Transcriptional Induction of Genes Encoding ER Resident Proteins Requires a Transmembrane Protein Ki

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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

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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
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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
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Scheme Followed
Identifying the possible gene that may be mutated
Cloning of IRE1-Fig. 3. Complementation of
mutant phenotypes high copy plasmid-pJC012. Constr
ucting 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
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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.

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Characterization of JC103 by S1 Nuclease
Protection Assay- (Fig. 1)

• Talk about the assay
• JC103 shows induction of KAR2 in response to
  Tunicamycin

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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

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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
  22 segregation ratio, implying mutation in a
  single gene.
• Same goes for PDI1.

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

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