Induction of the Unfolded Protein Response at High Temperature in Saccharomyces cerevisiae

Abstract

Ire1 is an endoplasmic reticulum (ER)-located endoribonuclease that is activated in response to ER stress. In yeast Saccharomyces cerevisiae cells, Ire1 promotes HAC1-mRNA splicing to remove the intron sequence from the HAC1u mRNA (“u” stands for “uninduced”). The resulting mRNA, which is named HAC1i mRNA (“i” stands for “induced”), is then translated into a transcription factor that is involved in the unfolded protein response (UPR). In this study, we designed an oligonucleotide primer that specifically hybridizes to the exon-joint site of the HAC1i cDNA. This primer allowed us to perform real-time reverse transcription-PCR to quantify HAC1i mRNA abundance with high sensitivity. Using this method, we detected a minor induction of HAC1-mRNA splicing in yeast cells cultured at their maximum growth temperature of 39 °C. Based on our analyses of IRE1-gene mutant strains, we propose that when yeast cells are cultured at or near their maximum growth temperature, protein folding in the ER is disturbed, leading to a minor UPR induction that supports cellular growth.

Endoplasmic stress sensor Ire1 is involved in cytosolic/nuclear protein quality control in Pichia pastoris cells independent of HAC1

“…Because, in general, high temperature impairs protein folding, heat stress likely induces both the HSR and UPR. Nevertheless, the UPR is only slightly induced at high temperatures in S. cerevisiae cells ( Hata et al, 2022 ). On the other hand, here we observed strong activation of Ire1 in heat-stressed P. pastoris cells ( Figure 10 ).…”

Induction of Endoplasmic Reticulum Stress by Prodigiosin in Yeast Saccharomyces cerevisiae

“…To check the splicing of HAC1 mRNA, total RNA samples were extracted from yeast cells using the hot phenol method and subjected to reverse transcription using a poly(dT) oligonucleotide primer, as described previously [ 27 ]. The resulting cDNA samples were subjected to real-time polymerase chain reaction (PCR) using the intercalator method with two different HAC1 primer sets [ 27 ].…”

“…Alternatively, the cDNA samples were subjected to PCR using the primer set to amplify the total HAC1 species (5′-TACAGGGATTTCCAGAGCACG-3′ and 5′-TGAAGTGATGAAGAAATCATTCAATTC-3′), as described previously [ 27 ]. The PCR products were separated by electrophoresis on 2% agarose containing 0.0001% ethidium bromide.…”

“…To check the splicing of HAC1 mRNA, total RNA samples were extracted from yeast cells using the hot phenol method and subjected to reverse transcription using a poly(dT) oligonucleotide primer, as described previously [27]. The resulting cDNA samples were subjected to real-time polymerase chain reaction (PCR) using the intercalator method with two different HAC1 primer sets [27]. One was used for amplification of the total HAC1 species (5 ′ -GCGTCGGACCAAGAGACTT-3 ′ and 5 ′ -TCGTCGACTCTGGTACATTTTC-3 ′ ), and the other was used for amplification of the spliced version (5 ′ -ACCTGCCGTAGACAACAACA-3 ′ and 5 ′ -ACCTGACTGCGCTTCTGGAT-3 ′ ).…”

“…One was used for amplification of the total HAC1 species (5 ′ -GCGTCGGACCAAGAGACTT-3 ′ and 5 ′ -TCGTCGACTCTGGTACATTTTC-3 ′ ), and the other was used for amplification of the spliced version (5 ′ -ACCTGCCGTAGACAACAACA-3 ′ and 5 ′ -ACCTGACTGCGCTTCTGGAT-3 ′ ). The HAC1 mRNA-splicing efficiency (%) was calculated using the following formula: 100 × [Abundance of spliced HAC1 mRNA]/[Abundance of total HAC1 mRNA] Alternatively, the cDNA samples were subjected to PCR using the primer set to amplify the total HAC1 species (5 ′ -TACAGGGATTTCCAGAGCACG-3 ′ and 5 ′ -TGAAGTGATGA AGAAATCATTCAATTC-3 ′ ), as described previously [27]. The PCR products were separated by electrophoresis on 2% agarose containing 0.0001% ethidium bromide.…”