The unfolded protein response: the dawn of a new field

Abstract

Originating from cancer research in mammalian cultured cells, the entirely new field of the unfolded protein response (UPR) was born in 1988. The UPR is a transcriptional induction program coupled with intracellular signaling from the endoplasmic reticulum (ER) to the nucleus to maintain the homeostasis of the ER, an organelle which controls the quality of proteins destined for the secretory pathway. Extremely competitive analyses using the budding yeast Saccharomyces cerevisiae revealed that although signaling from both the ER and cell surface is initiated by activation of a transmembrane protein kinase, the mechanism downstream of ER-resident Ire1p, a sensor molecule of the UPR, is unique. Thus, unconventional spliceosome-independent mRNA splicing is utilized to produce the highly active transcription factor Hac1p. This is the autobiographical story of how a young and not yet independent scientist competed with a very famous full professor in the early days of UPR research, which ultimately lead to their sharing Lasker Basic Medical Research Award in 2014.

Figure 1.

Schematic view of the UPR. The ER is an organelle in which newly synthesized secretory and transmembrane proteins are folded with the assistance of ER chaperones, and which controls the quality of these proteins. Accumulation of unfolded proteins in the ER results in enhanced transcription of the KAR2 gene encoding yeast BiP via the cis-acting UPRE in the nucleus. This culminates in the augmentation of productive folding capacity. This homeostatic response, termed the UPR, requires three mechanisms, namely ER stress sensing (1), transcriptional induction (2) and their connection (3).

Figure 2.

Genetic identification of the IRE1 gene. Temperature-sensitive sec53 mutant cells carrying the reporter gene (UPRE-containing KAR2 promoter fused to the β-galactosidase gene in a plasmid vector) were randomly mutagenized and grown on agar plates containing X-Gal at 23 ℃. White colonies turned blue after temperature upshift to 30 ℃ if the UPR was functional, but remained white if the UPR was not functional. Three upr mutant cells were selected from 100,000 independent colonies and recovered from replica plates because ER stress burdened at 30 ℃ was detrimental to cells. The defective gene was identified by complementation with a genomic library (white colonies turned blue again after temperature upshift to 30 ℃) as the IRE1 gene which encodes the ER stress sensor Ire1p. The domain structure predicted that Ire1p is activated by ER stress-induced oligomerization and trans-autophosphorylation, and Peter demonstrated that this is indeed the case.⁴³⁾

Figure 3.

Two-hybrid screening versus one-hybrid screening. In two-hybrid screening, the gene encoding protein X is fused with the gene encoding the GAL4 DNA-binding domain (DBD). The cDNA library contains various genes fused to the gene encoding the GAL4 activation domain (AD). If protein Y expressed from the cDNA library associates with protein X in a yeast, this two-hybrid has all the function of an active transcription factor that enhances transcription of the reporter gene constitutively via binding to its cis-acting element, called the GAL4 binding site. In one-hybrid screening, if my transcription factor is expressed from the cDNA library as a fusion with GAL4 AD, this one hybrid should activate transcription of the reporter gene constitutively via binding to the cis-acting UPRE.

Figure 4.

Ire1p-dependent splicing of HAC1 mRNA. In wild-type (ERN⁺) cells, HAC1 mRNA is constitutively expressed as a 1.4 kb precursor, which includes an intron of 252 nucleotides containing bulky stem-loop structures (SL1–SL4), but Hac1p is not detected. Upon ER stress (TM, +), the intron is spliced out, resulting in the detection of 1.2 kb mature mRNA and Hac1p as well as the induction of KAR2 mRNA: TM (tunicamycin) evokes ER stress by blocking N-glycosylation of newly synthesized proteins in the ER. These changes in HAC1 mRNA size, Hac1p level and KAR2 mRNA level do not occur in cells deficient in Ire1p (ire1Δ). Because the 5′ end of the intron is located within the HAC1 open reading frame, the Hac1p C-terminus is replaced from 10aa to 18aa without affecting the Hac1p N-terminus of 220aa, as a result of mRNA splicing. Thus, Hac1p of 230aa (220aa + 10aa) is translated from HAC1 precursor mRNA (1.4 kb), whereas Hac1p of 238aa (220aa + 18aa) is translated from HAC1 mature mRNA (1.2 kb).

Figure 5.

Unconventional mechanism of HAC1 mRNA splicing. Ire1p activated in response to ER stress directly cleaves 5′ (SL1) and 3′ (SL4) splice sites between the third and fourth nucleotides of the seven-nucleotide loop via the action of the tail domain immediately C-terminal to the protein kinase domain. The two cleaved exons are joined by the action of Rlg1, a tRNA ligase.