The effects of Vps74p on cell wall integrity are not due to the mislocalization of glycosyltransferases.
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(A) BY4741 wild-type, kre2Δ, and vps74Δ mutants were transformed with the empty vector pVT101U (Vec). BY4741 vps74Δ was transformed with HA-tagged Vps74, Vps74p-dN66, Vps74p-dC83, and Vps74p-3pm. Lysates prepared from these cells were subjected to Western blot analyses using an anti-Gas1p antibody. (B) and (C) The localization of Kre2p-GFP in vps74Δ mutant cells. HA-VPS74 and HA-VPS74-dN66, under the control of the ADH promoter in a 2μ plasmid, was transformed into vps74Δ mutant strains expressing Kre2p-GFP from its native locus. Transformants were grown in selection medium and the GFP signals were observed. Expression of Arf1p-mRFP was used as a Golgi marker. FM4-64 tracer dye was used for vacuole localization. (D) BY4741 was transformed with the empty vector pVT101U (Vec), and BY4741 vps74Δ yeast were transformed with pVT101U (Vec) and HA-tagged Vps74p, Vps74p-dN66, Vps74p-dN90, Vps74p-dN122, Vps74p-dC83, and Vps74p-3pm. These cells were cultured to mid-log phase. Subsequently, 10-fold serial dilutions were spotted onto Ura minus plates containing 2% glucose (left panel), YPD plates containing 50 µg/ml Congo red (middle panel), or YPD plates containing 50 µg/ml Congo red and 1.2 M sorbitol (right panel).