Down-regulation of Ace2 by Amn1.

(a) RNA and Protein levels of ACE2 in YL1C and Δamn1 mutant. (b) Budding index of YL1C and YL1C-368V cells. (c) Protein levels of Ace2 and Amn1 in synchronized YL1C and YL1C-368V strains. Immunoblotting was performed every 10 minutes after cells were released from G2/M arrest. The bands of Ace2/Amn1/Histone H3/Cln2 were quantified using Quantity One (BioRAD) and the data was shown after normalization. Cln2 and H3 were used as a cell cycle marker and loading control, respectively. (d) The protein levels of Amn1368D (or Amn1368V) and Ace2 in YL1C background strains. AMN1 was driven by PGAL10, and the cells were sampled at 0, 2, 4 and 6 hours after adding galactose. (e) Nuclear accumulation of Ace2-GFP in YL1C and the Δamn1 mutant. Ace2-GFP is shown in the up panel, nuclear DNA stained with DAPI (4',6-diamidino-2-phenylindole) in the middle panel, and the corresponding DIC views in the lower panel. Cells for the assay were collected and assessed at 50-, 70- and 90- minute time points after α-factor release.