golgi transport
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Traffic ◽  
2022 ◽  
Author(s):  
Liying Guan ◽  
Yongzhi Yang ◽  
Jing Jing Liang ◽  
Yue Miao ◽  
Ang Yang Shang ◽  
...  

Cells ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 15
Author(s):  
Azumi Yoshimura ◽  
Stéphanie Miserey-Lenkei ◽  
Evelyne Coudrier ◽  
Bruno Goud

In the early secretory pathway, the delivery of anterograde cargoes from the endoplasmic reticulum (ER) exit sites (ERES) to the Golgi apparatus is a multi-step transport process occurring via the ER-Golgi intermediate compartment (IC, also called ERGIC). While the role microtubules in ER-to-Golgi transport has been well established, how the actin cytoskeleton contributes to this process remains poorly understood. Here, we report that Arp2/3 inhibition affects the network of acetylated microtubules around the Golgi and induces the accumulation of unusually long RAB1/GM130-positive carriers around the centrosome. These long carriers are less prone to reach the Golgi apparatus, and arrival of anterograde cargoes to the Golgi is decreased upon Arp2/3 inhibition. Our data suggest that Arp2/3-dependent actin polymerization maintains a stable network of acetylated microtubules, which ensures efficient cargo trafficking at the late stage of ER to Golgi transport.


2021 ◽  
Author(s):  
Marije Kat ◽  
Ellie Karampini ◽  
Arie Johan Hoogendijk ◽  
Petra Bürgisser ◽  
Aat A. Mulder ◽  
...  

AbstractVon Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells (ECs). VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPBs), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde ER-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometrybased approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in ECs using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted ECs exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion-incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. Taken together, our study has identified SNARE protein STX5 as a novel regulator of WPB biogenesis.


eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Rania Elsabrouty ◽  
Youngah Jo ◽  
Seonghwan Hwang ◽  
Dong-Jae Jun ◽  
Russell A DeBose-Boyd

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4. The prenyltransferase has emerged as a key regulator of sterol-accelerated, endoplasmic reticulum (ER)-associated degradation (ERAD) of HMG CoA reductase, the rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids including GGpp. Sterols induce binding of UBIAD1 to reductase, inhibiting its ERAD. Geranylgeraniol (GGOH), the alcohol derivative of GGpp, disrupts this binding and thereby stimulates ERAD of reductase and translocation of UBIAD1 to Golgi. We now show that overexpression of Type 1 polyisoprenoid diphosphate phosphatase (PDP1), which dephosphorylates GGpp and other isoprenyl pyrophosphates to corresponding isoprenols, abolishes protein geranylgeranylation as well as GGOH-induced ERAD of reductase and Golgi transport of UBIAD1. Conversely, these reactions are enhanced in the absence of PDP1. Our findings indicate PDP1-mediated hydrolysis of GGpp significantly contributes to a feedback mechanism that maintains optimal intracellular levels of the nonsterol isoprenoid.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4652-4652
Author(s):  
Faezeh Darbaniyan ◽  
Caleb Class ◽  
Guillermo Montalban-Bravo ◽  
Rashmi Kanagal-Shamanna ◽  
Marcos Estecio ◽  
...  

Abstract INTRODUCTION: Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) are heterogeneous myeloid neoplastic disorders characterized by ineffective hematopoiesis leading to cytopenias and increased risk of transformation to acute myeloid leukemia (AML) . The hypomethylating agents (HMA) azacitidine (AZA) and decitabine (DAC) improve the natural history of MDS and CMML patients (Fenaux et al 2009). However, over half of the cases experience primary failure defined by a lack of response to HMA treatment which is associated with poor prognosis and a median survival of 4-6 months (Garcia-Manero et al. 2016, Jabbour et al. 2010). The highly heterogeneous pathophysiology of myeloid neoplasms and poorly understood mechanisms underlying therapeutic action of HMAs pose substantial challenges in understanding the biology of HMA failure in MDS and CMML. METHODS: We established a cohort of baseline bone marrow (BM) cells that were collected from 17 CMML and 34 MDS patients with excessive blasts (MDS-EB) prior to their HMA based therapies (Figure 1). RNA-Seq based transcriptomic analysis was performed in CD34+ BM hematopoietic stem and progenitor cells (HSPCs) of patients, known to be the cellular origin of these diseases, to identify biological signatures of primary HMA resistance. RESULTS: Similar to the recent reports regarding the lack of common gene expression signatures in HMA resistant AML cell lines (Leung et al. 2019) , RNA-Seq in patient cohort detected fewer than 0.5% of the total number of differentially expressed genes in non-responders in common for both AZA and DAC. Of note, all the AZA and a large portion of DAC associated genes with down-regulations in non-responders encode immunoglobulins, which is consistent with several recent findings indicating impaired differentiation B cells in association with unfavorable outcomes in MDS and CMML (Ribeiro et al. 2006 and Kahn et al. 2015). We performed flow cytometry analysis in BM cells available for 13 patients prior to start of HMA treatments (5 non-responders and 8 responders), and detected that HMA non-responders had a strong tendency (P=0.07) of decreased baseline frequencies of B cells in their BM than HMA responders. GSEA analysis based on leading edge genes identified over 200 and 300 biological signaling pathways to be associated with AZA and DAC failure respectively, with 78 pathways commonly correlated with treatment failure of both drugs (28 up-regulated and 60 down-regulated, Table 1). Clustering of these commonly altered pathways based on biological functions revealed that most of them are known to have a role in MDS and CMML pathogenesis and/ or drug resistance. For instance, neurotransmitter, olfactory pathways, and associated G-protein coupled receptor signals was recently reported to play a role in regulating the maintenance and differentiation of BM HSPCs (Shao et al. 2021 and Shim et al. 2013), whereas cell junction signaling that involves integrins and increased MAP2K-MAPK signaling were also reported to be associated with AZA resistance in MDS and CMML (Unnikrishnan et al. 2017). Among commonly down-regulated biological pathways in non-responders of AZA and DAC, there were clusters of immunoglobulin-associated signals, protein translation regulatory pathways that involve ribosomal proteins, cell cycle signaling, respiratory chain, and Golgi transport signals. Decreased expression of ribosomal proteins and related impairment of ribosomal functions were known mechanism in MDS and CMML development (Ebert et al. 2008 and Schneider et al. 2016). Furthermore, decreased respiratory chain and Golgi transport signals were also identified by transcriptomic investigation in the DAC resistant TF1-RES cell lines. In addition to the commonly altered signals for both AZA and DAC resistance, innate immune signaling pathways including interferon and toll-like receptor signals were significantly up-regulated in DAC non-responders but down-regulated in AZA non-responders. CONCLUSIONS: In this study, the transcriptomic data between responders and non-responders to AZA and DAC were investigated separately, thereby drug-specific as well as the common biomarkers associated with treatment failures of both drugs could be identified. The relatively low proportion of genes and pathways shared by AZA and DAC non-responders suggest the difference underlying biological mechanisms of AZA and DAC failure. Figure 1 Figure 1. Disclosures Sasaki: Novartis: Consultancy, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees. Wei: Daiichi Sanko: Research Funding.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Peter T. A. Linders ◽  
Eveline C. F. Gerretsen ◽  
Angel Ashikov ◽  
Mari-Anne Vals ◽  
Rinse de Boer ◽  
...  

AbstractThe SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein syntaxin-5 (Stx5) is essential for Golgi transport. In humans, the STX5 mRNA encodes two protein isoforms, Stx5 Long (Stx5L) from the first starting methionine and Stx5 Short (Stx5S) from an alternative starting methionine at position 55. In this study, we identify a human disorder caused by a single missense substitution in the second starting methionine (p.M55V), resulting in complete loss of the short isoform. Patients suffer from an early fatal multisystem disease, including severe liver disease, skeletal abnormalities and abnormal glycosylation. Primary human dermal fibroblasts isolated from these patients show defective glycosylation, altered Golgi morphology as measured by electron microscopy, mislocalization of glycosyltransferases, and compromised ER-Golgi trafficking. Measurements of cognate binding SNAREs, based on biotin-synchronizable forms of Stx5 (the RUSH system) and Förster resonance energy transfer (FRET), revealed that the short isoform of Stx5 is essential for intra-Golgi transport. Alternative starting codons of Stx5 are thus linked to human disease, demonstrating that the site of translation initiation is an important new layer of regulating protein trafficking.


2021 ◽  
Vol 220 (10) ◽  
Author(s):  
Lawrence G. Welch ◽  
Sew-Yeu Peak-Chew ◽  
Farida Begum ◽  
Tim J. Stevens ◽  
Sean Munro

The fidelity of Golgi glycosylation is, in part, ensured by compartmentalization of enzymes within the stack. The COPI adaptor GOLPH3 has been shown to interact with the cytoplasmic tails of a subset of Golgi enzymes and direct their retention. However, other mechanisms of retention, and other roles for GOLPH3, have been proposed, and a comprehensive characterization of the clientele of GOLPH3 and its paralogue GOLPH3L is lacking. GOLPH3’s role is of particular interest as it is frequently amplified in several solid tumor types. Here, we apply two orthogonal proteomic methods to identify GOLPH3+3L clients and find that they act in diverse glycosylation pathways or have other roles in the Golgi. Binding studies, bioinformatics, and a Golgi retention assay show that GOLPH3+3L bind the cytoplasmic tails of their clients through membrane-proximal positively charged residues. Furthermore, deletion of GOLPH3+3L causes multiple defects in glycosylation. Thus, GOLPH3+3L are major COPI adaptors that impinge on most, if not all, of the glycosylation pathways of the Golgi.


2021 ◽  
Author(s):  
Lawrence G Welch ◽  
Sew-Yeu Peak-Chew ◽  
Farida Begum ◽  
Tim J Stevens ◽  
Sean Munro

Glycosylation is a diverse and abundant modification of proteins, lipids and RNA. The fidelity of glycosylation is, in part, assured by the correct compartmentalisation of Golgi-resident glycosylation enzymes within the Golgi stack. The COPI adaptor GOLPH3 has been shown to interact with the cytoplasmic tails of a subset of Golgi enzymes and direct their retention in the Golgi. However, other mechanisms of retention, and other roles for GOLPH3, have been proposed, and a comprehensive characterisation of the clientele of GOLPH3 and its paralogue GOLPH3L has been lacking. The role of GOLPH3 is of particular interest as it is frequently amplified in several solid tumour types. Here, we combine two orthogonal proteomic analyses to identify a diverse range of GOLPH3+3L clients and find that they act in a wide spectrum of glycosylation pathways, or have other roles in the Golgi. Using binding studies, bioinformatics and an in vivo Golgi retention assay, we show that GOLPH3+3L interact with the cytoplasmic tails of their clients through membrane-proximal positively-charged residues. Furthermore, deletion of GOLPH3+3L causes diverse defects in glycosylation. Thus, GOLPH3+3L are major COPI adaptors that impinge on most, if not all, of the glycosylation pathways of the Golgi.


Toxins ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 424
Author(s):  
Andrey S. Selyunin ◽  
Karinel Nieves-Merced ◽  
Danyang Li ◽  
Stanton F. McHardy ◽  
Somshuvra Mukhopadhyay

Shiga toxin 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol of cells where they target ribosomes. As retrograde trafficking is essential for disease, inhibiting STx1/STx2 trafficking is therapeutically promising. Recently, we discovered that the chemotherapeutic drug tamoxifen potently inhibits the trafficking of STx1/STx2 at the critical early endosome-to-Golgi step. We further reported that the activity of tamoxifen against STx1/STx2 is independent of its selective estrogen receptor modulator (SERM) property and instead depends on its weakly basic chemical nature, which allows tamoxifen to increase endolysosomal pH and alter the recruitment of retromer to endosomes. The goal of the current work was to obtain a better understanding of the mechanism of action of tamoxifen against the more disease-relevant toxin STx2, and to differentiate between the roles of changes in endolysosomal pH and retromer function. Structure activity relationship (SAR) analyses revealed that a weakly basic amine group was essential for anti-STx2 activity. However, ability to deacidify endolysosomes was not obligatorily necessary because a tamoxifen derivative that did not increase endolysosomal pH exerted reduced, but measurable, activity. Additional assays demonstrated that protective derivatives inhibited the formation of retromer-dependent, Golgi-directed, endosomal tubules, which mediate endosome-to-Golgi transport, and the sorting of STx2 into these tubules. These results identify retromer-mediated endosomal tubulation and sorting to be fundamental processes impacted by tamoxifen; provide an explanation for the inhibitory effect of tamoxifen on STx2; and have important implications for the therapeutic use of tamoxifen, including its development for treating Shiga toxicosis.


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