Endoplasmic reticulum-translocation is essential for APOL1 cellular toxicity.
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ABSTRACT:
SUBMITTER: Kruzel-Davila E
PROVIDER: S-EPMC8762391 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The rough endoplasmic reticulum is a major site of protein biosynthesis in all eukaryotic cells, serving as the entry point for the secretory pathway and as the initial integration site for the majority of cellular integral membrane proteins. The core components of the protein translocation machinery have been identified, and high-resolution structures of the targeting components and the transport channel have been obtained. Research in this area is now focused on obtaining a better understanding of the molecular mechanism of protein translocation and membrane protein integration.
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Project description:Two coding sequence variants (G1 and G2) of Apolipoprotein L1 (APOL1) gene have been implicated as a higher risk factor for chronic kidney diseases (CKD) in African Americans when compared with European Americans. Previous studies have suggested that the APOL1 G1 and G2 variant proteins are more toxic to kidney cells than the wild-type APOL1 G0, but the underlying mechanisms are poorly understood. To determine whether endoplasmic reticulum (ER) stress contributes to podocyte toxicity, we generated human podocytes (HPs) that stably overexpressed APOL1 G0, G1, or G2 (Vec/HPs, G0/HPs, G1/HPs, and G2/HPs). Propidium iodide staining showed that HP overexpressing the APOL1 G1 or G2 variant exhibited a higher rate of necrosis when compared with those overexpressing the wild-type G0 counterpart. Consistently, the expression levels of nephrin and podocin proteins were significantly decreased in the G1- or G2-overexpressing cells despite the maintenance of their mRNA expressions levels. In contrast, the expression of the 78-kDa glucose-regulated protein ((GRP78), also known as the binding Ig protein, BiP) and the phosphorylation of the eukaryotic translation initiation factor 1 (eIF1) were significantly elevated in the G1/HPs and G2/HPs, suggesting a possible occurrence of ER stress in these cells. Furthermore, ER stress inhibitors not only restored nephrin protein expression, but also provided protection against necrosis in G1/HPs and G2/HPs, suggesting that APOL1 risk variants cause podocyte injury partly through enhancing ER stress.
| S-EPMC6131197 | biostudies-literature
Project description:Lhs1p is an Hsp70-related chaperone localized in the endoplasmic reticulum (ER) lumen. Deltalhs1 mutant cells are viable but are constitutively induced for the unfolded protein response (UPR). Here, we demonstrate a severe growth defect in Deltaire1Deltalhs1 double mutant cells in which the UPR can no longer be induced. In addition, we have identified a UPR- regulated gene, SIL1, whose overexpression is sufficient to suppress the Deltaire1Deltalhs1 growth defect. SIL1 encodes an ER-localized protein that interacts directly with the ATPase domain of Kar2p (BiP), suggesting some role in modulating the activity of this vital chaperone. SIL1 is a non-essential gene but the Deltalhs1Deltasil1 double mutation is lethal and correlates with a complete block of protein translocation into the ER. We conclude that the IRE1-dependent induction of SIL1 is a vital adaptation in Deltalhs1 cells, and that the activities associated with the Lhs1 and Sil1 proteins constitute an essential function required for protein translocation into the ER. The Sil1 protein appears widespread amongst eukaryotes, with homologues in Yarrowia lipolytica (Sls1p), Drosophila and mammals.
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Project description:Production and trafficking of proteins entering the secretory pathway of eukaryotic cells is coordinated at the endoplasmic reticulum (ER) in a process that begins with protein translocation via the membrane-embedded ER translocon. The same complex is also responsible for the co-translational integration of membrane proteins and orchestrates polypeptide modifications that are often essential for protein function. We now show that the previously identified inhibitor of ER-associated degradation (ERAD) eeyarestatin 1 (ES(I)) is a potent inhibitor of protein translocation. We have characterised this inhibition of ER translocation both in vivo and in vitro, and provide evidence that ES(I) targets a component of the Sec61 complex that forms the membrane pore of the ER translocon. Further analyses show that ES(I) acts by preventing the transfer of the nascent polypeptide from the co-translational targeting machinery to the Sec61 complex. These results identify a novel effect of ES(I), and suggest that the drug can modulate canonical protein transport from the cytosol into the mammalian ER both in vitro and in vivo.
| S-EPMC2779136 | biostudies-literature
Project description:Misfolded secretory proteins are transported across the endoplasmic reticulum (ER) membrane into the cytosol for degradation by proteasomes. A large fraction of proteasomes in a cell is associated with the ER membrane. We show here that binding of proteasomes to ER membranes is salt sensitive, ATP dependent, and mediated by the 19S regulatory particle. The base of the 19S particle, which contains six AAA-ATPases, binds to microsomal membranes with high affinity, whereas the 19S lid complex binds weakly. We demonstrate that ribosomes and proteasomes compete for binding to the ER membrane and have similar affinities for their receptor. Ribosomes bind to the protein conducting channel formed by the Sec61 complex in the ER membrane. We co-precipitated subunits of the Sec61 complex with ER-associated proteasome 19S particles, and found that proteoliposomes containing only the Sec61 complex retained proteasome binding activity. Collectively, our data suggest that the Sec61 channel is a principal proteasome receptor in the ER membrane.
| S-EPMC1173161 | biostudies-literature
Project description:PBN1 was identified as a gene required for production of protease B (PrB) activity in Saccharomyces cerevisiae. PBN1 encodes an endoplasmic reticulum (ER)-localized, type I membrane glycoprotein and is essential for cell viability. To study the essential function(s) of Pbn1p, we constructed a strain with PBN1 under control of the GAL promoter. Depletion of Pbn1p in this strain abrogates processing of the ER precursor forms of PrB, Gas1p, and Pho8p. Depletion of Pbn1p does not affect exit of proprotease A or procarboxypeptidase Y from the ER, indicating that Pbn1p is not required for global exit from the ER. Depleting Pbn1p leads to a significant increase in the unfolded protein response pathway, accompanied by an expansion of bulk ER membrane, indicating that there is a defect in protein folding in the ER. pbn1-1, a nonlethal allele of PBN1, displays synthetic lethality with the ero1-1 allele (ERO1 is required for oxidation in the ER) and synthetic growth defects with the cne1Delta allele (CNE1 encodes calnexin). ER-associated degradation of a lumenal substrate, CPY*, is blocked in the absence of Pbn1p. These results suggest that Pbn1p is required for proper folding and/or the stability of a subset of proteins in the ER. Thus, Pbn1p is an essential chaperone-like protein in the ER of yeast.
| S-EPMC1347969 | biostudies-literature
Project description:Mannose-phosphate-dolichol (MPD) is a multifunctional glycolipid that is synthesized on the cytoplasmic face of the endoplasmic reticulum (ER) and used on the opposite side of the membrane in the ER lumen as a mannose donor for protein N-glycosylation, glycosylphosphatidylinositol-anchoring, and C- and O-mannosylation. For this, it must be translocated, i.e., flipped, across the ER membrane. The molecular identity of the MPD translocator (MPD flippase) is not known. Here we show that MPD-flippase activity can be reconstituted in large unilamellar proteoliposomes prepared from phosphatidylcholine and Triton X-100-solubilized rat liver ER-membrane proteins. Using carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl NO(+) as a topological probe to selectively oxidize MPD molecules in the outer leaflet of the reconstituted vesicles, we demonstrate rapid, protein-dependent, ATP-independent transbilayer translocation of MPD from the inner to the outer leaflet. MPD flipping is highly specific. A stereoisomer of MPD was weakly translocated (> 10-fold lower rate) compared with natural MPD. Competition experiments with water-soluble isoprenyl monophosphates showed that MPD flippase recognizes the dolichol chain of MPD, preferring a saturated alpha-isoprene to unsaturated trans- or cis- alpha-isoprene units. Chromatography of the detergent-solubilized ER protein mixture prior to reconstitution indicated that MPD flippase (i) is not a Con A-binding glycoprotein and (ii) can be resolved from the oligosaccharide-diphosphate dolichol flippase that translocates Man(5)GlcNAc(2)-PP-dolichol, a lipid intermediate of N-glycosylation. These data provide a mechanistic framework for understanding MPD flipping, as well as a biochemical basis for identifying MPD flippase.
| S-EPMC2895134 | biostudies-literature
Project description:The oligosaccharide donor for protein N-glycosylation, Glc(3)Man(9)GlcNAc(2)-PP-dolichol, is synthesized via a multistep pathway that starts on the cytoplasmic face of the endoplasmic reticulum (ER) and ends in the lumen where the glycosylation reaction occurs. This necessitates transbilayer translocation or flipping of the lipid intermediate Man(5)GlcNAc(2)-PP-dolichol (M5-DLO) across the ER membrane. The mechanism by which M5-DLO-or any other lipid-is flipped across the ER is unknown, except that specific transport proteins or flippases are required. We recently demonstrated M5-DLO flipping activity in proteoliposomes reconstituted from detergent-solubilized ER membrane proteins and showed that it was ATP-independent and required a trypsin-sensitive protein that sedimented at approximately 4S. By using an activity-enriched fraction devoid of glycerophospholipid flippase activity, we now report that M5-DLO is rapidly flipped in the reconstituted system with a time constant tau <2 min, whereas its triantennary structural isomer is flipped slowly with tau >200 min. DLOs larger than M5-DLO are also poorly translocated, with tau ranging from approximately 10 min to >200 min. We conclude that (i) the number and arrangement of mannoses in the DLO glycan has a profound effect on the ability of the DLO to be translocated by the flippase, (ii) glycan size per se does not dictate whether a DLO will be flipped, and (iii) the flippase is highly specific for M5-DLO. Our results suggest a simple structural model for the interaction between the DLO head group and the flippase.
| S-EPMC2630073 | biostudies-literature
Project description:BackgroundCalreticulin (CRT) is major Ca 2+ -binding chaperone mainly resident in the endoplasmic reticulum (ER) lumen. Recently, it has been shown that non-ER CRT regulates a wide array of cellular responses. We previously found that CRT was up-regulated during hypoxia/reoxygenation (H/R) and this study was aimed to investigate whether CRT nuclear translocation aggravates ER stress (ERS)-associated apoptosis during H/R injury in neonatal rat cardiomyocytes.MethodsApoptosis rate and lactate dehydrogenase (LDH) leakage in culture medium were measured as indices of cell injury. Immunofluorescence staining showed the morphological changes of ER and intracellular translocation of CRT. Western blotting or reverse transcription polymerase chain reaction was used to detect the expression of target molecules.ResultsCompared with control, H/R increased apoptosis rate and LDH activity. The ER became condensed and bubbled, and CRT translocated to the nucleus. Western blotting showed up-regulation of CRT, Nrf2, activating transcription factor 4 (ATF4), CHOP and caspase-12 expression after H/R. Exogenous CRT overexpression induced by plasmid transfection before H/R increased cell apoptosis, LDH leakage, ER disorder, CRT nuclear translocation and the expression of ERS-associated molecules. However, administration of the ERS inhibitor, taurine, or CRT siRNA alleviated cell injury, ER disorder, and inhibited ERS-associated apoptosis.ConclusionsOur results indicated that during H/R stress, CRT translocation increases cell apoptosis and LDH leakage, aggravates ER disorder, up-regulates expression of nuclear transcription factors, Nrf2 and ATF4, and activates ERS-associated apoptosis.
| S-EPMC4837866 | biostudies-literature
Project description:Translocon clogging at the endoplasmic reticulum (ER) as a result of translation stalling triggers ribosome UFMylation, activating Translocation-Associated Quality Control (TAQC) to degrade clogged substrates. How cells sense ribosome UFMylation to initiate TAQC is unclear. We conduct a genome-wide CRISPR/Cas9 screen to identify an uncharacterized membrane protein named SAYSD1 that facilitates TAQC. SAYSD1 associates with the Sec61 translocon, and also recognizes both ribosome and UFM1 directly, engaging a stalled nascent chain to ensure its transport via the TRAPP complex to lysosomes for degradation. Like UFM1 deficiency, SAYSD1 depletion causes the accumulation of translocation-stalled proteins at the ER and triggers ER stress. Importantly, disrupting UFM1- and SAYSD1-dependent TAQC in Drosophila leads to intracellular accumulation of translocation-stalled collagens, defective collagen deposition, abnormal basement membranes, and reduced stress tolerance. Thus, SAYSD1 acts as a UFM1 sensor that collaborates with ribosome UFMylation at the site of clogged translocon, safeguarding ER homeostasis during animal development.
2023-01-20 | GSE222104 | GEO