Mitochondrial TRAP1 regulates the unfolded protein response in the endoplasmic reticulum

Stress in mitochondria or the endoplasmic reticulum (ER) independently causes cell death. Recently, it was reported that ER stress causes mitochondrial dysfunction via p53-upregulated modulator of apoptosis (PUMA). However, little is known regarding the mitochondria molecules that mediate ER dysfunction. The present study revealed that tumor necrosis factor receptor-associated protein 1 (TRAP1), which localizes in the mitochondria, is associated with the unfolded protein response (UPR) in the ER. TRAP1 knockdown activated the ER-resident caspase-4, which is activated by ER stress, to induce cell death in humans. However, TRAP1 knockdown cells did not show a significant increase in the level of cell death at least within 24 h after early phase of ER stress in comparison with that of the control cells. This finding could be attributed to a number of reasons. TRAP1 knockdown failed to activate caspase-9, which is activated by activated caspase-4. In addition, TRAP1 knockdown increased the basal level of GRP78/BiP expression, which protects cells, and decreased the basal level of C/EBP homologous protein (CHOP) expression, which induces cell death, even under ER stress. Thus, the present study revealed that mitochondria could be a potential regulator of the UPR in the ER through mitochondrial TRAP1.     

Protein quality control in the endoplasmic reticulum

Protein folding and quality control in the endoplasmic reticulum (ER) are synchronized mechanisms ensuring that only properly folded proteins are integrated in the plasma membrane or secreted from the cell. These mechanisms act in close collaboration with the molecular machinery involved in retrograde-translocation and degradation of non-native proteins and with the ER-stress activated signalling systems. The common goal of these mechanisms is to prevent expression and secretion of misfolded proteins. Protein misfolding can be detrimental to the cell and contributes to the disease mechanism in several inherited disorders, e.g. cystic fibrosis, familial hypercholesterolemia and diabetes insipidus. This review outlines the molecular mechanisms in protein quality control occurring in the ER, signalling caused by ER stress, and finally ER associated protein degradation.     

Protein secretion and the endoplasmic reticulum

In a complex multicellular organism, different cell types engage in specialist functions, and as a result, the secretory output of cells and tissues varies widely. Whereas some quiescent cell types secrete minor amounts of proteins, tissues like the pancreas, producing insulin and other hormones, and mature B cells, producing antibodies, place a great demand on their endoplasmic reticulum (ER). Our understanding of how protein secretion in general is controlled in the ER is now quite sophisticated. However, there remain gaps in our knowledge, particularly when applying insight gained from model systems to the more complex situations found in vivo. This article describes recent advances in our understanding of the ER and its role in preparing proteins for secretion, with an emphasis on glycoprotein quality control and pathways of disulfide bond formation.     

Protein-tyrosine phosphatase 1B potentiates IRE1 signaling during endoplasmic reticulum stress

Protein-tyrosine phosphatase 1B (PTP-1B) is the prototypic tyrosine phosphatase whose function in insulin signaling and metabolism is well established. Although the role of PTP-1B in dephosphorylating various cell surface receptor tyrosine kinases is clear, the mechanisms by which it modulates receptor function from the endoplasmic reticulum (ER) remains an enigma. Here, we provide evidence that PTP-1B has an essential function in regulating the unfolded protein response in the ER compartment. The absence of PTP-1B caused impaired ER stress-induced IRE1 signaling. More specifically, JNK activation, XBP-1 splicing, and EDEM (ER degradation-enhancing alpha-mannosidase-like protein) gene induction, as well as ER stress-induced apoptosis, were attenuated in PTP-1B knock-out mouse embryonic fibroblasts in response to two ER stressors, tunicamycin and azetidine-2 carboxylic acid. We demonstrate that PTP-1B is not just a passive resident of the ER but on the contrary has an essential role in potentiating IRE1-mediated ER stress signaling pathways.     

Involvement of the endoplasmic reticulum in peroxisome formation

The traditional view holds that peroxisomes are autonomous organelles multiplying by growth and division. More recently, new observations have challenged this concept. Herein, we present evidence supporting the involvement of the endoplasmic reticulum (ER) in peroxisome formation by electron microscopy, immunocytochemistry and three-dimensional image reconstruction of peroxisomes and associated compartments in mouse dendritic cells. We found the peroxisomal membrane protein Pex13p and the ATP-binding cassette transporter protein PMP70 present in specialized subdomains of the ER that were continuous with a peroxisomal reticulum from which mature peroxisomes arose. The matrix proteins catalase and thiolase were only detectable in the reticula and peroxisomes. Our results suggest the existence of a maturation pathway from the ER to peroxisomes and implicate the ER as a major source from which the peroxisomal membrane is derived.     

Autophagy regulates endoplasmic reticulum stress in ischemic preconditioning

Recent studies have suggested that autophagy plays a prosurvival role in ischemic preconditioning (IPC). This study was taken to assess the linkage between autophagy and endoplasmic reticulum (ER) stress during the process of IPC. The effects of IPC on ER stress and neuronal injury were determined by exposure of primary cultured murine cortical neurons to 30 min of OGD 24 h prior to a subsequent lethal OGD. The effects of IPC on ER stress and ischemic brain damage were evaluated in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The results showed that both IPC and lethal OGD increased the LC3-II expression and decreased p62 protein levels, but the extent of autophagy activation was varied. IPC treatment ameliorated OGD-induced cell damage in cultured cortical neurons, whereas 3-MA (5–20 mM) and bafilomycin A₁ (75–150 nM) suppressed the neuroprotection induced by IPC. 3-MA, at the dose blocking autophagy, significantly inhibited IPC-induced HSP70, HSP60 and GRP78 upregulation; meanwhile, it also aggregated the ER stress and increased activated caspase-12, caspase-3 and CHOP protein levels both in vitro and in vivo models. The ER stress inhibitor Sal (75 pmol) recovered IPC-induced neuroprotection in the presence of 3-MA. Rapamycin 50–200 nM in vitro and 35 pmol in vivo 24 h before the onset of lethal ischemia reduced ER stress and ischemia-induced neuronal damage. These results demonstrated that pre-activation of autophagy by ischemic preconditioning can boost endogenous defense mechanisms to upregulate molecular chaperones, and hence reduce excessive ER stress during fatal ischemia.     

Protein transport across the endoplasmic reticulum membrane

A decisive step in the biosynthesis of many eukaryotic proteins is their partial or complete translocation across the endoplasmic reticulum membrane. A similar process occurs in prokaryotes, except that proteins are transported across or are integrated into the plasma membrane. In both cases, translocation occurs through a protein-conducting channel that is formed from a conserved, heterotrimeric membrane protein complex, the Sec61 or SecY complex. Structural and biochemical data suggest mechanisms that enable the channel to function with different partners, to open across the membrane and to release laterally hydrophobic segments of membrane proteins into lipid.     

Autophagy regulates endoplasmic reticulum stress in ischemic preconditioning

Recent studies have suggested that autophagy plays a prosurvival role in ischemic preconditioning (IPC). This study was taken to assess the linkage between autophagy and endoplasmic reticulum (ER) stress during the process of IPC. The effects of IPC on ER stress and neuronal injury were determined by exposure of primary cultured murine cortical neurons to 30 min of OGD 24 h prior to a subsequent lethal OGD. The effects of IPC on ER stress and ischemic brain damage were evaluated in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The results showed that both IPC and lethal OGD increased the LC3-II expression and decreased p62 protein levels, but the extent of autophagy activation was varied. IPC treatment ameliorated OGD-induced cell damage in cultured cortical neurons, whereas 3-MA (5-20 mM) and bafilomycin A 1 (75-150 nM) suppressed the neuroprotection induced by IPC. 3-MA, at the dose blocking autophagy, significantly inhibited IPC-induced HSP70, HSP60 and GRP78 upregulation; meanwhile, it also aggregated the ER stress and increased activated caspase-12, caspase-3 and CHOP protein levels both in vitro and in vivo models. The ER stress inhibitor Sal (75 pmol) recovered IPC-induced neuroprotection in the presence of 3-MA. Rapamycin 50-200 nM in vitro and 35 pmol in vivo 24 h before the onset of lethal ischemia reduced ER stress and ischemia-induced neuronal damage. These results demonstrated that pre-activation of autophagy by ischemic preconditioning can boost endogenous defense mechanisms to upregulate molecular chaperones, and hence reduce excessive ER stress during fatal ischemia.     

BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress

Bax inhibitor-1 (BI-1) is an evolutionarily conserved endoplasmic reticulum (ER) protein that suppresses cell death in both animal and plant cells. We characterized mice in which the bi-1 gene was ablated. Cells from BI-1-deficient mice, including fibroblasts, hepatocytes, and neurons, display selective hypersensitivity to apoptosis induced by ER stress agents (thapsigargin, tunicamycin, brefeldin A), but not to stimulators of mitochondrial or TNF/Fas-death receptor apoptosis pathways. Conversely, BI-1 overexpression protects against apoptosis induced by ER stress. BI-1-mediated protection from apoptosis induced by ER stress correlated with inhibition of Bax activation and translocation to mitochondria, preservation of mitochondrial membrane potential, and suppression of caspase activation. BI-1 overexpression also reduces releasable Ca(2+) from the ER. In vivo, bi-1(-/-) mice exhibit increased sensitivity to tissue damage induced by stimuli that trigger ER stress, including stroke and tunicamycin injection. Thus, BI-1 regulates a cell death pathway important for cytopreservation during ER stress.     

Rapid in vitro formation of smooth endoplasmic reticulum aggregates within peptide-producing islet cells

We report here that heptanol (3.5 mM) induces in vitro a rapid formation of smooth endoplasmic reticulum aggregates (SERA) within isolated islets of Langerhans. SERA appeared after only 15 min of exposure to the alkanol and increased in number during the first 30 min of incubation. At that time, SERA represented 2% and 6% of the volume of B- and non-B-cells, respectively. Removal of heptanol resulted in the rapid disappearance of SERA, whereas reintroduction of the alkanol rapidly induced these structures again. SERA formation was seen in different types of endocrine and nonendocrine islet cells. In the insulin-producing B-cells, SERA formation was not modified by conditions known to alter the secretory activity and the microtubular-microfilament network or to inhibit protein synthesis. By contrast, SERA formation was inhibited by low temperature and by conditions depleting the energy sources of the cells. Similar observations were made in the presence of either octanol (1 mM) or nonanol (1 mM) but not of shorter chain alkanols, alkanes, oxidative derivates of either heptanol or octanol, and of other unrelated lipid-soluble compounds. Incubations in the presence of long-chain alkanols provide, therefore, a unique model to study in vitro the formation and disposal of smooth endoplasmic reticulum, as well as a system in which rapid membrane biogenesis is amenable to direct experimental testing.     

Protein folding during cotranslational translocation in the endoplasmic reticulum

To test how far into the protein-conducting channel of the translocon complex a nascent polypeptide domain must move before it can fold, we analyzed the folding of in vitro translated products of truncated mRNAs encoding the Semliki Forest virus capsid protease domain (Cp) during translocation into microsomes. Cp folded when the C-terminal linker connecting it to the peptidyltransferase center was 64 amino acids or longer. This means that to fold, Cp must exit the translocon channel. With an uncleaved signal sequence, about one out of four of the Cp domains could undergo folding with a C-terminal linker of only 38-66 amino acids. This suggested that the constraint imposed on folding by the translocon complex may be less stringent for signal-anchored membrane proteins.     

Protein folding and quality control in the endoplasmic reticulum

The endoplasmic reticulum (ER) is a highly versatile protein factory that is equipped with chaperones and folding enzymes essential for protein folding. ER quality control guided by these chaperones is essential for life. Whereas correctly folded proteins are exported from the ER, misfolded proteins are retained and selectively degraded. At least two main chaperone classes, BiP and calnexin/calreticulin, are active in ER quality control. Folding factors usually are found in complexes. Recent work emphasises more than ever that chaperones act in concert with co-factors and with each other.     

Protein sorting upon exit from the endoplasmic reticulum

It is currently thought that all secretory proteins travel together to the Golgi apparatus where they are sorted to different destinations. However, the specific requirements for transport of GPI-anchored proteins from the endoplasmic reticulum to the Golgi apparatus in yeast could be explained if protein sorting occurs earlier in the pathway. Using an in vitro assay that reconstitutes a single round of budding from the endoplasmic reticulum, we found that GPI-anchored proteins and other secretory proteins exit the endoplasmic reticulum in distinct vesicles. Therefore, GPI-anchored proteins are sorted from other proteins, in particular other plasma membrane proteins, at an early stage of the secretory pathway. These results have wide implications for the mechanism of protein exit from the endoplasmic reticulum.     

Contribution of the endoplasmic reticulum to peroxisome formation

How peroxisomes are formed in eukaryotic cells is unknown but important for insight into a variety of diseases. Both human and yeast cells lacking peroxisomes due to mutations in PEX3 or PEX19 genes regenerate the organelles upon reintroduction of the corresponding wild-type version. To evaluate how and from where new peroxisomes are formed, we followed the trafficking route of newly made YFP-tagged Pex3 and Pex19 proteins by real-time fluorescence microscopy in Saccharomyces cerevisiae. Remarkably, Pex3 (an integral membrane protein) could first be observed in the endoplasmic reticulum (ER), where it concentrates in foci that then bud off in a Pex19-dependent manner and mature into fully functional peroxisomes. Pex19 (a farnesylated, mostly cytosolic protein) enriches first at the Pex3 foci on the ER and then on the maturing peroxisomes. This trafficking route of Pex3-YFP is the same in wild-type cells. These results demonstrate that peroxisomes are generated from domains in the ER.     

Protein phosphatase type 1 regulates ion homeostasis in Saccharomyces cerevisiae

Protein phosphatase type 1 (PP1) is encoded by the essential gene GLC7 in Saccharomyces cerevisiae. glc7-109 (K259A, R260A) has a dominant, hyperglycogen defect and a recessive, ion and drug sensitivity. Surprisingly, the hyperglycogen phenotype is partially retained in null mutants of GAC1, GIP2, and PIG1, which encode potential glycogen-targeting subunits of Glc7. The R260A substitution in GLC7 is responsible for the dominant and recessive traits of glc7-109. Another mutation at this residue, glc7-R260P, confers only salt sensitivity, indicating that the glycogen and salt traits of glc7-109 are due to defects in distinct physiological pathways. The glc7-109 mutant is sensitive to cations, aminoglycosides, and alkaline pH and exhibits increased rates of l-leucine and 3,3'-dihexyloxacarbocyanine iodide uptake, but it is resistant to molar concentrations of sorbitol or KCl, indicating that it has normal osmoregulation. KCl suppresses the ion and drug sensitivities of the glc7-109 mutant. The CsCl sensitivity of this mutant is suppressed by recessive mutations in PMA1, which encodes the essential plasma membrane H(+)ATPase. Together, these results indicate that Glc7 regulates ion homeostasis by controlling ion transport and/or plasma membrane potential, a new role for Glc7 in budding yeast.     

The chaperone environment at the cytoplasmic face of the endoplasmic reticulum can modulate rhodopsin processing and inclusion formation

The human DnaJ (Hsp40) proteins HSJ1a and HSJ1b are type II DnaJ proteins with different C termini generated by alternate splicing. Both protein isoforms can regulate the ATPase activity and substrate binding of Hsp70. In this study, we have confirmed the neuronal expression of HSJ1a and HSJ1b proteins and localized their expression in human neural retina using isoform-specific antisera. HSJ1a and HSJ1b were enriched in photoreceptors, particularly the inner segments, but had different intracellular localization due to the prenylation of HSJ1b by a geranylgeranyl moiety. Because of their enrichment at the site of rhodopsin production, we investigated the effect of HSJ1 isoforms on the cellular processing of wild-type and mutant rhodopsin apoprotein in SK-N-SH cells. The expression of HSJ1b, but not HSJ1a, inhibited the normal cellular processing of wild-type rhodopsin-GFP, which co-localized with HSJ1b at the ER. HSJ1b expression also increased the incidence of inclusion formation by the wild-type protein. Both isoforms were recruited to mutant P23H rhodopsin inclusions, but only HSJ1b enhanced inclusion formation. Investigation of a prenylation-null mutant showed that the modulation of rhodopsin processing and inclusion formation was dependent on the correct subcellular targeting of HSJ1b to the cytosolic face of the ER. An Hsp70 interaction-null mutant of HSJ1b had the same effect as HSJ1b, suggesting that these phenomena were independent of Hsp70 and, furthermore, overexpression of Hsp70 with HSJ1b did not modulate the HSJ1b effect on inclusion formation, showing that Hsp70 was not limiting. The data provide evidence that cytoplasmic chaperones when targeted to the ER can influence the folding and processing of a GPCR and show that DnaJ protein function can be further specialized by alternative splicing and post-translational modification.     

In vitro studies on the translocation of acid phosphatase into the endoplasmic reticulum of the yeast Saccharomyces cerevisiae

We demonstrate here the in vitro translocation of yeast acid phosphatase into rough endoplasmic reticulum. The precursor of the repressible acid phosphatase from Saccharomyces cerevisiae encoded by the PHO5 gene, was synthesized in a yeast lysate programmed with in vitro transcribed PHO5 mRNA. In the presence of yeast rough microsomes up to 16% of the acid phosphatase synthesized was found to be translocated into the microsomes, as judged by proteinase resistance, and fully core-glycosylated. The translocation efficiency however, decreased to 3% if yeast rough microsomes were added after synthesis of acid phosphatase had been terminated. When a wheat-germ extract was used for in vitro synthesis, the precursor of acid phosphatase was translocated into canine pancreatic rough microsomes and thereby core-glycosylated in a signal-recognition-particle-dependent manner. Replacing canine with yeast rough microsomes in the wheat-germ translation system, however, resulted in a significant decrease in the ability to translocate and glycosylate the precursor. Translocation and glycosylation were partially restored by a high-salt extract prepared from yeast ribosomes. The results presented here suggest that yeast-specific factors are needed to translocate and glycosylate acid phosphatase efficiently in vitro.