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. 2020 Dec 22;220(1):e202001116. doi: 10.1083/jcb.202001116

Figure 2.

Figure 2.

A suppressor screen identified clusters on Ypq1 that are important in its degradation. (A) Design of the suppressor screen using constitutively degrading Ypq1 (*, S14D, L70D, or M73D). Vac, vacuole; Ura, uracil. (B) Summary of the genuine suppressor residues and their corresponding mutations. (C) Subcellular localization of Ypq1M73D-GFP-Ura3 with mutations on 139DEE141. Scale bar, 2 µm. (D) The suppressor mutations also blocked the Lys withdrawal-triggered Ypq1-GFP internalization. Scale bar, 2 µm. (E) Heat map of mutant Ypq1-GFP degradation defect based on flow cytometry. Negative control, Ypq1WT-GFP in ssh4Δ strain, defined as 100%. Positive control, Ypq1WT-GFP in WT strain. (F) CoIP of WT Ypq1-GFP with overexpressed PPxY mutant Ssh4 (bait) in Lys-starved conditions. Pho8, negative control; St, starting material; Ft, flow-through; El, elution; MW, molecular weight marker; * and **, nonspecific bands. (G) CoIP of Ypq1-GFP mutants with overexpressed PPxY mutant Ssh4 in Lys-starved conditions. (H) Critical regions on Ypq1 based on suppressor screen mapped on the conserved architecture of PQ-loop proteins. (I) Critical residues mapped on the 3D model of Ypq1. TM4 shown as a reference point.