Endoplasmic reticulum export, subcellular distribution, and fibril formation by Pmel17 require an intact N-terminal domain junction.
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ABSTRACT:
SUBMITTER: Leonhardt RM
PROVIDER: S-EPMC2871485 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Sphingomyelin synthase (SMS) is the key enzyme for cross-talk between bioactive sphingolipids and glycerolipids. In mammals, SMS consists of two isoforms: SMS1 is localized in the Golgi apparatus, whereas SMS2 is localized in both the Golgi and plasma membranes. SMS2 seems to exert cellular functions through protein-protein interactions; however, the existence and functions of quaternary structures of SMS1 and SMS2 remain unclear. Here we demonstrate that both SMS1 and SMS2 form homodimers. The SMSs have six membrane-spanning domains, and the N and C termini of both proteins face the cytosolic side of the Golgi apparatus. Chemical cross-linking and bimolecular fluorescence complementation revealed that the N- and/or C-terminal tails of the SMSs were in close proximity to those of the other SMS in the homodimer. Homodimer formation was significantly decreased by C-terminal truncations, SMS1-ΔC22 and SMS2-ΔC30, indicating that the C-terminal tails of the SMSs are primarily responsible for homodimer formation. Moreover, immunoprecipitation using deletion mutants revealed that the C-terminal tail of SMS2 mainly interacted with the C-terminal tail of its homodimer partner, whereas the C-terminal tail of SMS1 mainly interacted with a site other than the C-terminal tail of its homodimer partner. Interestingly, homodimer formation occurred in the endoplasmic reticulum (ER) membrane before trafficking to the Golgi apparatus. Reduced homodimerization caused by C-terminal truncations of SMSs significantly reduced ER-to-Golgi transport. Our findings suggest that the C-terminal tails of SMSs are involved in homodimer formation, which is required for efficient transport from the ER.
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Project description:The Endoplasmic Reticulum–Mitochondria Encounter Structure (ERMES) is a protein complex that tethers the two organelles and creates the physical basis for communication between them. ERMES functions in lipid and calcium exchange between the ER and mitochondria, mitochondrial protein import and maintenance of mitochondrial morphology and genome. Here we report that ERMES is also required for iron homeostasis. Loss of ERMES components activates an Aft1-dependent iron deficiency response even in iron-replete conditions, leading to accumulation of excess iron inside the cell. This function is independent of ERMES known roles in calcium regulation, phospholipid biosynthesis or mitochondrial biology. A mutation in the vacuolar protein sorting 13 (VPS13) gene that rescues the glycolytic phenotype of ERMES mutants suppresses the iron deficiency response and iron accumulation. Our study reveals that proper communication between the ER and mitochondria is required for appropriate maintenance of cellular iron levels.
2017-06-20 | GSE99499 | GEO
Project description:The endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES) is a protein complex that physically tethers the two organelles to each other and creates the physical basis for communication between them. ERMES functions in lipid exchange between the ER and mitochondria, protein import into mitochondria, and maintenance of mitochondrial morphology and genome. Here, we report that ERMES is also required for iron homeostasis. Loss of ERMES components activates an Aft1-dependent iron deficiency response even in iron-replete conditions, leading to accumulation of excess iron inside the cell. This function is independent of known ERMES roles in calcium regulation, phospholipid biosynthesis, or effects on mitochondrial morphology. A mutation in the vacuolar protein sorting 13 (VPS13) gene that rescues the glycolytic phenotype of ERMES mutants suppresses the iron deficiency response and iron accumulation. Our findings reveal that proper communication between the ER and mitochondria is required for appropriate maintenance of cellular iron levels.
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Project description:Background: Circulating polymers of alpha1-antitrypsin (α1AT) are chemo-attractant for neutrophils and contribute to inflammation in pulmonary, vascular and adipose tissues. Cellular factors affecting the intracellular itinerary of mutant polymerogenic α1AT remain obscure. Methods: Here, we report on an unbiased genome-wide CRISPR/Cas9 screen for regulators of trafficking of the polymerogenic α1AT-H334D variant. Single guide RNAs targeting genes whose inactivation enhanced accumulation of polymeric α1AT were enriched by iterative construction of CRISPR libraries based on genomic DNA from fixed cells selected for high polymer content by fluorescence-activated cell sorting. This approach bypassed the limitation to conventional enrichment schemes imposed by cell fixation. Results: Our screen identified 121 genes involved in polymer retention at false discovery rate < 0.1. From that set of genes, the pathway ‘cargo loading into COPII-coated vesicles’ was overrepresented with 16 significant genes, including two transmembrane cargo receptors, LMAN1 (ERGIG-53) and SURF4. LMAN1 and SURF4-disrupted cells displayed a secretion defect extended beyond α1AT monomers to polymers, whose low-level secretion was especially dependent on SURF4 and correlated with SURF4-α1AT-H334D physical interaction and with enhanced co-localisation of polymeric α1AT-H334D with the endoplasmic reticulum (ER). Conclusions: These findings suggest that ER cargo receptors co-ordinate intracellular progression of α1AT out of the ER and modulate the accumulation of polymeric α1AT not only by controlling the concentration of precursor monomers but also through a previously-unrecognised role in secretion of the polymers themselves.
2021-06-02 | GSE158574 | GEO
Project description:Cellubrevin is a ubiquitously expressed membrane protein that is localized to endosomes throughout the endocytotic pathway and functions in constitutive exocytosis. We report that cellubrevin binds with high specificity to BAP31, a representative of a highly conserved family of integral membrane proteins that has recently been discovered to be binding proteins of membrane immunoglobulins. The interaction between BAP31 and cellubrevin is sensitive to high ionic strength and appears to require the transmembrane regions of both proteins. No other proteins of liver membrane extracts copurified with BAP31 on immobilized recombinant cellubrevin, demonstrating that the interaction is specific. Synaptobrevin I bound to BAP31 with comparable affinity, whereas only weak binding was detectable with synaptobrevin II. Furthermore, a fraction of BAP31 and cellubrevin was complexed when each of them was quantitatively immunoprecipitated from detergent extracts of fibroblasts (BHK 21 cells). During purification of clathrin-coated vesicles or early endosomes, BAP31 did not cofractionate with cellubrevin. Rather, the protein was enriched in ER-containing fractions. When BHK cells were analyzed by immunocytochemistry, BAP31 did not overlap with cellubrevin, but rather colocalized with resident proteins of the ER. In addition, immunoreactive vesicles were clustered in a paranuclear region close to the microtubule organizing center, but different from the Golgi apparatus. When microtubules were depolymerized with nocodazole, this accumulation disappeared and BAP31 was confined to the ER. Truncation of the cytoplasmic tail of BAP31 prevented export of cellubrevin, but not of the transferrin receptor from the ER. We conclude that BAP31 represents a novel class of sorting proteins that controls anterograde transport of certain membrane proteins from the ER to the Golgi complex.
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Project description:Two independent functions of cTAGE5 have been reported in collagen VII export from the endoplasmic reticulum (ER). cTAGE5 not only forms a cargo receptor complex with TANGO1, but it also acts as a scaffold to recruit Sec12, a guanine-nucleotide exchange factor for Sar1 GTPase, to ER exit sites. However, the relationship between the two functions remains unclear. Here we isolated point mutants of cTAGE5 that lost Sec12-binding ability but retained binding to TANGO1. Although expression of the mutant alone could not rescue the defects in collagen VII secretion mediated by cTAGE5 knockdown, coexpression with Sar1, but not with the GTPase-deficient mutant, recovered secretion. The expression of Sar1 alone failed to rescue collagen secretion in cTAGE5-depleted cells. Taken together, these results suggest that two functionally irreplaceable and molecularly separable modules in cTAGE5 are both required for collagen VII export from the ER. The recruitment of Sec12 by cTAGE5 contributes to efficient activation of Sar1 in the vicinity of ER exit sites. In addition, the GTPase cycle of Sar1 appears to be responsible for collagen VII exit from the ER.
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Project description:The mammalian endoplasmic reticulum (ER) is an organelle that maintains a complex, compartmentalized organization of interconnected cisternae and tubules while supporting a continuous flow of newly synthesized proteins and lipids to the Golgi apparatus. Using a phenotypic screen, we identify a small molecule, dispergo, that induces reversible loss of the ER cisternae and extensive ER tubulation, including formation of ER patches comprising densely packed tubules. Dispergo also prevents export from the ER to the Golgi apparatus, and this traffic block results in breakdown of the Golgi apparatus, primarily due to maintenance of the constitutive retrograde transport of its components to the ER. The effects of dispergo are reversible, since its removal allows recovery of the ER cisternae at the expense of the densely packed tubular ER patches. This recovery occurs together with reactivation of ER-to-Golgi traffic and regeneration of a functional Golgi with correct morphology. Because dispergo is the first small molecule that reversibly tubulates the ER and inhibits its export function, it will be useful in studying these complex processes.
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Project description:Circulating polymers of α1-antitrypsin (α1AT) are neutrophil chemo-attractants and contribute to inflammation, yet cellular factors affecting their secretion remain obscure. We report on a genome-wide CRISPR-Cas9 screen for genes affecting trafficking of polymerogenic α1ATH334D. A CRISPR enrichment approach based on recovery of single guide RNA (sgRNA) sequences from phenotypically selected fixed cells reveals that cells with high-polymer content are enriched in sgRNAs targeting genes involved in "cargo loading into COPII-coated vesicles," where "COPII" is coat protein II, including the cargo receptors lectin mannose binding1 (LMAN1) and surfeit protein locus 4 (SURF4). LMAN1- and SURF4-disrupted cells display a secretion defect extending beyond α1AT monomers to polymers. Polymer secretion is especially dependent on SURF4 and correlates with a SURF4-α1ATH334D physical interaction and with their co-localization at the endoplasmic reticulum (ER). These findings indicate that ER cargo receptors co-ordinate progression of α1AT out of the ER and modulate the accumulation of polymeric α1AT not only by controlling the concentration of precursor monomers but also by promoting secretion of polymers.
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Project description:Endoplasmic reticulum (ER) homeostasis is essential for cell function. Increasing evidence indicates that, efficient protein ER export is important for ER homeostasis. However, the consequence of impaired ER export remains largely unknown. Herein, we found that defective ER protein transport caused by either Sar1 mutants or brefeldin A impaired proinsulin oxidative folding in the ER of β-cells. Misfolded proinsulin formed aberrant disulfide-linked dimers and high molecular weight proinsulin complexes, and induced ER stress. Limiting proinsulin load to the ER alleviated ER stress, indicating that misfolded proinsulin is a direct cause of ER stress. This study revealed significance of efficient ER export in maintaining ER protein homeostasis and native folding of proinsulin. Given the fact that proinsulin misfolding plays an important role in diabetes, this study suggests that enhancing ER export may be a potential therapeutic target to prevent/delay β-cell failure caused by proinsulin misfolding and ER stress.
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Project description:BACKGROUND: Endoplasmic reticulum (ER) quality control mechanisms are part of a comprehensive system to manage cell stress. The flux of molecules is monitored to retain folding intermediates and target misfolded molecules to ER-associated degradation (ERAD) pathways. The mechanisms of sorting remain unclear. While some proteins are retained statically, the classical model substrate CPY* is found in COPII transport vesicles, suggesting a retrieval mechanism for retention. However, its management can be even more dynamic. If ERAD is saturated under stress, excess CPY* traffics to the vacuole for degradation. These observations suggest that misfolded proteins might display different signals for their management. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report the existence of a functional ER exit signal in the pro-domain of CPY*. Compromising its integrity causes ER retention through exclusion from COPII vesicles. The signal co-exists with other signals used for retention and degradation. Physiologically, the export signal is important for stress tolerance. Disabling it converts a benign protein into one that is intrinsically cytotoxic. CONCLUSIONS/SIGNIFICANCE: These data reveal the remarkable interplay between opposing signals embedded within ERAD substrate molecules and the mechanisms that decipher them. Our findings demonstrate the diversity of mechanisms deployed for protein quality control and maintenance of protein homeostasis.
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