Generation of the AMD1WT, AMD1TGG, AMD1TAA, and PRp27-AMD1TGG transformants

For complementation assays, the entire AMD1 gene including its promoter region was amplified with primers 094-NF and 094-R (Table ^S3) and co-transformed with XhoI-digested pFL2 (carrying the geneticin resistance marker) into yeast strain XK1–25 by the gap repair approach^{17, 32}. The AMD1 ^WT-GFP fusion construct was rescued from Trp⁺ yeast transformants and confirmed by sequencing analysis. The same yeast gap repair approach was used to generate the AMD1 ^TGG-GFP, P_RP27 -AMD1 ^TGG-GFP, and AMD1 ^TAA-GFP constructs. To introduce the A632G and G633A mutations, AMD1 was amplified with primer pairs 094E-F /094E-R and 094S-F /094S-R (Table ^S3), respectively. All the resulting mutant alleles of AMD1 were verified by sequencing and transformed into the amd1 mutant. Transformants of amd1 expressing the AMD1 ^WT-, AMD1 ^TGG -, P_RP27 -AMD1 ^TGG -, and AMD1 ^TAA -GFP constructs were identified by PCR and examined for GFP signals by epifluorescence microscopy.