is not a substrate for Srt1. Uptake of 14 C-maltose (closed circles) was determined in parallel with the uptake of 14 C-sucrose (open circles) in the same srt1 -expressing S. cerevisiae cells that had been used to determine transport in. The extracellular pH was 5.0, substrate concentration was 1 mM. Although 14 C-maltose transport was analyzed for much longer than the transport of 14 C-sucrose (see also ), no significant import of 14 C-maltose into srt1 -expressing cells could be observed. Error bars represent standard error ( n = 3)."
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