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. 2013 Jun 3;8(6):e65717.
doi: 10.1371/journal.pone.0065717. Print 2013.

Agp2, a member of the yeast amino acid permease family, positively regulates polyamine transport at the transcriptional level

Mustapha Aouida  1 , Marta Rubio-Texeira, Johan M Thevelein, Richard Poulin, Dindial Ramotar
Affiliations

Agp2, a member of the yeast amino acid permease family, positively regulates polyamine transport at the transcriptional level

Mustapha Aouida et al. PLoS One. .

Erratum in

  • PLoS One. 2013;8(7). doi:10.1371/annotation/ff0ad3b6-fff4-4783-8c2e-968a95283ab8. Rubio Texeira, Marta [corrected to Rubio-Texeira, Marta]

Abstract

Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3Δ nor sam3Δ single mutant is defective in polyamine transport, while the dur3Δ sam3Δ double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2Δ mutant. Studies of Agp2 localization indicate that in the double mutant dur3Δ sam3Δ, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cellular responses of the parent and the isogenic sam3Δ and dur3Δ single and double mutants towards polyamine.
A and C) spot test analysis. Exponentially growing cells of OD 600 ∼0.6 were serially diluted as indicated and 5 µl spotted onto YPD agar containing various concentrations of spermine (SPM), spermidine (SPD), bleomycin-A5 (BLM), and the control drug 4-nitroquinoline-1-oxide (4-NQO). Plates were photographed after 48 h of incubation at 30°C. B and E) [14C]-spermidine uptake. Yeast strains were incubated with 10 µM of labelled [14C]-spermidine and samples withdrawn to monitor the uptake level. The data is the result of three independent analyses. D) dur3Δ sam3Δ double mutant is less tolerant to SPM as compared to the agp2Δ mutant in liquid media. Overnight cultures were diluted at a low density of OD600 ∼0.15 into fresh YDP media containing 1 mM SPM and the OD600 monitored over the indicated time, and F) assessment of AGP2 expression by RT-PCR analysis. Total RNA (5 µg) from the indicated exponentially growing cells was used for the RT-PCR reaction. The ACT1 gene was used as a control. Results are representative of three independent experiments.
Figure 2
Figure 2. Agp2 is a ubiquitous protein localized at plasma membrane and internal compartments.
A) Agp2-GFP localization in early exponential cells growing in selective medium and co-expressing ER marker Sec66-mRFP (above) or endosome marker DsRed-FYVE (below). DAPI staining is shown to localize nuclei in the same cells. B) Agp2-GFP is shown in agp2Δ or agp2Δ dur3Δ sam3Δ growing in the same conditions as in A), before, and 4 h after addition of spermidine 5 mM. C) Localization of Agp2-GFP in agp2Δ, agp2Δ dur3Δ sam3Δ, or agp2Δ end3Δ cells 24 h-starved for nitrogen.
Figure 3
Figure 3. agp2Δ mutant downregulates DUR3, SAM3 and HNM1 expression and Hnm1 is required for L-carnitine uptake.
A) RT-PCR analysis of the indicated transporter genes. Total RNA (2 µg) derived from the parent and agp2Δ mutant was analyzed for expression of the polyamine transporter genes DUR3, SAM3, as well as the choline transporter HNM1 (see Materials and Methods). ACT1 served as the control gene. The RT-PCR reaction was performed for 25 cycles and the expected size for each fragment is indicated by an arrow. B) agp2Δ and hnm1Δ single mutants are completely and partially defective, respectively, in L-carnitine uptake. Yeast strains were incubated with 10 µM of labelled [14C]-L-carnitine and samples withdrawn at the indicated time to monitor the uptake level. The data is the result of two independent analyses.
Figure 4
Figure 4. Simultaneous analysis of gene expression in the parent and agp2Δ mutant.
GeXP multiplex expression analysis of 24 genes was performed from exponentially growing cultures under normal conditions. The data is derived from two independent experiments and expressed relative to ACT1 and the internal PCR control KanR.
Figure 5
Figure 5. Validation of key Agp2 targeted and non-targeted genes by RT-PCR analysis.
Total RNA (2 µg) derived from the parent and agp2Δ mutant was analyzed for gene expression as described for Fig. 3. A) expression analysis of Agp2-targeted genes. B) expression analysis of Agp2-non-targeted genes. C) The plasmid pAGP2 rescues expression of Agp2-targeted genes in the agp2Δ mutant. Total RNA (2 µg) derived from the parent, agp2Δ and the agp2Δ carrying the plasmid pAGP2-HIS was analyzed for gene expression as in panel A. ACT1 served as the control gene. The size of the expected fragments are indicated by arrows.
Figure 6
Figure 6. DUR3 and SAM3 expression are not affected in the sky1Δ mutant or vice versa.
Total RNA (2 µg) derived from the indicated strains was analyzed for expression of DUR3 and SAM3 (panel A) and SKY1 (panel B). The RT-PCR conditions were the same as in Fig. 5, but the FMS1 gene was used as a control.
Figure 7
Figure 7. A model illustrating Agp2 function in regulating polyamine uptake.
Agp2 could act to sense several nutrients such as polyamines and maintain the transcriptional expression of many genes including the regulatory kinase Sky1 and the two high affinity polyamine transporters Dur3 and Sam3. Similar to Agp2, Sky1 controls both high and low affinity polyamine transport. Since Sky1 does not affect DUR3 or SAM3 expression, we propose that it may activate these transporters by post-translational modification, e.g., by phosphorylation. Furthermore, Sky1 has been shown to negatively regulate the Trk1 and 2 potassium transporters, perhaps via activation of the Ppz1 and 2 phosphatases, leading to polyamine uptake. ppz1Δ ppz2Δ mutants activate Trk1, 2 potassium uptake and contribute to polyamine resistance. The transcription factors (TFs) responsible for conveying Agp2 sensory function and culminating in gene activation remain to be identified.
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