Eukaryotic cells respond to unfolded proteins in their endoplasmic reticulum (ER stress), amino acid starvation, or oxidants by phosphorylating the alpha subunit of translation initiation factor 2 (eIF2alpha). This adaptation inhibits general protein synthesis while promoting translation and expression of the transcription factor ATF4. Atf4(-/-) cells are impaired in expressing genes involved in amino acid import, glutathione biosynthesis, and resistance to oxidative stress. Perk(-/-) cells, lacking an upstream ER stress-activated eIF2alpha kinase that activates Atf4, accumulate endogenous peroxides during ER stress, whereas interference with the ER oxidase ERO1 abrogates such accumulation. A signaling pathway initiated by eIF2alpha phosphorylation protects cells against metabolic consequences of ER oxidation by promoting the linked processes of amino acid sufficiency and resistance to oxidative stress.
Unfolded and malfolded client proteins impose a stress on the endoplasmic reticulum (ER), which contributes to cell death in pathophysiological conditions. The transcription factor C/EBP homologous protein (CHOP) is activated by ER stress, and CHOP deletion protects against its lethal consequences. We find that CHOP directly activates GADD34, which promotes ER client protein biosynthesis by dephosphorylating phosphoSer 51 of the ␣-subunit of translation initiation factor 2 (eIF2␣) in stressed cells. Thus, impaired GADD34 expression reduces client protein load and ER stress in CHOP −/− cells exposed to perturbations that impair ER function. CHOP −/− and GADD34 mutant cells accumulate less high molecular weight protein complexes in their stressed ER than wild-type cells. Furthermore, mice lacking GADD34-directed eIF2␣ dephosphorylation, like CHOP −/− mice, are resistant to renal toxicity of the ER stress-inducing drug tunicamycin. CHOP also activates ERO1␣, which encodes an ER oxidase. Consequently, the ER of stressed CHOP −/− cells is relatively hypo-oxidizing. Pharmacological and genetic manipulations that promote a hypo-oxidizing ER reduce abnormal high molecular weight protein complexes in the stressed ER and protect from the lethal consequences of ER stress. CHOP deletion thus protects cells from ER stress by decreasing ER client protein load and changing redox conditions within the organelle.[Keywords: Protein folding; chaperones; membranes; secretion; protein phosphorylation; gene expression] Supplemental material is available at http://www.genesdev.org.
Elimination of misfolded proteins from the endoplasmic reticulum (ER) by retro-translocation is an important physiological adaptation to ER stress. This process requires recognition of a substrate in the ER lumen and its subsequent movement through the membrane by the cytosolic p97 ATPase. Here we identify a p97-interacting membrane protein complex in the mammalian ER that links these two events. The central component of the complex, Derlin-1, is a homologue of Der1, a yeast protein whose inactivation prevents the elimination of misfolded luminal ER proteins. Derlin-1 associates with different substrates as they move through the membrane, and inactivation of Derlin-1 in C. elegans causes ER stress. Derlin-1 interacts with US11, a virally encoded ER protein that specifically targets MHC class I heavy chains for export from the ER, as well as with VIMP, a novel membrane protein that recruits the p97 ATPase and its cofactor.
The unfolded protein response (UPR) counteracts stress caused by unprocessed ER client proteins. A genome-wide survey showed impaired induction of many UPR target genes in xbp-1 mutant Caenorhabditis elegans that are unable to signal in the highly conserved IRE1-dependent UPR pathway. However a family of genes, abu (activated in blocked UPR), was induced to higher levels in ER-stressed xbp-1 mutant animals than in ER-stressed wild-type animals. RNA-mediated interference (RNAi) inactivation of a representative abu family member, abu-1 (AC3.3), activated the ER stress marker hsp-4::gfp in otherwise normal animals and killed 50% of ER-stressed ire-1 and xbp-1 mutant animals. Abu-1(RNAi) also enhanced the effect of inactivation of sel-1, an ER-associated protein degradation gene. The nine abu genes encode highly related type I transmembrane proteins whose lumenal domains have sequence similarity to a mammalian cell surface scavenger receptor of endothelial cells that binds chemically modified extracellular proteins and directs their lysosomal degradation. Our findings that ABU-1 is an intracellular protein located within the endomembrane system that is induced by ER stress in xbp-1 mutant animals suggest that ABU proteins may interact with abnormal ER client proteins and this function may be particularly important in animals with an impaired UPR.
Mammalian serine and arginine–rich (SR) proteins play important roles in both constitutive and regulated splicing, and SR protein–specific kinases (SRPKs) are conserved from humans to yeast. Here, we demonstrate a novel function of the single conserved SR protein kinase Sky1p in nuclear import in budding yeast. The yeast SR-like protein Npl3p is known to enter the nucleus through a composite nuclear localization signal (NLS) consisting of a repetitive arginine- glycine-glycine (RGG) motif and a nonrepetitive sequence. We found that the latter is the site for phosphorylation by Sky1p and that this phosphorylation regulates nuclear import of Npl3p by modulating the interaction of the RGG motif with its nuclear import receptor Mtr10p. The RGG motif is also methylated on arginine residues, but methylation does not affect the Npl3p–Mtr10p interaction in vitro. Remarkably, arginine methylation interferes with Sky1p-mediated phosphorylation, thereby indirectly influencing the Npl3p–Mtr10p interaction in vivo and negatively regulating nuclear import of Npl3p. These results suggest that nuclear import of Npl3p is coordinately influenced by methylation and phosphorylation in budding yeast, which may represent conserved components in the dynamic regulation of RNA processing in higher eukaryotic cells.
The ER's capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58 IPK /DNAJC3, a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58 IPK recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58 IPKÀ/À mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58 IPK is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells.
LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology. However, a clear understanding of L1’s lifecycle and the processes involved in restricting its insertion and intragenomic spread remains elusive. Here we identify modes of L1 proteins’ entrance into the nucleus, a necessary step for L1 proliferation. Using functional, biochemical, and imaging approaches, we also show a clear cell cycle bias for L1 retrotransposition that peaks during the S phase. Our observations provide a basis for novel interpretations about the nature of nuclear and cytoplasmic L1 ribonucleoproteins (RNPs) and the potential role of DNA replication in L1 retrotransposition.
SignificanceRetrotransposons replicate through RNA intermediates that are reverse transcribed and inserted at new genomic locations. LINE-1 (L1) elements constitute ∼17% of the human genome, making them the most successful retrotransposons in the human genome by mass. The activity of L1s was shown first in the germline or during early embryogenesis. More recent studies demonstrate a wider prevalence of L1 expression in somatic cells including neurons, aging cells, and different types of cancer. In this study, we developed the MapRRCon pipeline and performed a comprehensive computational analysis of L1 transcriptional regulators using ENCODE ChIP-seq datasets. We revealed the binding of various transcription factors, including Myc and CTCF, to the 5′ UTR promoter of the youngest human L1 family (L1HS) and their potential functional impact on L1HS expression.
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