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Summary Nutrient availability influences the production and degradation of materials that are required for cell growth and survival. Autophagy is a nutrient-regulated process that is used to degrade cytoplasmic materials and has been associated with human diseases. Solute transporters influence nutrient availability and sensing, yet we know little about how transporters influence autophagy. Here, we screen for solute transporters that are required for autophagy-dependent cell death and identify CG11665/hermes. We show that hermes is required for both autophagy during steroid-triggered salivary gland cell death and TNF-induced non-apoptotic eye cell death. hermes encodes a proton-coupled monocarboxylate transporter that preferentially transports pyruvate over lactate. mTOR signaling is elevated in hermes mutant cells, and decreased mTOR function suppresses the hermes salivary gland cell death phenotype. Hermes is most similar to human SLC16A11, a protein that was recently implicated in type 2 diabetes, thus providing a link between pyruvate, mTOR, autophagy, and possibly metabolic disorders.

The kinases PERK and IRE1 alleviate endoplasmic reticulum (ER) stress by orchestrating the unfolded protein response (UPR). If stress mitigation fails, PERK promotes cell death by activating pro-apoptotic genes, including death receptor 5 (DR5). Conversely, IRE1-which harbors both kinase and endoribonuclease (RNase) modules-blocks apoptosis through regulated IRE1-dependent decay (RIDD) of DR5 mRNA. Under irresolvable ER stress, PERK activity persists, whereas IRE1 paradoxically attenuates, by mechanisms that remain obscure. Here, we report that PERK governs IRE1's attenuation through a phosphatase known as RPAP2 (RNA polymerase II-associated protein 2). RPAP2 reverses IRE1 phosphorylation, oligomerization, and RNase activation. This inhibits IRE1-mediated adaptive events, including activation of the cytoprotective transcription factor XBP1s, and ER-associated degradation of unfolded proteins. Furthermore, RIDD termination by RPAP2 unleashes DR5-mediated caspase activation and drives cell death. Thus, PERK attenuates IRE1 via RPAP2 to abort failed ER-stress adaptation and trigger apoptosis.

The clearance of mitochondria by autophagy, mitophagy, is important for cell and organism health [1], and known to be regulated by ubiquitin. During Drosophila intestine development, cells undergo a dramatic reduction in cell size and clearance of mitochondria that depends on autophagy, the E1 ubiquitin-activating enzyme Uba1, and ubiquitin [2]. Here we screen a collection of putative ubiquitin-binding domain-encoding genes for cell size reduction and autophagy phenotypes. We identify the endosomal sorting complex required for transport (ESCRT) components TSG101 and Vps36, as well as the novel gene Vps13D. Vps13D is an essential gene that is necessary for autophagy, mitochondrial size, and mitochondrial clearance in Drosophila. Interestingly, a similar mitochondrial phenotype is observed in VPS13D mutant human cells. The ubiquitin-associated (UBA) domain of Vps13D binds K63 ubiquitin chains, and mutants lacking the UBA domain have defects in mitochondrial size and clearance and exhibit semi-lethality, highlighting the importance of Vps13D ubiquitin binding in both mitochondrial health and development. VPS13D mutant cells possess phosphorylated DRP1 and mitochondrial fission factor (MFF) as well as DRP1 association with mitochondria, suggesting that VPS13D functions downstream of these known regulators of mitochondrial fission. In addition, the large Vps13D mitochondrial and cell size phenotypes are suppressed by decreased mitochondrial fusion gene function. Thus, these results provide a previously unknown link between ubiquitin, mitochondrial size regulation, and autophagy.

Rad is a Ras-related small GTPase shown to inhibit cancer cell migration, and its expression is frequently lost in lung cancer cells. Here we provide evidence that Rad can negatively regulate the NFκB pathway. Overexpressing Rad in cells lowered both the basal and TNFα-stimulated transcriptional activity of NFκB. Compared with control cells, Rad-overexpressing cells displayed more cytoplasmic distribution of the NFκB subunit RelA/p65, while Rad-knockdown cells had higher levels of nuclear RelA/p65. Depleting Rad did not affect the kinetics of TNFα-induced IκB degradation, suggesting that Rad-mediated regulation of NFκB was through an IκB-independent mechanism. Expression of a nucleus-localized mutant Rad was sufficient to inhibit the NFκB transcriptional activity, whereas expressing the scaffolding protein 14-3-3γ to retain Rad in the cytoplasm alleviated the suppressive effect of Rad on NFκB. GST pull-down assays showed that Rad could directly bind to RelA/p65, and co-immunoprecipitation demonstrated that the Rad-p65 interaction primarily occurred in the nucleus. Adding Rad-containing nuclear extracts or purified GST-Rad in the electrophoretic mobility shift assays dose-dependently decreased the binding of RelA/p65 to an oligonucleotide probe containing the NFκB response element, suggesting that Rad may directly impede the interaction between RelA/p65 and DNA. Rad depletion altered the expression of an array of NFκB target genes, including upregulating MMP9. Knockdown of Rad expression in cells increased both basal and TNFα-stimulated MMP9 activities and cell invasion. Collectively, our results disclose a novel role of nuclear Rad in inhibiting the NFκB pathway function.

Autophagy is a conserved process that delivers components of the cytoplasm to lysosomes for degradation. The E1 and E2 enzymes encoded by Atg7 and Atg3 are thought to be essential for autophagy involving the ubiquitin-like protein Atg8. Here, we describe an Atg7- and Atg3-independent autophagy pathway that facilitates programmed reduction of cell size during intestine cell death. Although multiple components of the core autophagy pathways, including Atg8, are required for autophagy and cells to shrink in the midgut of the intestine, loss of either Atg7 or Atg3 function does not influence these cellular processes. Rather, Uba1, the E1 enzyme used in ubiquitylation, is required for autophagy and reduction of cell size. Our data reveal that distinct autophagy programs are used by different cells within an animal, and disclose an unappreciated role for ubiquitin activation in autophagy.

Autophagy has been implicated in both cell survival and programmed cell death (PCD), and this may explain the apparently complex role of this catabolic process in tumourigenesis. Our previous studies have shown that caspases have little influence on Drosophila larval midgut PCD, whereas inhibition of autophagy severely delays midgut removal. To assess upstreamsignals that regulate autophagy and larval midgut degradation, we have examined the requirement of growth signalling pathways. Inhibition of the class I phosphoinositide-3-kinase (PI3K) pathway prevents midgut growth, whereas ectopic PI3K and Ras signalling results in larger cells with decreased autophagy and delayed midgut degradation. Furthermore, premature induction of autophagy is sufficient to induce early midgut degradation. These data indicate that autophagy and the growth regulatory pathways have an important relationship during midgut PCD. Despite the roles of autophagy in both survival and death, our findings suggest that autophagy induction occurs in response to similar signals in both scenarios. Refereed/Peer-reviewed

The p53 tumor suppressor exerts its function mainly as a transcriptional activator. Here we show that the Ras-related small GTPase Rad, an inhibitor of Rho kinase, is a direct transcriptional target of p53. Expression of Rad messenger RNA (mRNA) and protein was induced by DNA damage in a p53-dependent manner. The −2934/−2905-bp Rad promoter region, to which p53 bound, was required for p53-mediated Rad gene activation. Treatment by DNA damaging agents increased p53 occupancy and histone acetylation in the region of Rad promoter containing the p53-binding site. Expression of Rad diminished the inhibitory phosphorylation at Ser3 of cofilin, a regulator of actin dynamics, and suppressed migration and invasiveness of cancer cells. Knockdown of Rad promoted cell migration and alleviated the p53-mediated migration suppression. Frequent loss of Rad mRNA and protein expression was observed in non-small cell lung carcinoma tissues. Together our results reveal a mechanism that p53 may inhibit cell migration by disrupting actin dynamics via Rad activation and implicate a tumor suppressor role of Rad in lung cancer.

The beta 4 integrin is expressed in epithelial cells, a few other cell types and in some carcinomas. Despite this restricted expression pattern and the functional importance of beta 4 integrin in epithelial and carcinoma biology, little is known about how its expression is regulated. Here, we assessed the epigenetic regulation of beta 4 integrin based on the presence of a large CpG island in the beta 4-integrin gene promoter. We separated basal (beta 4+) and luminal (beta 4-) epithelial cells from the mammary glands of K14-eGFP mice and demonstrated that the beta 4-integrin promoter is unmethylated in basal cells and methylated in luminal cells. We also observed that expression of beta 4 integrin and E-cadherin is lost during the epithelial-to-mesenchymal transition (EMT) of mammary gland cells induced by transforming growth factor beta (TGFbeta), which is coincident with de novo DNA methylation, a decrease in active histone modifications (H3K9Ac and H3K4me3) and an increase in the repressive histone modification H3K27me3. Furthermore, TGFbeta withdrawal promotes a mesenchymal-to-epithelial transition (MET) and triggers the re-expression of beta 4 integrin and E-cadherin. Intriguingly, demethylation at either promoter is not obligatory for transcriptional reactivation after TGFbeta withdrawal. However, both H3K9Ac and H3K4me3 modifications are restored during the MET, and H3K27me3 is reduced, strongly suggesting that reversible histone modifications rather than DNA demethylation are the predominant factors in reactivating expression of these genes. Our data indicate that complex epigenetic modifications contribute to the regulation of the beta 4 integrin and E-cadherin.