BioID performed on Golgi-enriched fractions identify C10orf76 as a GBF1 Binding Protein essential for Golgi maintenance and secretion
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF (Thermo)
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Brian Raught
PROVIDER: MSV000083866 | MassIVE | Mon May 27 18:12:00 BST 2019
SECONDARY ACCESSION(S): PXD014024
REPOSITORIES: MassIVE
Similar Datasets
Project description:The Golgi-specific Brefeldin-A resistance factor 1 (GBF1) is the only large GEF that regulates Arf activation at the cis-Golgi and is actively recruited to membranes on an increase in Arf-GDP. Recent studies have revealed that GBF1 recruitment requires one or more heat-labile and protease-sensitive protein factor(s) (Quilty et al., 2018, J. Cell Science, 132). Proximity-dependent biotinylation (BioID) and mass spectrometry from enriched Golgi fractions identified GBF1 proximal proteins that may regulate its recruitment. Knockdown studies revealed C10orf76 to be involved in Golgi maintenance. We find that C10orf76 interacts with GBF1 and rapidly cycles on and off GBF1-positive Golgi structures. More importantly, its depletion causes Golgi fragmentation, alters GBF1 recruitment, and impairs secretion. Homologs were identified in most species, suggesting its presence in the last eukaryotic common ancestor.
| S-EPMC6823846 | biostudies-literature
Project description:We explore the transcriptional response of mammalian cells undergoing various insults to Golgi homeostasis. HEK293 cells (Flp-In T-REx 293 cells) stably containing a doxycycline-inducible Golgi-localized HaloTag2 construct (GA-HT2) were treated with the ionophore nigericin, the glycosylation inhibitor xyloside, or were induced by doxycycline and treated with the hydrophobic tag HyT36 to induce destabilization of GA-HT2. We found that while nigericin and xyloside induce global transcriptional changes, destabilization of GA-HT2 induces a Golgi-specific response.
2018-06-21 | GSE99490 | GEO
Project description:The process of aging is associated with disturbed mineral homeostasis, such as zinc deficiency. Simultaneously, cellular response to external stimuli, including receptor signaling and DNA repair response diminishes, and epigenetic dysregulation occurs. The Golgi apparatus plays various roles in the cell; however, the effect of aging on the morphology and function of the Golgi apparatus and the impact of Golgi senescence in cellular activity remains unknown. In the present study, we found that Golgi stress is associated with senescent cell irresponsiveness to external stimuli. The senescent Golgi was associated with a disassembled microtubule network in the Golgi and perinuclear areas. These effects could be reproduced by disruption of the Golgi-associated microtubules or zinc depletion. To clarify the importance of Golgi-zinc homeostasis in more detail, the implications of the Golgi-zinc transporter ZIP13 were analyzed; Zip13-deficient mice Golgi exhibited morphology similar to that of senescent Golgi. Additionally, fibroblasts from Zip13-deficient mice showed dysregulation of DNA repair and cellular acetylation with downregulated nuclear translocation of p300, HDAC1/2, and p53 proteins, which are reminiscent characteristics of senescent cells. Our findings demonstrate the underlying reason for senescent cell irresponsiveness to various stimuli and highlight the importance of a zinc-rich diet during aging.
2024-08-26 | GSE235450 | GEO
Project description:We investigated the interaction network of human PKD2 in the cytosol as well as in Golgi-enriched subcellular protein fractions, using an affinity enrichment strategy combined with chemical cross-linking/mass spectrometry (MS). Analysis of the subproteomes revealed the presence of distinct proteins in the cytosolic and Golgi fractions. The covalent fixation of transient or weak interactors by chemical cross-linking allowed capturing interaction partners that might otherwise vanish during conventional pull-down experiments. In total, 31 interaction partners were identified for PKD2.
2016-09-27 | PXD003913 | Pride
Project description:Developing xylem from strong and mild compression wood of Pinus radiata were harvested and Golgi membranes enriched by density centrifugation. Enriched membranes were further purified by free-flow electrophoresis. A total of 3 fractions were collected (from each tissue) and analyzed by tandem mass spectromtery.
2014-04-14 | PXD000557 | Pride
Project description:Androgen receptor (AR) plays an essential role in normal prostate development and prostate cancer (PCa) progression. To understand the role of AR at the single-cell level, we performed single-cell transcriptome analysis on PCa cells stimulated with androgen and antiandrogen to reconstruct the dynamic and direct AR transcriptional network. Our work reveals that androgen stimulates the ER and Golgi stress response , promoting secreting protein trafficking, and inhibiting cell apoptosis. Moreover, we identify an ER-to-Golgi protein vesicle-mediated transport gene signature essential for maximal androgen-mediated ER-Golgi trafficking, cell proliferation, and association with PCa prognosis and progression. Notably, we show that AR collaborates with CREB3L2, XXX, to coordinately promote ER-Golgi trafficking of Golgi enzyme Mannosidase II and PCa cell survival. Finally, we show the inhibition of the ER-Golgi transport process with Brefeldin A leads to tumor regression. Our study collectively reveals the heterogeneity of PCa cell transcriptional response to androgen stimulation, demonstrates a functional role for increased ER-Golgi trafficking process, and provides a mechanism for how the process is augmented in PCa as well as the potential of targeting may provide novel treatment strategies.
2021-10-10 | GSE165562 | GEO
Project description:Abnormal distribution of cellular cholesterol is associated with numerous diseases, including cardiovascular and neurodegenerative diseases. Regulated transport of cholesterol is critical for maintaining its proper distribution in the cell, yet the underlying mechanisms remain unclear. Here, we show that lipid transfer proteins, namely ORP9, OSBP, and GRAMD1s/Asters (GRAMD1a/GRAMD1b/GRAMD1c), control non-vesicular cholesterol transport at points of contact between the ER and the trans-Golgi network (TGN), thereby maintaining cellular cholesterol distribution. ORP9 localizes to the TGN via interaction between its tandem α-helices and ORP10/ORP11. ORP9 extracts PI4P from the TGN to prevent its overaccumulation and suppresses OSBP-mediated PI4P-driven cholesterol transport to the Golgi. By contrast, GRAMD1s transport excess cholesterol from the Golgi to the ER, thereby preventing its build-up. Cells lacking ORP9 exhibit accumulation of cholesterol at the Golgi, which is further enhanced by additional depletion of GRAMD1s with major accumulation in the plasma membrane. This is accompanied by chronic activation of the SREBP-2 signalling pathway. Our findings reveal the importance of regulated lipid transport at ER-Golgi contacts for maintaining cellular cholesterol distribution and homeostasis.
2023-09-24 | GSE240960 | GEO
Project description:The Golgi complex is a central hub for intracellular protein trafficking and glycosylation. Steady-state localization of glycosylation enzymes is achieved by a combination of mechanisms involving retention and recycling, but the machinery governing these mechanisms is poorly understood. Herein we show that the Golgi-associated retrograde protein (GARP) complex is a critical component of this machinery. Using multiple human cell lines, we show that depletion of GARP subunits impairs Golgi modification of N- and O-glycans and reduces the stability of glycoproteins and Golgi enzymes. Moreover, GARP-knockout (KO) cells exhibit reduced retention of glycosylation enzymes in the Golgi. A RUSH assay shows that, in GARP-KO cells, the enzyme beta-1,4-galactosyltransferase 1 is not retained at the Golgi complex but instead is missorted to the endolysosomal system. We propose that the endosomal system is part of the trafficking itinerary of Golgi enzymes or their recycling adaptors and that the GARP complex is essential for recycling and stabilization of the Golgi glycosylation machinery. [Media: see text].
| S-EPMC8351751 | biostudies-literature
Project description:Identification of TBC1D23 interacting proteins by IP-MS; TBC1D23 preferentially regulates the phosphorylation of Golgi-localized proteins
2023-03-13 | PXD040801 |
Project description:The Golgi is a membrane-bound organelle that is essential for protein and lipid biosynthesis. It represents a central trafficking hub that sorts proteins and lipids to various destinations or for secretion from the cell. The Golgi has emerged as a docking platform for cellular signaling pathways including LRRK2 kinase whose deregulation leads to Parkinson disease. Golgi dysfunction is associated with a broad spectrum of diseases including cancer, neurodegeneration, and cardiovascular diseases. To allow the study of the Golgi at high resolution, we report a rapid Golgi immunoprecipitation technique (Golgi-IP) to isolate intact Golgi mini-stacks for subsequent analysis of their content. By fusing the Golgi-resident protein TMEM115 to three tandem HA epitopes (GolgiTAG), we purified the Golgi using Golgi-IP with minimal contamination from other compartments. We then established an analysis pipeline using liquid chromatography coupled with mass spectrometry to characterize the human Golgi proteome, metabolome, and lipidome. Subcellular proteomics confirmed known Golgi proteins and identified proteins not previously associated with the Golgi. Metabolite profiling established the human Golgi metabolome and revealed the enrichment of uridine-diphosphate (UDP) sugars and their derivatives, which is consistent with their roles in protein and lipid glycosylation. Furthermore, targeted metabolomics validated SLC35A2 as the subcellular transporter for UDP-hexose. Finally, lipidomics analysis showed that phospholipids including phosphatidylcholine, phosphatidylinositol, and phosphatidylserine are the most abundant Golgi lipids and that glycosphingolipids are enriched in this compartment. Altogether, our work establishes a comprehensive molecular map of the human Golgi and provides a powerful method to study the Golgi with high precision in health and disease.
2023-06-27 | MTBLS6511 | MetaboLights