Reduced amino acid availability attenuates mRNA translation in cells and helps to extend lifespan in model organisms. The amino acid deprivation–activated kinase GCN2 mediates this response in part by phosphorylating eIF2α. In addition, the cap-dependent translational inhibitor 4E-BP is transcriptionally induced to extend lifespan in Drosophila melanogaster, but through an unclear mechanism. Here, we show that GCN2 and its downstream transcription factor, ATF4, mediate 4E-BP induction, and GCN2 is required for lifespan extension in response to dietary restriction of amino acids. The 4E-BP intron contains ATF4-binding sites that not only respond to stress but also show inherent ATF4 activity during normal development. Analysis of the newly synthesized proteome through metabolic labeling combined with click chemistry shows that certain stress-responsive proteins are resistant to inhibition by 4E-BP, and gcn2 mutant flies have reduced levels of stress-responsive protein synthesis. These results indicate that GCN2 and ATF4 are important regulators of 4E-BP transcription during normal development and aging.

The ubiquitin-proteasome system and the autophagy-lysosome system are two major intracellular proteolytic pathways in eukaryotes. Although several biochemical mechanisms underlying the crosstalk between them have been suggested, little is known about the effect of enhanced proteasome activity on autophagic flux. Here, we found that upregulation of proteasome activity, which was achieved through the inhibition of USP14, significantly impaired cellular autophagic flux, especially at the autophagosome-lysosome fusion step. UVRAG appeared to function as a crucial checkpoint for the proper progression of autophagic flux. Although proteasome activation through USP14 inhibition facilitated the clearance of microtubule-associated protein tau (MAPT) and reduced the amount of its oligomeric forms, the same conditions increased the formation of inclusion bodies from nonproteasomal substrates such as huntingtin with long polyglutamine repeats. Our results collectively indicate that USP14 may function as a common denominator in the compensatory negative feedback between the two major proteolytic processes in the cell.

Eukaryotic cells have evolved signaling pathways that help to restore cellular homeostasis in response to various physiological or pathological conditions. ATF4 is a transcription factor whose mRNA translation is stimulated in response to stress-activated eIF2alpha kinases. Established conditions that activate eIF2alpha phosphorylation and ATF4 translation include excessive stress in the endoplasmic reticulum (ER) and amino acid deprivation. ATF4 is activated through a unique translational activation mechanism that involves multiple upstream open reading frames (uORFs) in the 5’-untranslated region (UTR), which is conserved from yeast to mammals. Taking advantage of this, we developed a translational activation reporter of ATF4 in Drosophila, in which the dsRed reporter coding sequence was placed downstream of the Drosophila ATF4 5’ UTR. This reporter remained inactive in most tissues under normal conditions, but showed dsRed expression when starved, or when challenged with conditions that imposed ER stress. In normally developing flies, a small number of cell types showed reporter expression even without exogenous stress, which included the salivary gland, gut, the male reproductive organ, and the photoreceptor cells, suggestive of inherent stress during the normal development of these cell types. These results establish a new tool to study ATF4-mediated stress response in Drosophila development and disease.

Expression of genes in the endoplasmic reticulum (ER) beyond its protein folding capacity activates signaling pathways that are collectively referred to as the Unfolded Protein Response (UPR). A major branch of the UPR pathway is mediated by IRE1, an ER-tethered endonuclease. Upon ER stress-induced activation, IRE1 splices the mRNA of XBP1, thereby generating an active isoform of this transcription factor. During normal Drosophila development, tissues with high protein secretory load show signs of IRE1/XBP1 activity indicative of inherent ER stress associated with those cell types. Here, we report that the XBP1 promoter activity itself is enhanced in secretory tissues of Drosophila, and it can be induced by excessive ER stress. Specifically, we developed a Drosophila XBP1 transcription reporter by placing dsRed under the control of the XBP1 intergenic sequence. DsRed expression in these xbp1_p>dsRed transgenic flies showed patterns similar to that of xbp1 transcript distribution. In healthy developing flies, the reporter expression was highest in salivary glands and the intestine. In the adult, the male reproductive organs showed high levels of dsRed. These tissues are known to have high protein secretory load. Consistently, the xbp1_p>dsRed reporter was induced by excessive ER stress caused by mutant Rhodopsin-1 overexpression. These results suggest that secretory cells suffer from inherent ER stress, and the xbp1_p>dsRed flies provide a useful tool in studying the function and homeostasis of those cells.

The mitochondrial outer membrane is a major site of apoptosis regulation across phyla. Human and C. elegans Bcl-2 family proteins and Drosophila Hid require the C-terminal tail-anchored (TA) sequence in order to insert into the mitochondrial membrane, but it remains unclear whether cytosolic proteins actively regulate the mitochondrial localization of these proteins. Here, we report that the cdk7 complex regulates the mitochondrial localization of Hid and its ability to induce apoptosis. We identified cdk7 through an in vivo RNAi screen of genes required for cell death. Although CDK7 is best known for its role in transcription and cell-cycle progression, a hypomorphic cdk7 mutant suppressed apoptosis without impairing these other known functions. In this cdk7 mutant background, Hid failed to localize to the mitochondria and failed to bind to recombinant inhibitors of apoptosis (IAPs). These findings indicate that apoptosis is promoted by a newly identified function of CDK7, which couples the mitochondrial localization and IAP binding of Hid.