A 11-residue domain of Ace2 as the binding target for Amn1 to mediate Ace2 proteolysis.

(a) RNA (by RT-qPCR assay) and protein (by western blotting assay) levels of SWI5 in YL1C and YL1C Δamn1 strains. (b) the diagram of Ace2*(where the 11-residue domain of Ace2 was replaced by the corresponding aligned region in Swi5). (c) Protein levels of Ace2 or Ace2* under YL1C background with an endogenous promoter of ACE2. (d) Microscope images of cell clumping phenotype of YL1C and Ace2* mutant. The corresponding RNA levels of DSE1, DSE2, SCW11 and CTS1 were presented above the images. (e) In YL1C/Ace2* mutant strains, Ace2 and Ace2* protein complexes were respectively captured by anti-Myc and detected by western blotting with anti-Myc for Ace2 and anti-Flag for Amn1. Ace2 and Amn1 were tagged with Myc and Flag, respectively. Untagged Ace2 was used as the IP control.