The Yeast Permease Agp2 Senses Cycloheximide and Undergoes Degradation That Requires the Small Protein Brp1-Cellular Fate of Agp2 in Response to Cycloheximide

Abstract

The Saccharomyces cerevisiae Agp2 is a plasma membrane protein initially reported to be an uptake transporter for L-carnitine. Agp2 was later rediscovered, together with three additional proteins, Sky1, Ptk2, and Brp1, to be involved in the uptake of the polyamine analogue bleomycin-A5, an anticancer drug. Mutants lacking either Agp2, Sky1, Ptk2, or Brp1 are extremely resistant to polyamines and bleomycin-A5, suggesting that these four proteins act in the same transport pathway. We previously demonstrated that pretreating cells with the protein synthesis inhibitor cycloheximide (CHX) blocked the uptake of fluorescently labelled bleomycin (F-BLM), raising the possibility that CHX could either compete for F-BLM uptake or alter the transport function of Agp2. Herein, we showed that the agp2Δ mutant displayed striking resistance to CHX as compared to the parent, suggesting that Agp2 is required to mediate the physiological effect of CHX. We examined the fate of Agp2 as a GFP tag protein in response to CHX and observed that the drug triggered the disappearance of Agp2 in a concentration- and time-dependent manner. Immunoprecipitation analysis revealed that Agp2-GFP exists in higher molecular weight forms that were ubiquitinylated, which rapidly disappeared within 10 min of treatment with CHX. CHX did not trigger any significant loss of Agp2-GFP in the absence of the Brp1 protein; however, the role of Brp1 in this process remains elusive. We propose that Agp2 is degraded upon sensing CHX to downregulate further uptake of the drug and discuss the potential function of Brp1 in the degradation process.

Keywords: a small protein; cycloheximide; drugs; protein degradation; protein synthesis inhibitor; sensor; ubiquitinylation; uptake transporter; yeast.

Figure 1

Protter representation of Agp2 transmembrane topology comprising 12 transmembrane helices. The ubiquitylation sites (Lysine 20, 22, 24, and 595) and the phosphorylation site (Serine 45) are indicated as purple square and orange diamond, respectively.

Figure 2

I-TASSER predicted model of Agp2 from different views. Extracellular is the top view from the outside of the lipid bilayer. The side view is from the front looking across the lipid bilayer, N and C denote N- and C-termini, respectively. The intracellular view is from the inside of the cell.

Figure 3

Docked complex structure representing Agp2 bound with CHX. (a,b) show the ribbon and surface representation of Agp2 in yellow and CHX in blue. (c) Ligplot interaction map of Agp2-CHX complex.

Figure 4

Survival analysis of the indicated strains showing that the agp2Δ mutant is resistant to the toxic effects of CHX. (A) Spot test analysis. Briefly, overnight cultures were serially diluted and spotted onto solid YPD plates without and with CHX. Plates were photographed after 72 h of incubation at 30 °C. (B–D) Overnight cultures were adjusted to OD_600nm of ~0.2 in fresh YPD liquid media without and with CHX and the growth of the cells was monitored automatically using a plate reader equipped with an orbital shaker. Panel B shows growth of the cells untreated. Panel C and D show growth of the cells in the presence of CHX, 0.1 and 0.14 µg/mL, respectively.

Figure 5

Immunoblot showing Agp2-GFP is disappearing upon exposure to CHX. (A) Immunoblot analysis of cells treated with increasing concentrations of CHX. Exponentially growing cells from the wild type (WT) (strain BY4741) expressing Agp2-GFP were treated with 0 to 10 μM CHX for 30 min, followed by crude plasma membrane preparation (see “Section 4”) and analysis by immunoblot probed with anti-GFP antibodies. Ponceau staining was performed to monitor for protein loading. (B) Quantification of the disappearance of Agp2-GFP relative to the untreated sample (lane 1). (C) Immunoblot analysis of cells treated with a fixed concentration of CHX at different times. The samples were processed as in panel (A), showing Ponceau staining below and (D) quantification of Agp2 levels. M, prestained protein markers in kDa. Results are representative of three independent experiments. Error bars indicate S.E. The data are representative of three biological replicates and analyzed by 2-way Anova Test. ** Is equivalent to p-value < 0.01, *** Is equivalent to p-value < 0.001, **** Is equivalent to p-value < 0.0001.

Figure 6

Comparison of Agp2-GFP levels between the WT (BY4741) and the brp1Δ and ptk2Δ mutants upon treatment with the indicated doses of CHX. (A) The crude plasma membrane fraction was prepared as in Figure 5A following treatment with 10 μM CHX for 0, 15 and 30 min and analyzed by immunoblot. Ponceau staining of the immunoblot to monitor for protein loading. (B) Quantification of the relative level of Agp2-GFP under the various treatment conditions. M, prestained protein markers in kDa. Error bars indicate S.E. The data are representative of three biological replicates and analyzed by 2-way Anova Test. **** Is equivalent to p-value < 0.0001.

Figure 7

Analysis of immunoprecipitated Agp2-GFP from exponentially growing cultures of the WT and the brp1Δ mutant. (A) Immunoprecipitation of Agp2-GFP showing its disappearance in the WT and not in the brp1Δ mutant following CHX treatment. Briefly, total extracts were prepared from exponentially growing cells treated with CHX, subjected to pull-down assay using anti-GFP magnetic beads and probed by immunoblot with anti-GFP antibodies. (B) The same amount of the pull-down samples from panel (A) were analyzed by immunoblot probed with anti-ubiquitin antibodies. (C) Quantification of the relative level of the immunoprecipitated Agp2-GFP under the indicated conditions. M, prestained protein markers in kDa. Error bars indicate S.E. The data are representative of three biological replicates and analyzed by 2-way Anova Test. **** Is equivalent to p-value < 0.0001.

Figure 8

Analysis of immunoprecipitated Agp2-GFP from saturated cultures of the WT and the brp1Δ mutant. (A) Immunoprecipitation of Agp2-GFP showing its disappearance in the WT and not in the brp1Δ mutant following CHX treatment. Briefly, total extracts were prepared from saturated cultures treated with CHX, subjected to pull-down assay using anti-GFP magnetic beads and probed by immunoblot with anti-GFP antibodies. (B) Total cell extracts were used to monitor the input level of Agp2-GFP. (C) The same amount of the pull-down samples from panel (A) were analyzed by immunoblot probed with anti-ubiquitin antibodies. (D,E) Quantification of the relative level of the immunoprecipitated Agp2-GFP under the indicated conditions from panels (A,C), respectively. M, prestained protein markers in kDa. Error bars indicate S.E. The data are representative of three biological replicates and analyzed by 2-way Anova Test. *** Is equivalent to p-value < 0.001, **** Is equivalent to p-value < 0.0001.

Figure 9

Re-introduction of the BRP1 gene into the brp1Δ mutant reinstates the disappearance of Agp2-GFP in response to CHX. (A) Exponentially growing cells of the brp1Δ mutant expressing AGP2-GFP and carrying either the empty vector or the BRP1-MYC plasmid were treated with 10 μM CHX followed by plasma membrane extraction and immunoblot analysis. Ponceau staining was performed to monitor for protein loading. (B) Quantification of the relative level of Agp2-GFP in the brp1Δ mutant carrying either the empty vector or the BRP1-MYC plasmid under the indicated treatment conditions. M, prestained protein markers in kDa. Error bars indicate S.E. The data are representative of three biological replicates and analyzed by 2-way Anova Test. ** Is equivalent to p-value < 0.01, *** Is equivalent to p-value < 0.001.

Figure 10

Confocal microscopy showing the distribution of Agp2-GFP in the WT and brp1Δ mutant in response to CHX. Briefly, exponentially growing cells were treated with CHX, washed, fixed, and imaged using confocal microscopy. (A,B,D–F) Cells were imaged at 100× magnification and panel (C) at 60× magnification. (G) Quantification of Agp2-GFP in the indicated strains following CHX treatment. Error bars indicate S.E. The data are representative of three biological replicates and analyzed by 2-way Anova Test. ** Is equivalent to p-value < 0.01, **** Is equivalent to p-value < 0.0001.