Protein folding by the endoplasmic reticulum (ER) is physiologically critical, while its disruption causes ER stress and augments disease. ER stress activates the unfolded protein response (UPR) to restore homeostasis. If stress persists, the UPR induces apoptotic cell death, but the mechanisms remain elusive. Here we find that unmitigated ER stress promotes apoptosis through cell-autonomous, UPR-controlled activation of death receptor 5 (DR5). ER stressors induced DR5 transcription via the UPR mediator CHOP; however, the UPR sensor IRE1α transiently catalyzed DR5 mRNA decay, allowing time for adaptation. Persistent ER stress built up intracellular DR5 protein, driving ligand-independent DR5 activation and apoptosis engagement via caspase-8. Thus, DR5 integrates opposing UPR signals to couple ER stress and apoptotic cell fate.

Glucose-dependent regulation of carbon metabolism is a subject of intensive studies. We have previously shown that the switch from gluconeogenesis to glycolysis is associated with ubiquitin-proteasome linked elimination of the key enzyme fructose-1,6-bisphosphatase. Seven glucose induced degradation deficient (Gid)-proteins found previously in a genomic screen were shown to form a complex that binds FBPase. One of the subunits, Gid2/Rmd5, contains a degenerated RING finger domain. In an in vitro assay, heterologous expression of GST-Gid2 leads to polyubiquitination of proteins. In addition, we show that a mutation in the degenerated RING domain of Gid2/Rmd5 abolishes fructose-1,6-bisphosphatase polyubiquitination and elimination in vivo. Six Gid proteins are present in gluconeogenic cells. A seventh protein, Gid4/Vid24, occurs upon glucose addition to gluconeogenic cells and is afterwards eliminated. Forcing abnormal expression of Gid4/Vid24 in gluconeogenic cells leads to fructose-1,6-bisphosphatase degradation. This suggests that Gid4/Vid24 initiates fructose-1,6-bisphosphatase polyubiquitination by the Gid complex and its subsequent elimination by the proteasome. We also show that an additional gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, is subject to Gid complex-dependent degradation. Our study uncovers a new type of ubiquitin ligase complex composed of novel subunits involved in carbohydrate metabolism and identifies Gid4/Vid24 as a major regulator of this E3.

Yeast is a powerful model for systems genetics. We present a versatile, time- and labor-efficient method to functionally explore the Saccharomyces cerevisiae genome using saturated transposon mutagenesis coupled to high-throughput sequencing. SAturated Transposon Analysis in Yeast (SATAY) allows one-step mapping of all genetic loci in which transposons can insert without disrupting essential functions. SATAY is particularly suited to discover loci important for growth under various conditions. SATAY (1) reveals positive and negative genetic interactions in single and multiple mutant strains, (2) can identify drug targets, (3) detects not only essential genes, but also essential protein domains, (4) generates both null and other informative alleles. In a SATAY screen for rapamycin-resistant mutants, we identify Pib2 (PhosphoInositide-Binding 2) as a master regulator of TORC1. We describe two antagonistic TORC1-activating and -inhibiting activities located on opposite ends of Pib2. Thus, SATAY allows to easily explore the yeast genome at unprecedented resolution and throughput.DOI: http://dx.doi.org/10.7554/eLife.23570.001

The unfolded protein response (UPR) monitors the protein folding capacity of the endoplasmic reticulum (ER). In all organisms analyzed to date, the UPR drives transcriptional programs that allow cells to cope with ER stress. The non-conventional splicing of Hac1 (yeasts) and XBP1 (metazoans) mRNA, encoding orthologous UPR transcription activators, is conserved and dependent on Ire1, an ER membrane-resident kinase/endoribonuclease. We found that the fission yeast Schizosaccharomyces pombe lacks both a Hac1/XBP1 ortholog and a UPR-dependent-transcriptional-program. Instead, Ire1 initiates the selective decay of a subset of ER-localized-mRNAs that is required to survive ER stress. We identified Bip1 mRNA, encoding a major ER-chaperone, as the sole mRNA cleaved upon Ire1 activation that escapes decay. Instead, truncation of its 3′ UTR, including loss of its polyA tail, stabilized Bip1 mRNA, resulting in increased Bip1 translation. Thus, S. pombe uses a universally conserved stress-sensing machinery in novel ways to maintain homeostasis in the ER.DOI: http://dx.doi.org/10.7554/eLife.00048.001