Fig 4. A multicopy plasmid carrying either the entire SAM3 or DUR3 gene rescues DOX uptake in the sam3Δdur3Δ double mutant but not in the agp2Δ single mutant.

(A) RT-PCR analysis showing the expression level of SAM3 and DUR3 in the sam3Δdur3Δ and the agp2Δ mutants with the indicated plasmids. Total RNA (1 μg) was reverse-transcribed and the expression level of either SAM3 or DUR3 was assessed from the resulting cDNA using gene specific primers [5]. (B) DOX uptake is mediated by the Sam3 and Dur3 transporters and depends on Agp2 function. The multicopy plasmid pSAM3 or pDUR3 carrying the entire SAM3 or DUR3 gene, respectively, with the endogenous promoter was introduced into either the sam3Δdur3Δ double mutant or the agp2Δ single mutant and the resulting transformants monitored for DOX uptake in low YNB using FACS analysis. (C) Epifluorescent microscopy showing that either pSAM3 or pDUR3 restores DOX uptake to the sam3Δdur3Δ double mutant, but not to the agp2Δ mutant. Microscopy was conducted as in Fig 3B.