GRASP depletion-mediated Golgi fragmentation impairs glycosaminoglycan synthesis, sulfation, and secretion.

Synthesis of glycosaminoglycans, such as heparan sulfate (HS) and chondroitin sulfate (CS), occurs in the lumen of the Golgi, but the relationship between Golgi structural integrity and glycosaminoglycan synthesis is not clear. In this study, we disrupted the Golgi structure by knocking out GRASP55 and GRASP65 and determined its effect on the synthesis, sulfation, and secretion of HS and CS. We found that GRASP depletion increased HS synthesis while decreasing CS synthesis in cells, altered HS and CS sulfation, and reduced both HS and CS secretion. Using proteomics, RNA-seq and biochemical approaches, we identified EXTL3, a key enzyme in the HS synthesis pathway, whose level is upregulated in GRASP knockout cells; while GalNAcT1, an essential CS synthesis enzyme, is robustly reduced. In addition, we found that GRASP depletion decreased HS sulfation via the reduction of PAPSS2, a bifunctional enzyme in HS sulfation. Our study provides the first evidence that Golgi structural defect may significantly alter the synthesis and secretion of glycosaminoglycans.

The Golgi-Associated PDZ Domain Protein Gopc/PIST Is Required for Synaptic Targeting of mGluR5.

In neuronal cells, many membrane receptors interact via their intracellular, C-terminal tails with PSD-95/discs large/ZO-1 (PDZ) domain proteins. Some PDZ proteins act as scaffold proteins. In addition, there are a few PDZ proteins such as Gopc which bind to receptors during intracellular transport. Gopc is localized at the trans-Golgi network (TGN) and binds to a variety of receptors, many of which are eventually targeted to postsynaptic sites. We have analyzed the role of Gopc by knockdown in primary cultured neurons and by generating a conditional Gopc knockout (KO) mouse line. In neurons, targeting of neuroligin 1 (Nlgn1) and metabotropic glutamate receptor 5 (mGlu5) to the plasma membrane was impaired upon depletion of Gopc, whereas NMDA receptors were not affected. In the hippocampus and cortex of Gopc KO animals, expression levels of Gopc-associated receptors were not altered, while their subcellular localization was disturbed. The targeting of mGlu5 to the postsynaptic density was reduced, coinciding with alterations in mGluR-dependent synaptic plasticity and deficiencies in a contextual fear conditioning paradigm. Our data imply Gopc in the correct subcellular sorting of its associated mGlu5 receptor in vivo.

Focused CRISPR-Cas9 genetic screening reveals USO1 as a vulnerability in B-cell acute lymphoblastic leukemia.

Post-transcriptional gene regulation, including that by RNA binding proteins (RBPs), has recently been described as an important mechanism in cancer. We had previously identified a set of RBPs that were highly dysregulated in B-cell acute lymphoblastic leukemia (B-ALL) with MLL translocations, which carry a poor prognosis. Here, we sought to functionally characterize these dysregulated RBP genes by performing a focused CRISPR dropout screen in B-ALL cell lines, finding dependencies on several genes including EIF3E, EPRS and USO1. Validating our findings, CRISPR/Cas9-mediated disruption of USO1 in MLL-translocated B-ALL cells reduced cell growth, promoted cell death, and altered the cell cycle. Transcriptomic analysis of USO1-deficient cells revealed alterations in pathways related to mTOR signaling, RNA metabolism, and targets of MYC. In addition, USO1-regulated genes from these experimental samples were significantly and concordantly correlated with USO1 expression in primary samples collected from B-ALL patients. Lastly, we found that loss of Uso1 inhibited colony formation of MLL-transformed in primary bone marrow cells from Cas9-EGFP mice. Together, our findings demonstrate an approach to performing focused sub-genomic CRISPR screens and highlight a putative RBP vulnerability in MLL-translocated B-ALL, thus identifying potential therapeutic targets in this disease.

The Golgin Protein RUD3 Regulates Fusarium graminearum Growth and Virulence.

Golgins are coiled-coil proteins that play prominent roles in maintaining the structure and function of the Golgi complex. However, the role of golgin proteins in phytopathogenic fungi remains poorly understood. In this study, we functionally characterized the Fusarium graminearum golgin protein RUD3, a homolog of ScRUD3/GMAP-210 in Saccharomyces cerevisiae and mammalian cells. Cellular localization observation revealed that RUD3 is located in the cis-Golgi. Deletion of RUD3 caused defects in vegetative growth, ascospore discharge, deoxynivalenol (DON) production, and virulence. Moreover, the Δrud3 mutant showed reduced expression of tri genes and impairment of the formation of toxisomes, both of which play essential roles in DON biosynthesis. We further used green fluorescent protein (GFP)-tagged SNARE protein SEC22 (SEC22-GFP) as a tool to study the transport between the endoplasmic reticulum (ER) and Golgi and observed that SEC22-GFP was retained in the cis-Golgi in the Δrud3 mutant. RUD3 contains the coiled coil (CC), GRAB-associated 2 (GA2), GRIP-related Arf binding (GRAB), and GRAB-associated 1 (GA1) domains, which except for GA1, are indispensable for normal localization and function of RUD3, whereas only CC is essential for normal RUD3-RUD3 interaction. Together, these results demonstrate how the golgin protein RUD3 mediates retrograde trafficking in the ER-to-Golgi pathway and is necessary for growth, ascospore discharge, DON biosynthesis, and pathogenicity in F. graminearumIMPORTANCEFusarium head blight (FHB) caused by the fungal pathogen Fusarium graminearum is an economically important disease of wheat and other small grain cereal crops worldwide, and limited effective control strategies are available. A better understanding of the regulation mechanisms of F. graminearum development, deoxynivalenol (DON) biosynthesis, and pathogenicity is therefore important for the development of effective control management of this disease. Golgins are attached via their extreme carboxy terminus to the Golgi membrane and are involved in vesicle trafficking and organelle maintenance in eukaryotic cells. In this study, we systematically characterized a highly conserved Golgin protein, RUD3, and found that it is required for vegetative growth, ascospore discharge, DON production, and pathogenicity in F. graminearum Our findings provide a comprehensive characterization of the golgin family protein RUD3 in plant-pathogenic fungus, which could help to identify a new potential target for effective control of this devastating disease.

GORASP2/GRASP55 collaborates with the PtdIns3K UVRAG complex to facilitate autophagosome-lysosome fusion.

It has been indicated that the Golgi apparatus contributes to autophagy, but how it is involved in autophagosome formation and maturation is not well understood. Here we show that amino acid starvation causes trans-Golgi derived membrane fragments to colocalize with autophagosomes. Depletion of the Golgi stacking protein GORASP2/GRASP55, but not GORASP1/GRASP65, increases both MAP1LC3 (LC3)-II and SQSTM1/p62 levels. We demonstrate that GORASP2 facilitates autophagosome-lysosome fusion by physically linking autophagosomes and lysosomes through the interactions with LC3 on autophagosomes and LAMP2 on late endosomes/lysosomes. Furthermore, we provide evidence that GORASP2 interacts with BECN1 to facilitate the assembly and membrane association of the phosphatidylinositol 3-kinase (PtdIns3K) UVRAG complex. These findings indicate that GORASP2 plays an important role in autophagosome maturation during amino acid starvation. Abbreviations: ATG14: autophagy related 14; BafA1: bafilomycin A1; BSA: bovine serum albumin; CQ: chloroquine; EBSS: earle's balanced salt solution; EM: electron microscopy; EEA1: early endosome antigen 1; GFP: green fluorescent protein; GORASP1/GRASP65: golgi reassembly stacking protein 1; GORASP2/GRASP55: golgi reassembly stacking protein 2; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; MAP1LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PBS: phosphate-buffered saline; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; PK: protease K; PNS: post-nuclear supernatant; RFP: red fluorescent protein; SD: standard deviation; TGN: trans-Golgi network; UVRAG: UV radiation resistance associated.

A role of GCC88 in the retrograde transport of CI-M6PR and the maintenance of lysosomal activity.

GCC88 is a golgin coiled-coil protein at the trans-Golgi (TGN) that functions as a tethering factor for the endosome-derived retrograde transport vesicles. Here, we demonstrate that GCC88 is required for the endosome-to-TGN retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-M6PR). The knockout of GCC88 perturbs the retrieval of CI-M6PR and decreases its cellular level at the steady state, which causes the improper processing of newly synthesized cathepsin-D, a lysosomal hydrolase dependent on CI-M6PR for its delivery to lysosomes. At the whole cell level, the knockout of GCC88 reduces the lysosomal proteolytic capacity but does not impair of the efficiency of autophagy within these cells.

GRASP depletion-mediated Golgi destruction decreases cell adhesion and migration via the reduction of α5ß1 integrin.

The Golgi apparatus is a membrane-bound organelle that serves as the center for trafficking and processing of proteins and lipids. To perform these functions, the Golgi forms a multilayer stacked structure held by GRASP55 and GRASP65 trans-oligomers and perhaps their binding partners. Depletion of GRASP proteins disrupts Golgi stack formation and impairs critical functions of the Golgi, such as accurate protein glycosylation and sorting. However, how Golgi destruction affects other cellular activities is so far unknown. Here, we report that depletion of GRASP proteins reduces cell attachment and migration. Interestingly, GRASP depletion reduces the protein level of α5ß1 integrin, the major cell adhesion molecule at the surface of HeLa and MDA-MB-231 cells, due to decreased integrin protein synthesis. GRASP depletion also increases cell growth and total protein synthesis. These new findings enrich our understanding on the role of the Golgi in cell physiology and provide a potential target for treating protein-trafficking disorders.

Golgins.

Muschalik and Munro introduce golgins and their roles as vesicle tethers and scaffolds at the Golgi.