Download Transcriptional Induction of Genes Encoding ER Resident Proteins

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
What do we know about the Functional Significance of
Ire1 today?
Cytoplasmic part of IRE1 cleaved in
response to UP
ERSE element
BiP gene
Ire1
ER
Nucleus
Unfolded Proteins
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
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
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.
Characterization of JC103 by S1 Nuclease
Protection Assay- (Fig. 1)
•Talk about the assay
•JC103 shows induction of KAR2 in response to
Tunicamycin
Isolation of Mutants defective in UPR
JC103 cells were genetically screened for 3
criteria:
1. Tunicamycin sensitivity
2. Induction of Lac Z from a second regulated
promoter
3. Induction of transcription of KAR2 gene
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.
Cloning IRE1 (Fig. 3)
Use figure to explain
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.
ire1 cells are Supersensitive to Tunicamycin and Mercaptoethanol. (Fig. 5)
Ire1 knockouts show greater sensitivity to Tunicamycin and
to mercaptoethanol.
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