酵母PMT1基因参与内质网应激的调控机制

【目的】内质网应激(Endoplasmic reticulum stress,ERS)可激活细胞保护性信号级联反应——未折叠蛋白质反应(Unfolded protein response,UPR)。研究表明,酵母细胞中的UPR信号通路由转录因子Hac1p和ERS感应因子Ire1p共同介导。前期研究发现:蛋白质-O-甘露糖转移酶1(Protein-O-mannosyltransferase 1,PMT1)基因缺失能延长酵母细胞的复制性寿命,其机制与上调UPR通路活性相关。本文进一步探讨PMT1基因缺失在酵母ERS反应中的作用。【方法】观察PMT1基因与IRE1或HAC1基因双缺失酵母菌株(pmt1?hac1?和pmt1?ire1?)在ERS反应条件下的克隆形成能力;通过比色法检测各菌株的细胞增殖活性;RT-PCR检测各菌株UPR通路下游部分靶基因的转录水平。【结果】与对照菌株比较,PMT1基因缺失菌株(pmt1?)在ERS反应条件下生长较慢,而HAC1和IRE1单基因缺失菌株(hac1?和ire1?)在ERS反应条件下无法存活;在hac1?或ire1?菌株的基础上进一步缺失PMT1基因,可以改善hac1?菌株在ERS反应条件下的生长状态;但缺失PMT1基因没有上调hac1?菌株UPR通路靶基因的转录水平。【结论】缺失PMT1基因可增强hac1?菌株对ERS诱导剂衣霉素的抗性,机制与已知的UPR通路不相关,提示可能存在其它途径参与ERS反应的调控。     

Characterization of two homologs of Ire1p,a kinase/endoribonuclease in yeast,in Arabidopsis thaliana

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) elicits an ER-to-nucleus signaling pathway known as the unfolded protein response (UPR) in eukaryotes. In yeast, Ire1p, a kinase/endoribonuclease in the ER membrane, plays a key role in the UPR signaling. We isolated two cDNA homologs of IRE1 gene from Arabidopsis (AtIre1a, AtIre1b). The two IRE1 homologs were predicted to form a type I transmembrane protein structure and contain kinase/endoribonuclease domains at their C-terminal halves. The expressions of the two genes were detected in various organ tissues of the Arabidopsis plant. The C-terminal half of the AtIre1a protein showed in vitro autophosphorylation activity. However, we could not detect endoribonuclease activity of the AtIre1a protein when we used yeast HAC1 RNA as the substrate in vivo.     

Attenuation of yeast UPR is essential for survival and is mediated by IRE1 kinase

The unfolded protein response (UPR) activates Ire1, an endoplasmic reticulum (ER) resident transmembrane kinase and ribonuclease (RNase), in response to ER stress. We used an in vivo assay, in which disappearance of the UPR-induced spliced HAC1 messenger ribonucleic acid (mRNA) correlates with the recovery of the ER protein-folding capacity, to investigate the attenuation of the UPR in yeast. We find that, once activated, spliced HAC1 mRNA is sustained in cells expressing Ire1 carrying phosphomimetic mutations within the kinase activation loop, suggesting that dephosphorylation of Ire1 is an important step in RNase deactivation. Additionally, spliced HAC1 mRNA is also sustained after UPR induction in cells expressing Ire1 with mutations in the conserved DFG kinase motif (D828A) or a conserved residue (F842) within the activation loop. The importance of proper Ire1 RNase attenuation is demonstrated by the inability of cells expressing Ire1-D828A to grow under ER stress. We propose that the activity of the Ire1 kinase domain plays a role in attenuating its RNase activity when ER function is recovered.     

Essential role of calcineurin in response to endoplasmic reticulum stress

Depletion of calcium ions (Ca2+) from the endoplasmic reticulum (ER) of yeast cells resulted in the activation of the unfolded protein response (UPR) signaling pathway involving Ire1p and Hac1p. The depleted ER also stimulated Ca2+ influx at the plasma membrane through the Cch1p-Mid1p Ca2+ channel and another system. Surprisingly, both Ca2+ influx systems were stimulated upon accumulation of misfolded proteins in the ER even in the presence of Ca2+. The ability of misfolded ER proteins to stimulate Ca2+ influx at the plasma membrane did not require Ire1p or Hac1p, and Ca2+ influx and signaling factors were not required for initial UPR signaling. However, activation of the Ca2+ channel, calmodulin, calcineurin and other factors was necessary for long-term survival of cells undergoing ER stress. A similar calcium cell survival (CCS) pathway operates in the pathogenic fungi and promotes resistance to azole antifungal drugs. These findings reveal an unanticipated new regulatory mechanism that couples ER stress to Ca2+ influx and signaling pathways, which help to prevent cell death and promote resistance to an important class of fungistatic drugs.     

Cross‐compartment proteostasis regulation during redox imbalance induced ER stress

Imbalance in protein homeostasis in specific subcellular organelles is alleviated through organelle‐specific stress response pathways. As a canonical example of stress activated pathway, accumulation of misfolded proteins in ER activates unfolded protein response (UPR) in almost all eukaryotic organisms. However, very little is known about the involvement of proteins of other organelles that help to maintain the cellular protein homeostasis during ER stress. In this study, using iTRAQ‐based LC–MS approach, we identified organelle enriched proteins that are differentially expressed in yeast (Saccharomyces cerevisiae) during ER stress in the absence of UPR sensor Ire1p. We have identified about 750 proteins from enriched organelle fraction in three independent iTRAQ experiments. Induction of ER stress resulted in the differential expression of 93 proteins in WT strains, 40 of which were found to be dependent on IRE1. Our study reveals a cross‐talk between ER‐ and mitochondrial proteostasis exemplified by an Ire1p‐dependent induction of Hsp60p, a mitochondrial chaperone. Thus, in this study, we show changes in protein levels in various organelles in response to ER stress. A large fraction of these changes were dependent on canonical UPR signalling through Ire1, highlighting the importance of interorganellar cross‐talk during stress.