Stress-Independent Activation of XBP1s and/or ATF6 Reveals Three Functionally Diverse ER Proteostasis Environments

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Inositol deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian PGAP1 and yeast Bst1p

The inositol moiety of mammalian glycosylphosphatidylinositol (GPI) is acylated at an early step in GPI biosynthesis. The inositol acylation is essential for the generation of mature GPI capable of attachment to proteins. However, the acyl group is usually absent from GPI-anchored proteins (GPI-APs) on the cell surface due to inositol deacylation that occurs in the endoplasmic reticulum (ER) soon after GPI-anchor attachment. Mammalian GPI inositol-deacylase has not been cloned, and the biological significance of the deacylation has been unclear. Here we report a GPI inositol-deacylase-deficient Chinese hamster ovary cell line established by taking advantage of resistance to phosphatidylinositol-specific phospholipase C and the gene responsible, which was termed PGAP1 for Post GPI Attachment to Proteins 1. PGAP1 encoded an ER-associated, 922-amino acid membrane protein bearing a lipase consensus motif. Substitution of a conserved putative catalytic serine with alanine resulted in a complete loss of function, indicating that PGAP1 is the GPI inositol-deacylase. The mutant cells showed a clear delay in the maturation of GPI-APs in the Golgi and accumulation of GPI-APs in the ER. Thus, the GPI inositol deacylation is important for efficient transport of GPI-APs from the ER to the Golgi.     

Sustained IRE1 and ATF6 signaling is important for survival of melanoma cells undergoing ER stress

Apoptosis triggered by endoplasmic reticulum (ER) stress is associated with rapid attenuation of the IRE1α and ATF6 pathways but persistent activation of the PERK branch of the unfolded protein response (UPR) in cells. However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that the kinetics and durations of activation of the UPR pathways are deregulated in melanoma cells undergoing ER stress. We show here that the IRE1α and ATF6 pathways are sustained along with the PERK signaling in melanoma cells subjected to pharmacological ER stress, and that this is, at least in part, due to increased activation of the MEK/ERK pathway. In contrast to an initial increase followed by rapid reduction in activation of IRE1α and ATF6 signaling in control cells that were relatively sensitive to ER stress-induced apoptosis, activation of IRE1α and ATF6 by the pharmacological ER stress inducer tunicamycin (TM) or thapsigargin (TG) persisted in melanoma cells. On the other hand, the increase in PERK signaling lasted similarly in both types of cells. Sustained activation of IRE1α and ATF6 signaling played an important role in protecting melanoma cells from ER stress-induced apoptosis, as interruption of IRE1α or ATF6 rendered melanoma cells sensitive to apoptosis induced by TM or TG. Inhibition of MEK partially blocked IRE1α and ATF6 activation, suggesting that MEK/ERK signaling contributed to sustained activation of IRE1α and ATF6. Taken together, these results identify sustained activation of the IRE1α and ATF6 pathways of the UPR driven by the MEK/ERK pathway as an important protective mechanism against ER stress-induced apoptosis in melanoma cells.     

Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control

Misfolded proteins retained in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation pathway. The mechanisms used to sort them from correctly folded proteins remain unclear. Analysis of substrates with defined folded and misfolded domains has revealed a system of sequential checkpoints that recognize topologically distinct domains of polypeptides. The first checkpoint examines the cytoplasmic domains of membrane proteins. If a lesion is detected, it is retained statically in the ER and rapidly degraded without regard to the state of its other domains. Proteins passing this test face a second checkpoint that monitors domains localized in the ER lumen. Proteins detected by this pathway are sorted from folded proteins and degraded by a quality control mechanism that requires ER-to-Golgi transport. Although the first checkpoint is obligatorily directed at membrane proteins, the second monitors both soluble and membrane proteins. Our data support a model whereby "properly folded" proteins are defined biologically as survivors that endure a series of distinct checkpoints.