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The unfolded protein response (UPR) is a complex homeostatic programme that balances the load of secretory protein synthesis with the folding capacity of the endoplasmic reticulum (ER). Although originally believed to function predominantly as a stress response pathway, growing evidence supports a role for the UPR in the regulation of development. The study of human diseases alongside work with transgenic mouse models has implicated the UPR in a wide range of developmental processes. This chapter examines the three distinct branches of the UPR and their importance during development.

Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium ionophores. Calcium-dependent protein kinase 3 (TgCDPK3) was previously identified as a key mediator in this process, as TgCDPK3 knockout (∆cdpk3) parasites fail to egress in a timely manner. Phosphoproteomic analysis comparing WT with ∆cdpk3 parasites revealed changes in the TgCDPK3-dependent phosphoproteome that included proteins important for regulating motility, but also metabolic enzymes, indicating that TgCDPK3 controls processes beyond egress. Here we have investigated a predicted direct target of TgCDPK3, ApiAT5-3, a putative transporter of the major facilitator superfamily, and show that it is rapidly phosphorylated at serine 56 after induction of calcium signalling. Conditional knockout of apiAT5-3 results in transcriptional upregulation of most ribosomal subunits, but no alternative transporters, and subsequent parasite death. Mutating the S56 to a non-phosphorylatable alanine leads to a fitness cost, suggesting that phosphorylation of this residue is beneficial, albeit not essential, for tyrosine import. Using a combination of metabolomics and heterologous expression, we confirmed a primary role in tyrosine import for ApiAT5-3. However, no significant differences in tyrosine import could be detected in phosphorylation site mutants showing that if tyrosine transport is affected by S56 phosphorylation, its regulatory role is subtle.

Developmental pathways must be responsive to the environment. Phosphorylation of eIF2α enables a family of stress sensing kinases to trigger the integrated stress response (ISR), which has pro-survival and developmental consequences. Mutations of the ISR kinase GCN2 have been implicated in the development of pulmonary arterial hypertension, a disorder known to be associated with defects of BMP signaling, but how the ISR and BMP signaling might interact is unknown. Here we show inDrosophilathat GCN2 antagonises BMP signaling through direct effects on translation and indirectly via the transcription factorcrc(dATF4). Expression of a constitutively active GCN2 or loss of the eIF2α phosphatase dPPP1R15 impair developmental BMP signaling in flies. In cells, inhibition of translation by GCN2 blocks downstream BMP signaling. Moreover, loss of d4E-BP, a target ofcrc, augments BMP signalingin vitroand rescues tissue developmentin vivo. These results identify a novel mechanism by which the ISR modulates BMP signaling during development. Since abnormalities of both GCN2 and BMP signaling lead to pulmonary hypertension, these findings may have wider relevance for the development of therapies for this disease.

α1-Antitrypsin is a serine protease inhibitor produced in the liver that is responsible for the regulation of pulmonary inflammation. The commonest pathogenic gene mutation yields Z-α1-antitrypsin, which has a propensity to self-associate forming polymers that become trapped in inclusions of endoplasmic reticulum (ER). It is unclear whether these inclusions are connected to the main ER network in Z-α1-antitrypsin-expressing cells. Using live cell imaging, we found that despite inclusions containing an immobile matrix of polymeric α1-antitrypsin, small ER resident proteins can diffuse freely within them. Inclusions have many features to suggest they represent fragmented ER, and some are physically separated from the tubular ER network, yet we observed cargo to be transported between them in a cytosol-dependent fashion that is sensitive to N-ethylmaleimide and dependent on Sar1 and sec22B. We conclude that protein recycling occurs between ER inclusions despite their physical separation.—Dickens, J. A., Ordóñez, A., Chambers, J. E., Beckett, A. J., Patel, V., Malzer, E., Dominicus, C. S., Bradley, J., Peden, A. A., Prior, I. A., Lomas, D. A., Marciniak, S. J. The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-α1-antitrypsin.

Alpha-1 antitrypsin deficiency is predominantly caused by point mutations that alter the protein's folding. These mutations fall into two broad categories: those that destabilize the protein dramatically and lead to its post-translational degradation and those that affect protein structure more subtly to promote protein polymerization within the endoplasmic reticulum (ER). This distinction is important because it determines the cell's response to each mutant. The severely misfolded mutants trigger an unfolded protein response (UPR) that promotes improved protein folding but can kill the cell in the chronic setting. In contrast, mutations that permit polymer formation fail to activate the UPR but instead promote a nuclear factor-κB-mediated ER overload response. The ability of polymers to increase a cell's sensitivity to ER stress likely explains apparent inconsistencies in the alpha-1 antitrypsin-signaling literature that have linked polymers with the UPR. In this review we discuss the use of mutant serpins to dissect each signaling pathway.

Four stress-sensing kinases phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) to activate the integrated stress response (ISR). In animals, the ISR is antagonised by selective eIF2α phosphatases comprising a catalytic protein phosphatase 1 (PP1) subunit in complex with a PPP1R15-type regulatory subunit. An unbiased search for additional conserved components of the PPP1R15-PP1 phosphatase identified monomeric G-actin. Like PP1, G-actin associated with the functional core of PPP1R15 family members and G-actin depletion, by the marine toxin jasplakinolide, destabilised the endogenous PPP1R15A-PP1 complex. The abundance of the ternary PPP1R15-PP1-G-actin complex was responsive to global changes in the polymeric status of actin, as was its eIF2α-directed phosphatase activity, while localised G-actin depletion at sites enriched for PPP1R15 enhanced eIF2α phosphorylation and the downstream ISR. G-actin's role as a stabilizer of the PPP1R15-containing holophosphatase provides a mechanism for integrating signals regulating actin dynamics with stresses that trigger the ISR. DOI: http://dx.doi.org/10.7554/eLife.04872.001, eLife digest For a cell to build a protein, it must first copy the instructions contained within a gene. A complex molecular machine called a ribosome then reads these instructions and translates them into a protein. This translation process involves a number of steps. Proteins called eukaryotic translation initiation factors (or eIFs for short) coordinate the first step in the process, which is known as ‘initiation’. The eIFs also provide the cell with ways to control how quickly it makes proteins. For example, when a cell is stressed, either by starvation or toxins, it adds a phosphate group onto part of an eIF protein, called eIF2α. This modification makes this eIF protein less able to initiate translation, and so the cell builds fewer proteins and conserves more of its resources during times of stress. Once the stressful conditions are over, the phosphate group is removed from eIF2α by an enzyme called a phosphatase. This phosphatase contains two subunits: one that recognizes eIF2α and another that removes the phosphate group. However, experiments that attempted to recreate this phosphatase activity using just these two subunits in a test tube failed to generate a working enzyme that specifically targeted the phosphate group of eIF2α. This suggests that in cells this enzyme contains an additional unknown subunit. Now, Chambers, Dalton et al. (and Chen et al.) report the identity of a ‘missing’ third subunit as a protein known as globular-actin or G-actin. Chambers, Dalton et al. engineered human and fruit fly cells to add ‘molecular handles’ on the two known subunits of the phosphatase enzyme. These handles could then be used to essentially pull these proteins out of the mixture of molecules within a cell and see what other proteins came along too. Both of the known subunits ‘pulled’ G-actin along with them; this suggested that it could be the missing part of the phosphatase enzyme. Further experiments confirmed that G-actin works together with the other two subunits to specifically remove the phosphate group from eIF2α in mouse cells that had been stressed using a harmful chemical. Individual G-actin proteins can bind together to form long filaments, and signals that encourage a cell to divide or move also trigger the formation of actin filaments. This reduces the activity of the phosphatase enzyme by depriving it of a crucial component, i.e., free G-actin proteins. As such, the new mechanism described by Chambers, Dalton et al. suggests how growth and movement signals might also change a cell's sensitivity to stress. These findings may hopefully enable stressed cells to be targeted by drugs to treat disease; but future work is needed to clarify under what circumstances the integration of such signals into the stress response is beneficial to the cell. DOI: http://dx.doi.org/10.7554/eLife.04872.002

Introduction and objectives The majority of inherited cases of pulmonary arterial hypertension (PAH) are caused by mutations that affect the Bone Morphogenetic Protein (BMP) receptor 2. Recently, patients with pulmonary veno-occlusive disease (PVOD), a rare subtype of pulmonary hypertension, were found to have mutations of EIF2AK4. This gene encodes the kinase GCN2, which regulates cellular responses to amino acid availability by phosphorylating the translation initiation factor eIF2a. Phosphorylation of eIF2a reduces protein synthesis and induces expression of a transcription factor called ATF4. It is unclear if BMP and GCN2 signalling are functionally related and so we set out to test this using the model organism Drosophila melanogaster. Methods We examined the development of wing veins in Drosophila because this is known to depend on BMP signalling. We manipulated the expression of dGCN2 and its antagonistic phosphatase dPPP1R15 by RNA interference. Morphology of the wing veins was examined in adult flies and early developmental signalling was measured in pupating animals. Similar experiments performed in animals with defective BMP signalling tested for a genetic interaction between eIF2a phosphorylation and BMP signalling. Results We observed that depletion of dGCN2 in the developing wing by RNA interference enhanced BMP signalling as evidenced by hyperphosphorylation of MAD, the Drosophila orthologue of SMAD1. This was associated with development of ectopic wing veins in the regions of RNAi expression. In contrast, knock down of the eIF2a phosphatase dPPP1R15 selectively impaired anterior crossvein formation. The effects of dPPP1R15 depletion were cell-autonomous suggesting an effect on downstream BMP signalling rather than on ligand secretion. The vein phenotypes were highly sensitive to manipulation of BMP signalling. Both in insect and mammalian cells, ATF4 expression inhibited BMP signalling suggesting a mechanism for the crosstalk between BMP and GCN2. Conclusion We have discovered in Drosophila that GCN2 activation modulates BMP signalling. This effect is mediated, at least in part, by the downstream transcription factor ATF4, which inhibits the phosphorylation of MAD (insect SMAD1). Our findings indicate that this pathway is conserved between insects and mammals and this model may shed light on the pathogenesis of PAH and PVOD.

The ontogeny of asialoglycoprotein receptor was investigated by electron microscopic cytochemistry in hepatocytes isolated from foetal and adult rat. The binding capacity for asialofetuin coupled to horseradish peroxidase was lacking before the 18th day of intrauterine life; it arises at this time and increases with developmental age. The ligand-receptor complexes form small patches. The distribution pattern of positivity is very similar in pre and postnatal age, covering the entire cell surface. These results indicate a rather late appearance of the galactose-binding capacity related to the asialoglycoprotein clearance function, which is typical of adult mammalian liver.

α1-Antitrypsin is a serine protease inhibitor produced in the liver that is responsible for the regulation of pulmonary inflammation. The commonest pathogenic gene mutation yields Z-α1-antitrypsin, which has a propensity to self-associate forming polymers that become trapped in inclusions of endoplasmic reticulum (ER). It is unclear whether these inclusions are connected to the main ER network in Z-α1-antitrypsin-expressing cells. Using live cell imaging, we found that despite inclusions containing an immobile matrix of polymeric α1-antitrypsin, small ER resident proteins can diffuse freely within them. Inclusions have many features to suggest they represent fragmented ER, and some are physically separated from the tubular ER network, yet we observed cargo to be transported between them in a cytosol-dependent fashion that is sensitive to N-ethylmaleimide and dependent on Sar1 and sec22B. We conclude that protein recycling occurs between ER inclusions despite their physical separation.-Dickens, J. A., Ordóñez, A., Chambers, J. E., Beckett, A. J., Patel, V., Malzer, E., Dominicus, C. S., Bradley, J., Peden, A. A., Prior, I. A., Lomas, D. A., Marciniak, S. J. The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-α1-antitrypsin.

The number and distribution of galactose-specific binding sites were investigated in rat liver cells during perinatal development. Ligand binding to hepatocytes, macrophages and endothelial cells was followed with in vitro and in situ experiments by electron microscopy, using lactosylated bovine serum albumin adsorbed onto 5 nm colloidal gold particles as ligand. Binding capacity, starting at a late stage of fetal development, is very low both on the hepatocyte and on the macrophage surface, which show single particles statistically distributed. By contrast, bound particles are absent from fetal endothelial cells, which also lack the typical coated regions. In vivo, experiments at 37 °C show that endocytosis occurs to some extent in prenatal life. These results indicate that the expression of galactose-specific receptors’ activity on the different liver cell types follows different developmental patterns, which are independently modulated.