Abstract
The yeast plasma membrane protein Agp2 belongs to the family of amino acid transporters. It acts as a regulator that controls the expression of several uptake transporter genes such as DUR3 and SAM3 encoding two high-affinity polyamine permeases. agp2Δ mutants display extreme resistance to several cationic compounds including polyamines, the anticancer agent bleomycin, and cationic antifungal peptides. We propose that Agp2 might be involved in regulating the uptake of other cationic anticancer drugs. To date, an uptake transporter has not been reported for anthracyclines, a family of chemotherapeutic agents that are used for treating adult patients with acute myeloid leukemia. Herein, we develop assay conditions to monitor the uptake of the anthracycline doxorubicin into yeast cells and demonstrate for the first time that Agp2 is required for the drug uptake. Deletion of both the DUR3 and SAM3 genes reduced doxorubicin uptake, but not the deletion of either gene alone, while the agp2Δ mutant was severely compromised, suggesting that Agp2 controls the drug uptake via Dur3 and Sam3 and at least one additional transporter. Overexpression of DUR3 or SAM3 from the endogenous promoter rescued doxorubicin uptake into the sam3Δdur3Δ double mutant, consistent with a role for these transporters in the uptake of anthracyclines. We further show by cross-species complementation analysis that expression of the Caenorhabditis elegans oct-1 gene encoding an organic cation transporter restored full doxorubicin uptake in the agp2Δ mutant. Four separate variants of CeOCT-1 derived by substituting the amino acid residues Gln15, Cys31, Gln109 and Lys300 with alanine were stably expressed, but did not mediate doxorubicin uptake into the agp2Δ mutant. Moreover, we show that overexpression of CeOCT-1 sensitized parent yeast cells to doxorubicin, suggesting that CeOCT-1 related members might be key transporters to facilitate entry of anthracyclines into human cells.
Highlights
The yeast Saccharomyces cerevisiae plasma membrane protein, Agp2, was initially identified as a transporter for L-carnitine, which serves as a carrier for acetyl-CoA from the peroxisomes to the mitochondria [1]
These include (i) L-carnitine, even at high concentrations, did not block the uptake of labeled spermidine into the cells or protected parent cells from the cytotoxic effects of polyamines, and (ii) a study by Uemura et al documented the existence of two high affinity polyamine transporters, Dur3 and Sam3, which exist on the plasma membrane of yeast cells, the link to Agp2 was not investigated [4, 5]
To confirm that C. elegans OCT-1 (CeOCT-1) is responsible for DOX uptake in the agp2Δ mutant, we examined the effect of four separate amino acid substitutions Q15A, C31A, Q109A and K300A within the transporter
Summary
The yeast Saccharomyces cerevisiae plasma membrane protein, Agp, was initially identified as a transporter for L-carnitine, which serves as a carrier for acetyl-CoA from the peroxisomes to the mitochondria [1]. We have shown that agp2Δ mutants are strikingly resistant to polyamines and completely blocked for the uptake of very low concentrations of spermine and spermidine [3] While these earlier studies strongly suggest that Agp might function as a high affinity transporter for L-carnitine, bleomycin and polyamines, subsequent findings dismissed this notion. These include (i) L-carnitine, even at high concentrations, did not block the uptake of labeled spermidine into the cells or protected parent cells from the cytotoxic effects of polyamines, and (ii) a study by Uemura et al documented the existence of two high affinity polyamine transporters, Dur and Sam, which exist on the plasma membrane of yeast cells, the link to Agp was not investigated [4, 5]. Consistent with this notion, an independent study revealed that Agp is involved in the uptake of the antifungal drug NaD1, but it is not known which of the transporters regulated by Agp is involved in NaD1 uptake [6]
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