FIG 1.

Expression of various kAE1 variants in S. cerevisiae. (A) Schematic overview of full-length and truncated kAE1 fusion proteins constructed in this study. Positions of GGGGS linker (Linker), yeGFP, and epitope tags (V5 or HA) are indicated. In the truncated kAE1 version (kAE1^B3Mem), the first 360 amino acids of the native AE1 sequence are deleted. (B) Expression profiles (left) and quantification (right, n = 2) of different V5-tagged kAE1 variants containing the original human (hkAE1) or yeast codon-optimized (ykAE1) DNA sequence. The ER signal sequence of Kar2p (Kar2^SS) was added to the N terminus of the human kAE1 sequence to improve its ER targeting (Kar2^SS-hkAE1). A protein standard (M) was used as a size determination marker, and phosphoglycerate kinase 1 (Pgk1p) served as a loading control. (C and D) Western blot analysis of yeast cells expressing yeast codon-optimized variants of untagged wild-type kAE1 (kAE1^WT), kAE1 containing an HA tag within the third extracellular loop between Asn⁵⁵⁶ and Val⁵⁵⁷ (kAE1^HA), truncated kAE1 (kAE1^B3Mem) (C), or kAE1 with an N-terminal fusion to yeast enhanced GFP (yeGFP-kAE1) (D). Pgk1p served as a loading control.