JS Cox, CE Shamu & P Walter. Cell . 1993 Presented by: Ashim Malhotra February 2, 2004

1. Transcriptional Induction of Genes Encoding ER Resident Proteins Requires a Transmembrane Protein Kinase JS Cox, CE Shamu & P Walter. Cell. 1993 Presented by: Ashim Malhotra February 2, 2004

2. What do we know about the Functional Significance of Ire1 today? ERSE element Cytoplasmic part of IRE1 cleaved in response to UP BiP gene Ire1 Nucleus ER Unfolded Proteins

3. Scheme Followed Construction of a lacZ reporter gene that is activated by the accumulation of UPR in ER lumen- JC103 Characterization of reporter strain JC103- Fig. 1 Induce mutation using EMS Isolate mutant phenotypes – Replica Plating Eliminate mutants that are not defective for KAR2 induction using genetic screens- CS171. Characterization of CS171-Fig. 2

4. Scheme Followed Identifying the possible gene that may be mutated – Cloning of IRE1-Fig. 3. Complementation of mutant phenotypes high copy plasmid-pJC012. Constructing low copy number plasmid-pCS110. Disrupting the chromosomal copy of the gene in the parent strain-JC103-construction of CS165. Confirming IRE1 deficiency in auxotrophic strains- Fig. 4 Functional Characterization of IRE1 in yeast using Viability Assays-Fig. 5

5. Construction of Lac Z Reporter Gene Activated by UPR • UPRE in KAR2 promoter can function as UAS when fused to a heterologous promoter. • UPRE from KAR2 was inserted upstream of a crippled CYC1 promoter that is transcriptionally silent in the absence of UAS. • Single copies of reporter construct were integrated at two different locations to create JC103 strain. • JC103 colonies turn blue when transferred to X-Gal-Tunicamycin indicator plates but are white in the absence of Tunicamycin.

6. Characterization of JC103 by S1 Nuclease Protection Assay- (Fig. 1) • Talk about the assay • JC103 shows induction of KAR2 in response to Tunicamycin

7. Isolation of Mutants defective in UPR • JC103 cells were genetically screened for 3 criteria: • Tunicamycin sensitivity • Induction of Lac Z from a second regulated promoter • Induction of transcription of KAR2 gene

8. Characterization of CS171 (Fig. 2) • S1 nuclease protection assay was used to characterize CS171. • CS171 showed lower induction of KAR2 than JC103. • Test cross between CS171 and JC104 shows restoration of KAR2 levels. • The trait is recessive and tetrad analysis shows 2:2 segregation ratio, implying mutation in a single gene. • Same goes for PDI1.

9. Cloning IRE1 (Fig. 3) Use figure to explain

10. Inositol Auxotrophy of ire-1 and ire1 Cells (Fig. 4) • Indicated yeast strains were strained fro single colonies on media containing either 100g/ml inositol or no inositol. • CS165 and CS171 show reduced growth, corresponding to mutations in IRE1.

11. ire1 cells are Supersensitive to Tunicamycin and -Mercaptoethanol. (Fig. 5) Ire1 knockouts show greater sensitivity to Tunicamycin and to mercaptoethanol.

12. Conclusions • Reduction in transcription of KAR2 in response to Tuincamycin treatment in mutated yeast strains occurs due to mutations in the IRE1 gene. • Ire1 is required for the proper functioning of the unfolded protein stress response pathway in yeast cells • Ire1 is probably a transmembrane protein kinase with a cytosolic domain and two possible mechanisms by which ER protein accumulation can be sensed