Human PERK rescues unfolded protein response-deficient yeast cells

Abstract

Protein folding and quality control is tightly regulated at the endoplasmic reticulum (ER), and its disruption is associated with many diseases. In eukaryotes, the accumulation of unfolded protein in the ER is sensed by the three sensors, IRE1, PERK, and ATF6 to activate the unfolded protein response (UPR) to restore ER homeostasis. However, uncoupling the sensing of each sensor and their respective downstream pathways has been challenging as the absence of one is compensated by the remaining two sensors. Here, we report a fully functional human PERK (hPERK) chimeric protein expressed in Saccharomyces cerevisiae that could be used for high throughput screen to identify new PERK inhibitory or activating compounds as well as to characterize the PERK stress sensing mechanisms.

Figure 1. Human PERK chimeric protein fully rescues yeast ire1 Δ growth defect upon ER stress

A , Strains were grown at 30ºC, and serial dilutions of the culture were spotted onto plates with or without tunicamycin (Tm). B , Immunoblot of phosphorylated Sui2 (Sui2-p) from ire1 Δ mutant cells expressing hPERK incubated 1h with Tm. C , Immunoblot of Sui2-p from hPERK-expressing yeast cells were incubated with Tm 1h followed by washout. D, Immunoblot of GFP levels driven by yeast promoter GCN4 in yeast cells expressing hPERK treated with Tm, when indicated. E , Microsomes were isolated from the indicated cells. A portion was kept as the total fraction (T), and the remaining was subjected to centrifugation at 30,000 x g . Supernatant (S) and membrane pellet (P) fractions were collected and analyzed by immunoblotting. F , Strains were grown at 30ºC, and serial dilutions of the culture were spotted onto plates with or without Tm. C, cytosolic domain, LD, luminal domain, TM, transmembrane domain.