ResearchPad - trafficking https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[GRAF2, WDR44, and MICAL1 mediate Rab8/10/11–dependent export of E-cadherin, MMP14, and CFTR ΔF508]]> https://www.researchpad.co/article/N88198355-9a3a-4f65-8c59-63c7abeb5ba9 In addition to the classical pathway of secretion, some transmembrane proteins reach the plasma membrane through alternative routes. Several proteins transit through endosomes and are exported in a Rab8-, Rab10-, and/or Rab11-dependent manner. GRAFs are membrane-binding proteins associated with tubules and vesicles. We found extensive colocalization of GRAF1b/2 with Rab8a/b and partial with Rab10. We identified MICAL1 and WDR44 as direct GRAF-binding partners. MICAL1 links GRAF1b/2 to Rab8a/b and Rab10, and WDR44 binds Rab11. Endogenous WDR44 labels a subset of tubular endosomes, which are closely aligned with the ER via binding to VAPA/B. With its BAR domain, GRAF2 can tubulate membranes, and in its absence WDR44 tubules are not observed. We show that GRAF2 and WDR44 are essential for the export of neosynthesized E-cadherin, MMP14, and CFTR ΔF508, three proteins whose exocytosis is sensitive to ER stress. Overexpression of dominant negative mutants of GRAF1/2, WDR44, and MICAL1 also interferes with it, facilitating future studies of Rab8/10/11–dependent exocytic pathways of central importance in biology.

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<![CDATA[MEF2C Silencing Attenuates Load-Induced Left Ventricular Hypertrophy by Modulating mTOR/S6K Pathway in Mice]]> https://www.researchpad.co/article/5989daeaab0ee8fa60bbee3f

Background

The activation of the members of the myocyte enhancer factor-2 family (MEF2A, B, C and D) of transcription factors promotes cardiac hypertrophy and failure. However, the role of its individual components in the pathogenesis of cardiac hypertrophy remains unclear.

Methodology/Principal Findings

In this study, we investigated whether MEF2C plays a role in mediating the left ventricular hypertrophy by pressure overload in mice. The knockdown of myocardial MEF2C induced by specific small interfering RNA (siRNA) has been shown to attenuate hypertrophy, interstitial fibrosis and the rise of ANP levels in aortic banded mice. We detected that the depletion of MEF2C also results in lowered levels of both PGC-1α and mitochondrial DNA in the overloaded left ventricle, associated with enhanced AMP:ATP ratio. Additionally, MEF2C depletion was accompanied by defective activation of S6K in response to pressure overload. Treatment with the amino acid leucine stimulated S6K and suppressed the attenuation of left ventricular hypertrophy and fibrosis in the aforementioned aortic banded mice.

Conclusion/Significance

These findings represent new evidences that MEF2C depletion attenuates the hypertrophic responses to mechanical stress and highlight the potential of MEF2C to be a target for new therapies to cardiac hypertrophy and failure.

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<![CDATA[EIPR1 controls dense-core vesicle cargo retention and EARP complex localization in insulin-secreting cells]]> https://www.researchpad.co/article/N4150200d-dbe1-487f-b3e6-6a59f823ef90

Dense-core vesicles (DCVs) are secretory vesicles found in neurons and endocrine cells. DCVs package and release cargoes including neuropeptides, biogenic amines, and peptide hormones. We recently identified the endosome-associated recycling protein (EARP) complex and the EARP-interacting-protein EIPR-1 as proteins important for controlling levels of DCV cargoes in Caenorhabditis elegans neurons. Here we determine the role of mammalian EIPR1 in insulinoma cells. We find that in Eipr1 KO cells, there is reduced insulin secretion, and mature DCV cargoes such as insulin and carboxypeptidase E (CPE) accumulate near the trans-Golgi network and are not retained in mature DCVs in the cell periphery. In addition, we find that EIPR1 is required for the stability of the EARP complex subunits and for the localization of EARP and its association with membranes, but EIPR1 does not affect localization or function of the related Golgi-associated retrograde protein (GARP) complex. EARP is localized to two distinct compartments related to its function: an endosomal compartment and a DCV biogenesis-related compartment. We propose that EIPR1 functions with EARP to control both endocytic recycling and DCV maturation.

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<![CDATA[Mitochondrial lipid droplet formation as a detoxification mechanism to sequester and degrade excessive urothelial membranes]]> https://www.researchpad.co/article/N1d39d702-ad9b-4b86-80cb-ec337332bdfe

The apical surface of the terminally differentiated mammalian urothelial umbrella cell is mechanically stable and highly impermeable, in part due to its coverage by urothelial plaques consisting of 2D crystals of uroplakin particles. The mechanism for regulating the uroplakin/plaque level is unclear. We found that genetic ablation of the highly tissue-specific sorting nexin Snx31, which localizes to plaques lining the multivesicular bodies (MVBs) in urothelial umbrella cells, abolishes MVBs suggesting that Snx31 plays a role in stabilizing the MVB-associated plaques by allowing them to achieve a greater curvature. Strikingly, Snx31 ablation also induces a massive accumulation of uroplakin-containing mitochondria-derived lipid droplets (LDs), which mediate uroplakin degradation via autophagy/lipophagy, leading to the loss of apical and fusiform vesicle plaques. These results suggest that MVBs play an active role in suppressing the excessive/wasteful endocytic degradation of uroplakins. Failure of this suppression mechanism triggers the formation of mitochondrial LDs so that excessive uroplakin membranes can be sequestered and degraded. Because mitochondrial LD formation, which occurs at a low level in normal urothelium, can also be induced by disturbance in uroplakin polymerization due to individual uroplakin knockout and by arsenite, a bladder carcinogen, this pathway may represent an inducible, versatile urothelial detoxification mechanism.

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<![CDATA[Degradation of integral membrane proteins modified with the photosensitive degron module requires the cytosolic endoplasmic reticulum–associated degradation pathway]]> https://www.researchpad.co/article/N749fe3ad-3118-4d0e-8b36-d0a964772d7e

Protein quality mechanisms are fundamental for proteostasis of eukaryotic cells. Endoplasmic reticulum–associated degradation (ERAD) is a well-studied pathway that ensures quality control of secretory and endoplasmic reticulum (ER)–resident proteins. Different branches of ERAD are involved in degradation of malfolded secretory proteins, depending on the localization of the misfolded part, the ER lumen (ERAD-L), the ER membrane (ERAD-M), and the cytosol (ERAD-C). Here we report that modification of several ER transmembrane proteins with the photosensitive degron (psd) module resulted in light-dependent degradation of the membrane proteins via the ERAD-C pathway. We found dependency on the ubiquitylation machinery including the ubiquitin-activating enzyme Uba1, the ubiquitin-­conjugating enzymes Ubc6 and Ubc7, and the ubiquitin–protein ligase Doa10. Moreover, we found involvement of the Cdc48 AAA-ATPase complex members Ufd1 and Npl4, as well as the proteasome, in degradation of Sec62-myc-psd. Thus, our work shows that ERAD-C substrates can be systematically generated via synthetic degron constructs, which facilitates future investigations of the ERAD-C pathway.

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<![CDATA[Vezatin is required for the maturation of the neuromuscular synapse]]> https://www.researchpad.co/article/Ne59223b1-a985-48ab-a6ff-b51fb4434c09

Key genes, such as Agrin, Lrp4, and MuSK, are required for the initial formation, subsequent maturation, and long-term stabilization of mammalian neuromuscular synapses. Additional molecules are thought to function selectively during the evolution and stabilization of these synapses, but these molecular players are largely unknown. Here, we used mass spectrometry to identify vezatin, a two-pass transmembrane protein, as an acetylcholine receptor (AChR)–associated protein, and we provide evidence that vezatin binds directly to AChRs. We show that vezatin is dispensable for the formation of synapses but plays a later role in the emergence of a topologically complex and branched shape of the synapse, as well as the stabilization of AChRs. In addition, neuromuscular synapses in vezatin mutant mice display premature signs of deterioration, normally found only during aging. Thus, vezatin has a selective role in the structural elaboration and postnatal maturation of murine neuromuscular synapses.

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<![CDATA[Uroplakins play conserved roles in egg fertilization and acquired additional urothelial functions during mammalian divergence]]> https://www.researchpad.co/article/5c8fb4e5d5eed0c484019e6b

Uroplakin (UP) tetraspanins and their associated proteins are major mammalian urothelial differentiation products that form unique two-dimensional crystals of 16-nm particles (“urothelial plaques”) covering the apical urothelial surface. Although uroplakins are highly expressed only in mammalian urothelium and are often referred to as being urothelium specific, they are also expressed in several mouse nonurothelial cell types in stomach, kidney, prostate, epididymis, testis/sperms, and ovary/oocytes. In oocytes, uroplakins colocalize with CD9 on cell-surface and multivesicular body-derived exosomes, and the cytoplasmic tail of UPIIIa undergoes a conserved fertilization-dependent, Fyn-mediated tyrosine phosphorylation that also occurs in Xenopus laevis eggs. Uroplakin knockout and antibody blocking reduce mouse eggs’ fertilization rate in in vitro fertilization assays, and UPII/IIIa double-knockout mice have a smaller litter size. Phylogenetic analyses showed that uroplakin sequences underwent significant mammal-specific changes. These results suggest that, by mediating signal transduction and modulating membrane stability that do not require two-dimensional-crystal formation, uroplakins can perform conserved and more ancestral fertilization functions in mouse and frog eggs. Uroplakins acquired the ability to form two-dimensional-crystalline plaques during mammalian divergence, enabling them to perform additional functions, including umbrella cell enlargement and the formation of permeability and mechanical barriers, to protect/modify the apical surface of the modern-day mammalian urothelium.

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<![CDATA[Human trafficking and labor exploitation: Toward identifying, implementing, and evaluating effective responses]]> https://www.researchpad.co/article/5c59ff0ed5eed0c4841359d5

In an Editorial, Ligia Kiss and Cathy Zimmerman discuss the need for research on the prevention of human trafficking and mitigation of its effects.

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<![CDATA[The apical protein Apnoia interacts with Crumbs to regulate tracheal growth and inflation]]> https://www.researchpad.co/article/5c478c97d5eed0c484bd34d1

Most organs of multicellular organisms are built from epithelial tubes. To exert their functions, tubes rely on apico-basal polarity, on junctions, which form a barrier to separate the inside from the outside, and on a proper lumen, required for gas or liquid transport. Here we identify apnoia (apn), a novel Drosophila gene required for tracheal tube elongation and lumen stability at larval stages. Larvae lacking Apn show abnormal tracheal inflation and twisted airway tubes, but no obvious defects in early steps of tracheal maturation. apn encodes a transmembrane protein, primarily expressed in the tracheae, which exerts its function by controlling the localization of Crumbs (Crb), an evolutionarily conserved apical determinant. Apn physically interacts with Crb to control its localization and maintenance at the apical membrane of developing airways. In apn mutant tracheal cells, Crb fails to localize apically and is trapped in retromer-positive vesicles. Consistent with the role of Crb in apical membrane growth, RNAi-mediated knockdown of Crb results in decreased apical surface growth of tracheal cells and impaired axial elongation of the dorsal trunk. We conclude that Apn is a novel regulator of tracheal tube expansion in larval tracheae, the function of which is mediated by Crb.

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<![CDATA[<i>PLoS Medicine</i> Issue Image | Vol. 16(1) January 2019]]> https://www.researchpad.co/article/5c5ca27bd5eed0c48441e474

Human trafficking and labor exploitation: Toward identifying, implementing, and evaluating effective responses

Worldwide, some 25 million people are estimated to be victims of human trafficking, including migrant workers who are subject to different forms of serious exploitation and abuse. In an Editorial, Ligia Kiss and Cathy Zimmerman discuss the need for researchers to move beyond documentation of the nature of trafficking phenomena and the contexts in which they occur, and aim to understand the different settings and relevant causal factors. Appropriate research methods can then be evaluated and used to investigate approaches aimed at the prevention of human trafficking and mitigation of its damaging and prolonged effects on the people and families involved.

Image Credit: Free-Photos, Pixabay

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<![CDATA[Tracking yeast pheromone receptor Ste2 endocytosis using fluorogen-activating protein tagging]]> https://www.researchpad.co/article/5c48f75ed5eed0c4841f64a5

To observe internalization of the yeast pheromone receptor Ste2 by fluorescence microscopy in live cells in real time, we visualized only those molecules present at the cell surface at the time of agonist engagement (rather than the total cellular pool) by tagging this receptor at its N-terminus with an exocellular fluorogen-activating protein (FAP). A FAP is a single-chain antibody engineered to bind tightly a nonfluorescent, cell-impermeable dye (fluorogen), thereby generating a fluorescent complex. The utility of FAP tagging to study trafficking of integral membrane proteins in yeast, which possesses a cell wall, had not been examined previously. A diverse set of signal peptides and propeptide sequences were explored to maximize expression. Maintenance of the optimal FAP-Ste2 chimera intact required deletion of two, paralogous, glycosylphosphatidylinositol (GPI)-anchored extracellular aspartyl proteases (Yps1 and Mkc7). FAP-Ste2 exhibited a much brighter and distinct plasma membrane signal than Ste2-GFP or Ste2-mCherry yet behaved quite similarly. Using FAP-Ste2, new information was obtained about the mechanism of its internalization, including novel insights about the roles of the cargo-selective endocytic adaptors Ldb19/Art1, Rod1/Art4, and Rog3/Art7.

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<![CDATA[Syntaxin clusters at secretory granules in a munc18-bound conformation]]> https://www.researchpad.co/article/5c48f74bd5eed0c4841f635a

Syntaxin (stx)-1 is an integral plasma membrane protein that is crucial for two distinct steps of regulated exocytosis, docking of secretory granules at the plasma membrane and membrane fusion. During docking, stx1 clusters at the granule docking site, together with the S/M protein munc18. Here we determined features of stx1 that contribute to its clustering at granules. In live insulin-secreting cells, stx1 and stx3 (but not stx4 or stx11) accumulated at docked granules, and stx1 (but not stx4) rescued docking in cells expressing botulinum neurotoxin-C. Using a series of stx1 deletion mutants and stx1/4 chimeras, we found that all four helical domains (Ha, Hb, Hc, SNARE) and the short N-terminal peptide contribute to recruitment to granules. However, only the Hc domain confers specificity, and it must be derived from stx1 for recruitment to occur. Point mutations in the Hc or the N-terminal peptide designed to interfere with binding to munc18-1 prevent stx1 from clustering at granules, and a mutant munc18 deficient in binding to stx1 does not cluster at granules. We conclude that stx1 is recruited to the docking site in a munc18-1–bound conformation, providing a rationale for the requirement for both proteins for granule docking.

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<![CDATA[Cargo induces retromer-mediated membrane remodeling on membranes]]> https://www.researchpad.co/article/5c48f76ad5eed0c4841f659b

Endosomes serve as a central sorting station of lipids and proteins that arrive via vesicular carrier from the plasma membrane and the Golgi complex. At the endosome, retromer complexes sort selected receptors and membrane proteins into tubules or vesicles that bud off the endosome. The mature endosome finally fuses with the lysosome. Retromer complexes consist of a cargo selection complex (CSC) and a membrane remodeling part (sorting nexin [SNX]-Bin/amphiphysin/Rvs [BAR], or Snx3 in yeast) and different assemblies of retromer mediate recycling of different cargoes. Due to this complexity, the exact order of events that results in carrier formation is not yet understood. Here, we reconstituted this process on giant unilamellar vesicles together with purified retromer complexes from yeast and selected cargoes. Our data reveal that the membrane remodeling activity of both Snx3 and the SNX-BAR complex is strongly reduced at low concentrations, which can be reactivated by CSC. At even lower concentrations, these complexes still associate with membranes, but only remodel membranes in the presence of their specific cargoes. Our data thus favor a simple model, where cargo functions as a specific trigger of retromer-mediated sorting on endosomes.

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<![CDATA[TOR-autophagy branch signaling via Imp1 dictates plant-microbe biotrophic interface longevity]]> https://www.researchpad.co/article/5bff0537d5eed0c484a3281f

Like other intracellular eukaryotic phytopathogens, the devastating rice blast fungus Magnaporthe (Pyricularia) oryzae first infects living host cells by elaborating invasive hyphae (IH) surrounded by a plant-derived membrane. This forms an extended biotrophic interface enclosing an apoplastic compartment into which fungal effectors can be deployed to evade host detection. M. oryzae also forms a focal, plant membrane-rich structure, the biotrophic interfacial complex (BIC), that accumulates cytoplasmic effectors for translocation into host cells. Molecular decision-making processes integrating fungal growth and metabolism in host cells with interface function and dynamics are unknown. Here, we report unanticipated roles for the M. oryzae Target-of-Rapamycin (TOR) nutrient-signaling pathway in mediating plant-fungal biotrophic interface membrane integrity. Through a forward genetics screen for M. oryzae mutant strains resistant to the specific TOR kinase inhibitor rapamycin, we discovered IMP1 encoding a novel vacuolar protein required for membrane trafficking, V-ATPase assembly, organelle acidification and autophagy induction. During infection, Δimp1 deletants developed intracellular IH in the first infected rice cell following cuticle penetration. However, fluorescently labeled effector probes revealed that interface membrane integrity became compromised as biotrophy progressed, abolishing the BIC and releasing apoplastic effectors into host cytoplasm. Growth between rice cells was restricted. TOR-independent autophagy activation in Δimp1 deletants (following infection) remediated interface function and cell-to-cell growth. Autophagy inhibition in wild type (following infection) recapitulated Δimp1. In addition to vacuoles, Imp1GFP localized to IH membranes in an autophagy-dependent manner. Collectively, our results suggest TOR-Imp1-autophagy branch signaling mediates membrane homeostasis to prevent catastrophic erosion of the biotrophic interface, thus facilitating fungal growth in living rice cells. The significance of this work lays in elaborating a novel molecular mechanism of infection stressing the dominance of fungal metabolism and metabolic control in sustaining long-term plant-microbe interactions. This work also has implications for understanding the enigmatic biotrophy to necrotrophy transition.

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<![CDATA[Dynamic changes in complexes of IRE1α, PERK, and ATF6α during endoplasmic reticulum stress]]> https://www.researchpad.co/article/5c0baf75d5eed0c4847566ea

The endoplasmic reticulum (ER) localized unfolded protein response (UPR) sensors, IRE1α, PERK, and ATF6α, are activated by the accumulation of misfolded proteins in the ER. It is unclear how the endogenous UPR sensors are regulated by both ER stress and the ER luminal chaperone BiP, which is a negative regulator of UPR sensors. Here we simultaneously examined the changes in the endogenous complexes of UPR sensors by blue native PAGE immunoblotting in unstressed and stressed cells. We found that all three UPR sensors exist as preformed complexes even in unstressed cells. While PERK complexes shift to large complexes, ATF6α complexes are reduced to smaller complexes on ER stress. In contrast, IRE1α complexes were not significantly increased in size on ER stress, unless IRE1α is overexpressed. Surprisingly, depletion of BiP had little impact on the endogenous complexes of UPR sensors. In addition, overexpression of BiP did not significantly affect UPR complexes, but suppressed ER stress mediated activation of IRE1α, ATF6α and, to a lesser extent, PERK. Furthermore, we captured the interaction between IRE1α and misfolded secretory proteins in cells, which suggests that the binding of unfolded proteins to preformed complexes of UPR sensors may be crucial for activation.

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<![CDATA[Control of insulin granule formation and function by the ABC transporters ABCG1 and ABCA1 and by oxysterol binding protein OSBP]]> https://www.researchpad.co/article/5c08b635d5eed0c48414d0a4

In pancreatic β-cells, insulin granule membranes are enriched in cholesterol and are both recycled and newly generated. Cholesterol’s role in supporting granule membrane formation and function is poorly understood. ATP binding cassette transporters ABCG1 and ABCA1 regulate intracellular cholesterol and are important for insulin secretion. RNAi inter­ference–induced depletion in cultured pancreatic β-cells shows that ABCG1 is needed to stabilize newly made insulin granules against lysosomal degradation; ABCA1 is also involved but to a lesser extent. Both transporters are also required for optimum glucose-stimulated insulin secretion, likely via complementary roles. Exogenous cholesterol addition rescues knockdown-induced granule loss (ABCG1) and reduced secretion (both transporters). Another cholesterol transport protein, oxysterol binding protein (OSBP), appears to act proximally as a source of endogenous cholesterol for granule formation. Its knockdown caused similar defective stability of young granules and glucose-stimulated insulin secretion, neither of which were rescued with exogenous cholesterol. Dual knockdowns of OSBP and ABC transporters support their serial function in supplying and concentrating cholesterol for granule formation. OSBP knockdown also decreased proinsulin synthesis consistent with a proximal endoplasmic reticulum defect. Thus, membrane cholesterol distribution contributes to insulin homeostasis at production, packaging, and export levels through the actions of OSBP and ABCs G1 and A1.

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<![CDATA[The dynamic Atg13-free conformation of the Atg1 EAT domain is required for phagophore expansion]]> https://www.researchpad.co/article/5c08b633d5eed0c48414d090

Yeast macroautophagy begins with the de novo formation of a double-membrane phagophore at the preautophagosomal structure/phagophore assembly site (PAS), followed by its expansion into the autophagosome responsible for cargo engulfment. The kinase Atg1 is recruited to the PAS by Atg13 through interactions between the EAT domain of the former and the tMIM motif of the latter. Mass-spectrometry data have shown that, in the absence of Atg13, the EAT domain structure is strikingly dynamic, but the function of this Atg13-free dynamic state has been unclear. We used structure-based mutational analysis and quantitative and superresolution microscopy to show that Atg1 is present on autophagic puncta at, on average, twice the stoichiometry of Atg13. Moreover, Atg1 colocalizes with the expanding autophagosome in a manner dependent on Atg8 but not Atg13. We used isothermal titration calorimetry and crystal structure information to design an EAT domain mutant allele ATG1DD that selectively perturbs the function of the Atg13-free state. Atg1DD shows reduced PAS formation and does not support phagophore expansion, showing that the EAT domain has an essential function that is separate from its Atg13-dependent role in autophagy initiation.

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<![CDATA[Commonly used trafficking blocks disrupt ARF1 activation and the localization and function of specific Golgi proteins]]> https://www.researchpad.co/article/5c0460a9d5eed0c4846ec618

Cold temperature blocks used to synchronize protein trafficking inhibit GBF1 function, leading to a decrease in ARF1-GTP levels and mislocalization of the ARF1 effector golgin-160. Several other, but not all, Golgi proteins including ARL1 also mislocalize. ARF1 activity and golgin-160 localization require more than 30 min to recover from these blocks.

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<![CDATA[Cdc48 and ubiquilins confer selective anterograde protein sorting and entry into the multivesicular body in yeast]]> https://www.researchpad.co/article/5c0460abd5eed0c4846ec63c

Cdc48/p97 and the ubiquilin family of UBA-UBL proteins are known for their role in the retrotranslocation of damaged proteins from the endoplasmic reticulum. We demonstrate that Cdc48 and the ubiquilin-like proteins in yeast also play a role in the anterograde trafficking of proteins, in this case the vacuolar protease, Cps1.

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<![CDATA[Membrane detachment is not essential for COG complex function]]> https://www.researchpad.co/article/5c0460add5eed0c4846ec666

COG is a multisubunit vesicle tethering complex in the Golgi. We demonstrate that both COG subcomplexes can be permanently attached to Golgi membranes and that major COG functions do not require cycling between the membrane and cytosol.

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