TGBp3 triggers the unfolded protein response and SKP1‐dependent programmed cell death

The Potato virus X (PVX) triple gene block protein 3 (TGBp3), an 8‐kDa membrane binding protein, aids virus movement and induces the unfolded protein response (UPR) during PVX infection. TGBp3 was expressed from the Tobacco mosaic virus (TMV) genome (TMV‐p3), and we noted the up‐regulation of SKP1 and several endoplasmic reticulum (ER)‐resident chaperones, including the ER luminal binding protein (BiP), protein disulphide isomerase (PDI), calreticulin (CRT) and calmodulin (CAM). Local lesions were seen on leaves inoculated with TMV‐p3, but not TMV or PVX. Such lesions were the result of TGBp3‐elicited programmed cell death (PCD), as shown by an increase in reactive oxygen species, DNA fragmentation and induction of SKP1 expression. UPR‐related gene expression occurred within 8 h of TMV‐p3 inoculation and declined before the onset of PCD. TGBp3‐mediated cell death was suppressed in plants that overexpressed BiP, indicating that UPR induction by TGBp3 is a pro‐survival mechanism. Anti‐apoptotic genes Bcl‐xl, CED‐9 and Op‐IAP were expressed in transgenic plants and suppressed N gene‐mediated resistance to TMV, but failed to alleviate TGBp3‐induced PCD. However, TGBp3‐mediated cell death was reduced in SKP1‐silenced Nicotiana benthamiana plants. The combined data suggest that TGBp3 triggers the UPR and elicits PCD in plants.     

Cell death triggered by the P25 protein in Potato virus X-associated synergisms results from endoplasmic reticulum stress in Nicotiana benthamiana

The synergistic interaction of Potato virus X (PVX) with a number of potyviruses results in systemic necrosis in Nicotiana spp. Previous investigations have indicated that the viral suppressor of RNA silencing (VSR) protein P25 of PVX triggers systemic necrosis in PVX-associated synergisms in a threshold-dependent manner. However, little is still known about the cellular processes that lead to this necrosis, and whether the VSR activity of P25 is involved in its elicitation. Here, we show that transient expression of P25 in the presence of VSRs from different viruses, including the helper component-proteinase (HC-Pro) of potyviruses, induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), which ultimately lead to ER collapse. However, the host RNA silencing pathway was dispensable for the elicitation of cell death by P25. Confocal microscopy studies in leaf patches co-expressing P25 and HC-Pro showed dramatic alterations in ER membrane structures, which correlated with the up-regulation of bZIP60 and several ER-resident chaperones, including the ER luminal binding protein (BiP). Overexpression of BiP alleviated the cell death induced by the potexviral P25 protein when expressed together with VSRs derived from different viruses. Conversely, silencing of the UPR master regulator, bZIP60, led to an increase in cell death elicited by the P25/HC-Pro combination as well as by PVX-associated synergism. In addition to its role as a negative regulator of P25-induced cell death, UPR partially restricted PVX infection. Thus, systemic necrosis caused by PVX-associated synergistic infections is probably the effect of an unmitigated ER stress following the overaccumulation of a viral protein, P25, with ER remodelling activity.     

Protein disulfide isomerase, but not binding protein, overexpression enhances secretion of a non-disulfide-bonded protein in yeast

In eukaryotes, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). Many heterologous proteins are retained in the ER due to suboptimal folding conditions. We previously reported that heterologous secretion of Pyrococcus furiosus beta-glucosidase in Saccharomyces cerevisiae resulted in the accumulation of a large fraction of inactive beta-glucosidase in the ER. In this work, we determine the effect of introducing additional genes of ER-resident yeast proteins, Kar2p (binding protein [BiP]) and protein disulfide isomerase (PDI), on relieving this bottleneck. Single-copy expression of BiP and PDI worked synergistically to improve secretion by reverse similar 60%. In an effort to optimize BiP and PDI interactions, we created a library of beta-glucosidase expression strains that incorporated four combinations of constitutively or inducibly-expressed BiP and PDI genes integrated to random gene copynumbers in the yeast chromosome. Approximately 15% of the transformants screened had secretion level improvements higher than that seen with single BiP/PDI gene overexpression, and the highest secreting strain had threefold higher beta-glucosidase levels than the control. Nineteen of the improved strains were re-examined for beta-glucosidase secretion as well as BiP and PDI levels. Within the improved transformants BiP and PDI levels ranged sevenfold and tenfold over the control, respectively. Interestingly, increasing BiP levels decreased beta-glucosidase secretion, whereas increasing PDI levels increased beta-glucosidase secretion. The action of PDI was unexpected because beta-glucosidase is not a disulfide-bonded protein. We suggest that PDI may be acting in a chaperone-like capacity or possibly creating mixed disulfides with the beta-glucosidase's lone cysteine residue during the folding and assembly process.     

The potato virus X TGBp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virus-infected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.     

Light enhances the unfolded protein response as measured by BiP2 gene expression and the secretory GFP-2SC marker in Arabidopsis

Disruption of the protein-folding capacity in the ER induces the accumulation of unfolded proteins and ER stress, which activate the unfolded protein response (UPR). Although UPR has been extensively studied in yeast and mammals, much less is known about UPR and its relationship with light in plants. Here, we examined the effects of chemically induced UPR and light on a molecular marker of UPR (binding protein, BiP2, gene expression) and a secretory green fluorescent protein marker (GFP-2SC) that is trafficked from the ER to vacuole in Arabidopsis thaliana (L). UPR, which was induced by DTT and tunicamycin (TM), increased Bip2 mRNA levels and decreased the levels of microsomal and vacuolar forms of GFP-2SC. Treatment with protease inhibitors lessened the effects of DTT and TM on GFP-2SC, indicating the decrease in GFP levels partially involved protein degradation. Light treatments synergistically enhanced the decrease in GFP levels in both the ER and vacuole and induced the expression of UPR marker genes for BiP2 and protein disulfide isomerase (PDI, EC 5.3.4.1). DTT and TM treatments required light for maximal induction of the UPR. Light-induced UPR occurred during the daily dark to light cycle and when dark-adapted plants were exposed to light. We propose that light activates the UPR to increase the protein folding capacity in the ER to accommodate an increase in translation during dark to light transitions.     

Analysis of unfolded protein response during single-chain antibody expression in Saccaromyces cerevisiae reveals different roles for BiP and PDI in folding

The production of recombinant proteins is a critical technology for biotechnology and biomedical research. Heterologous expression of secreted proteins can saturate the cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. The overexpression of chaperone binding protein (BiP) and disulfide bond isomerase (PDI) in Saccaromyces cerevisiae can effectively increase protein production levels of single-chain antibody (scFv) 4-4-20. These studies show that overexpression of BiP did not reduce the UPR activated by heterologous protein expression; however, overexpression of PDI or co-overexpression of BiP and PDI could reduce the UPR. We observed that co-overexpression of BiP and PDI led to the greatest secretion of scFv from the cell, but BiP and PDI appear to interact with the newly synthesized scFv at different stages in the folding process, as determined by pulse-chase analysis. We propose that BiP acts primarily to facilitate translocation and retain unfolded or partially folded scFv, and PDI actively folds the scFv through its functions as a catalyst, and/or an isomerase, of disulfide bonds. Free BiP is released when scFv is folded, stabilizing Ire1p, and leading to the reduced UPR.     

Zinc homeostasis is involved in unfolded protein response under salt stress

Accumulation of unfolded protein or misfolded protein causes endoplasmic reticulum (ER) stress. Increased salt concentration activates a stress response pathway in the ER in Arabidopsis thaliana to induce the expression of several salt stress response genes, leading to a more optimal protein folding environment in the ER. In addition, some salt stress-regulated proteins require zinc for their activity, including some zinc-dependent DNA binding proteins and zinc-finger proteins. In a recent study, we reported that ZTP29, a putative zinc transporter at the ER membrane, is involved in the response to salt stress through regulation of zinc level in the ER to induce the UPR pathway. In this addendum, we propose a testable hypothesis for the role of ZTP29 in the response to salt stress via the regulation of zinc levels in the ER.Key words: zinc, ER stress, unfolded protein response, salt stress, arabidopsisHigh salinity is a common abiotic stress that adversely affects plant growth and crop production.¹ Plants must sense the stress and transduce stress signals to activate response pathways leading to adaptation to, or tolerance of, the abiotic stress in salt environment.² Salt stress activates a stress response pathway in the endoplasmic reticulum (ER) in Arabidopsis thaliana, indicating that the adaptation of plants to salt stress involves ER stress signal regulation.^3,4 There is limited understanding of molecular mechanisms on ER stress in plants, as compared to yeast and mammalian cells. bZIP60, bZIP28, bZIP17 are three membrane-associated basic domain/leucine zipper (bZIP) factors, which have been reported as candidates for ER-folding proteins in plants.^5–7 BiP acts as a general chaperone in the ER lumen, due to its ability to discriminate between properly folded and unfolded protein structures.⁸ Unfolded or misfolded proteins are retained in the ER and form stable complexes with BiP and other ER resident chaperones.⁹ Zinc deficiency induces unfolded protein response (UPR) in most eukaryotes.¹⁰ Zinc is an important trace element, which participates in physiological and biochemical process in vivo. The requirement of zinc for proper ER function is evolutionarily conserved.     

Linear remodeling of helical virus by movement protein binding

Previously we have shown that encapsidated potato virus X (PVX) RNA was nontranslatable in vitro, but could be converted into a translatable form by binding of the PVX-coded movement protein (termed TGBp1) to one end of a polar helical PVX virion. We reported that binding of TGBp1 to coat protein (CP) subunits located at one extremity of the helical particles induced a linear destabilization of the CP helix, which was transmitted along the whole particle. Two model structures were used: (i) native PVX and (ii) artificial polar helical PVX-like particles lacking intact RNA (PVX(RNA-DEG)). Binding of TGBp1 to the end of either of these particles led to their destabilization, but no disassembly of the CP helix occurred. Influence of additional factors was required to trigger rapid disassembly of TGBp1-PVX and TGBp1-PVX(RNA-DEG) complexes. Thus: (i) no disassembly was observed unless TGBp1-PVX complex was translated. A novel phenomenon of TGBp1-dependent, ribosome-triggered disassembly of PVX was described: initiation of translation and few translocation steps were needed to trigger rapid (and presumably cooperative) disassembly of TGBp1-PVX into protein subunits and RNA. Importantly, the whole of the RNA molecule (including its 3'-terminal region) was released. The TGBp1-induced linear destabilization of CP helix was reversible, suggesting that PVX in TGBp1-PVX complex was metastable; (ii) entire disassembly of the TGBp1-PVX(RNA-DEG) complex (but not of the TGBp1-free PVX(RNA-DEG) particles) into 2.8S subunits was triggered under influence of a centrifugal field. To our knowledge, transmission of the linear destabilization along the polar helical protein array induced by a foreign protein binding to the end of the helix represents a novel phenomenon. It is tempting to suggest that binding of TGBp1 to the end of the PVX CP helix induced conformational changes in terminal CP subunits that can be linearly transferred along the whole helical particle, i.e. that intersubunit conformational changes may be transferred along the CP helix.     

Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

In Pichia pastoris, secretion of the A33 single-chain antibody fragment (A33scFv) was shown to reach levels of approximately 4 g l⁻¹ in fermentor cultures. In this study, we investigated whether manipulating chaperone and foldase levels in P. pastoris could further increase secretion of A33scFv. Cells were engineered to cooverexpress immunoglobulin binding protein (BiP) and/or protein disulfide isomerase (PDI) with A33scFv during growth in methanol as the sole carbon and energy source. Cooverexpression of BiP resulted in increased secretion levels of A33scFv by approximately threefold. In contrast, cooverexpression of PDI had no apparent effect on secretion of A33scFv. In cells cooverexpressing BiP and PDI, A33scFv secretion did not increase and protein levels remained the same as the control strain. We believe that secretion of A33scFv is increased by cooverexpression of BiP as a result of an increase in folding capacity inside the endoplasmic reticulum (ER). In addition, lack of increased single-chain secretion when PDI is coexpressed was unexpected due to the presence of disulfide bonds in A33scFv. We also show that during PDI cooverexpression with the single-chain there is a sixfold increase in BiP levels, indicating that the former is possibly inducing an unfolded protein response due to excess chaperone and recombinant protein in the ER.     

The putative Arabidopsis zinc transporter ZTP29 is involved in the response to salt stress

Salt stress leads to a stress response, called the unfolded protein response (UPR), in the endoplasmic reticulum (ER). UPR is also induced in a wide range of organisms by zinc deficiency. However, it is not clear whether regulation of zinc levels is involved in the initiation of the UPR in plant response to salt stress. In this study, a putative zinc transporter, ZTP29, was identified in Arabidopsis thaliana. ZTP29 localizes to the ER membrane and is expressed primarily in hypocotyl and cotyledon tissues, but its expression can be induced in root tissue by salt stress. T-DNA insertion into the ZTP29 gene led to NaCl hypersensitivity in seed germination and seedling growth, leaf etiolation, and widening of cells in the root elongation zone. In addition, in ztp29 mutant plants, salt stress-induced upregulation of the UPR pathway genes BiP2 and bZIP60 was inhibited. Furthermore, under conditions of salt stress, upregulation of BiP2 and bZIP60 was inhibited by treatment with high concentrations of zinc in both control and ztp29 plants. However, zinc chelation restored salt stress-induced BiP2 and bZIP60 upregulation in ztp29 mutant plants. These experimental results suggest that ZTP29 is involved in the response to salt stress, perhaps through regulation of zinc levels required to induce the UPR pathway.     

Endoplasmic reticulum-quality control chaperones facilitate the biogenesis of Cf receptor-like proteins involved in pathogen resistance of tomato

Cf proteins are receptor-like proteins (RLPs) that mediate resistance of tomato (Solanum lycopersicum) to the foliar pathogen Cladosporium fulvum. These transmembrane immune receptors, which carry extracellular leucine-rich repeats that are subjected to posttranslational glycosylation, perceive effectors of the pathogen and trigger a defense response that results in plant resistance. To identify proteins required for the functionality of these RLPs, we performed immunopurification of a functional Cf-4-enhanced green fluorescent protein fusion protein transiently expressed in Nicotiana benthamiana, followed by mass spectrometry. The endoplasmic reticulum (ER) heat shock protein70 binding proteins (BiPs) and lectin-type calreticulins (CRTs), which are chaperones involved in ER-quality control, were copurifying with Cf-4-enhanced green fluorescent protein. The tomato and N. benthamiana genomes encode four BiP homologs and silencing experiments revealed that these BiPs are important for overall plant viability. For the three tomato CRTs, virus-induced gene silencing targeting the plant-specific CRT3a gene resulted in a significantly compromised Cf-4-mediated defense response and loss of full resistance to C. fulvum. We show that upon knockdown of CRT3a the Cf-4 protein accumulated, but the pool of Cf-4 protein carrying complex-type N-linked glycans was largely reduced. Together, our study on proteins required for Cf function reveals an important role for the CRT ER chaperone CRT3a in the biogenesis and functionality of this type of RLP involved in plant defense.     

Thapsigargin down-regulates protein levels of GRP78/BiP in INS-1E cells

Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) and express large amounts of chaperones and protein disulfide isomerases (PDI) to meet the high demand for synthesis of proteins. We have observed an unexpected decrease in chaperone protein level in the β-cell model INS-1E after exposure to the ER stress inducing agent thapsigargin. As these cells are a commonly used model for primary β-cells and has been shown to be vulnerable to ER stress, we hypothesize these cells are incapable of mounting a chaperone defense upon activation of ER stress. To investigate the chaperone expression during an ER stress response, induced by thapsigargin in INS-1E cells, we used quantitative mass spectrometry based proteomics. The results displayed a decrease of GRP78/BiP, PDIA3 and PDIA6. Decrease of GRP78/BiP was verified by Western blot and occurred in parallel with enhanced levels of p-eIF2α and CHOP. In contrast to INS-1E cells, GRP78/BiP was not decreased in MIN6 cell or rat and mouse islets after thapsigargin exposure. Investigation of the decreased protein levels of GRP78/BiP indicates that this is not a consequence of reduced mRNA expression. Rather the reduction results from the combined effect of reduced protein synthesis and enhanced proteosomal degradation and possibly also degradation via autophagy. Induction of ER stress with thapsigargin leads to lower protein levels of GRP78/BiP, PDIA3 and PDIA6 in INS-1E cells which may contribute to the susceptibility of ER stress in this β-cell model.