N-glycosylation of SnRK2s affects NADPH maintenance in peroxisomes during prolonged ABA signalling.

Unfavourable conditions, such as prolonged drought and high salinity, pose a threat to the survival and agricultural yield of plants. The phytohormone ABA plays a key role in the regulation of plant stress adaptation and is often maintained at high levels for extended periods. While much is known about ABA signal perception and activation in the early signalling stage, the molecular mechanism underlying desensitization of ABA signalling remains largely unknown. Here we demonstrate that in the endoplasmic reticulum (ER)-Golgi network, the key regulators of ABA signalling, SnRK2.2/2.3, undergo N-glycosylation, which promotes their redistribution from the nucleus to the peroxisomes in Arabidopsis roots and influences the transcriptional response in the nucleus during prolonged ABA signalling. On the peroxisomal membrane, SnRK2s can interact with glucose-6-phosphate (G6P)/phosphate translocator 1 (GPT1) to maintain NADPH homeostasis through increased activity of the peroxisomal oxidative pentose phosphate pathway (OPPP). The resulting maintenance of NADPH is essential for the modulation of hydrogen peroxide (H2O2) accumulation, thereby relieving ABA-induced root growth inhibition. The subcellular dynamics of SnRK2s, mediated by N-glycosylation suggest that ABA responses transition from transcriptional regulation in the nucleus to metabolic processes in the peroxisomes, aiding plants in adapting to long-term environmental stress.

The ORP9-ORP11 dimer promotes sphingomyelin synthesis.

Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.

Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells.

Small GTPases are essential in various cellular signaling pathways, and detecting their activation within living cells is crucial for understanding cellular processes. The current methods for detecting GTPase activation using fluorescent proteins rely on the interaction between the GTPase and its effector. Consequently, these methods are not applicable to factors, such as Sar1, where the effector also functions as a GTPase-activating protein. Here, we present a novel method, the Small GTPase ActIvitY ANalyzing (SAIYAN) system, for detecting the activation of endogenous small GTPases via fluorescent signals utilizing a split mNeonGreen system. We demonstrated Sar1 activation at the endoplasmic reticulum (ER) exit site and successfully detected its activation state in various cellular conditions. Utilizing the SAIYAN system in collagen-secreting cells, we discovered activated Sar1 localized both at the ER exit sites and ER-Golgi intermediate compartment (ERGIC) regions. Additionally, impaired collagen secretion confined the activated Sar1 at the ER exit sites, implying the importance of Sar1 activation through the ERGIC in collagen secretion.

In Vitro Reconstitution of Plant COPII Vesicles.

In vitro reconstitution studies enable the controllable and stepwise investigation of complicated biochemical processes. In yeast and mammals, in vitro reconstitution of COPII vesicles marked a pivotal point in characterizing the endoplasmic reticulum-to-Golgi anterograde trafficking route and revealed how vesicles mediate the selective and reliable transportation among topologically equivalent compartments. By providing the necessary physiological conditions in a cell-free environment, it enables the dissection of essential components required for the vesicle formation. To enrich and purify the small amount in vivo membrane-bounded compartments, it simplifies the evaluation of vesicle regulation by distinct external stimuli or upstream signals. Here, we describe the preparation of plant microsomes and cytosol for the reconstitution of plant COPII vesicles. Purified vesicles can be used for further biochemical or microscopical analyses.

Genetic Screening of Factors in the Plant Protein Secretion.

The endomembrane system in plants is composed of interconnected membrane organelles that contribute to intracellular structure and function. These organelles include the endoplasmic reticulum (ER), Golgi apparatus, vacuole, trans-Golgi network, and prevacuolar compartment or multivesicular body. Through vesicle-mediated transport, secreted proteins are synthesized in the ER and subsequently transported along the secretory pathway to the vacuole or outside of cells to fulfill specialized functions. Genetic screening is a crucial method for studying plant protein secretion. It entails identifying phenotypic differences resulting from genetic mutations, such as ethyl methanesulfonate, T-DNA insertion, and RNAi, to investigate gene function and discover mutants with specific traits or gene functions. Significant progress has been achieved in the study of plant protein secretion through genetic screening. In this protocol, we provide a step-by-step guide to studying the protein secretion pathway using a genetic screen approach. We use the example of the free 1 suppressor of Arabidopsis thaliana and oil body mutants of Marchantia polymorpha. Additionally, we offer an overview of genetic screening and briefly summarize the emerging technologies in the field of protein secretion research.

Globoside Is an Essential Intracellular Factor Required for Parvovirus B19 Endosomal Escape.

Human parvovirus B19 (B19V), like most parvoviruses, possesses phospholipase A2 (PLA2) activity, which is thought to mediate endosomal escape by membrane disruption. Here, we challenge this model and find evidence for a mechanism of B19V entry mediated by the glycosphingolipid globoside without endosome disruption and retrograde transport to the Golgi. We show that B19V PLA2 activity requires specific calcium levels and pH conditions that are not optimal in endosomes. Accordingly, endosomal membrane integrity was maintained during B19V entry. Furthermore, endosomes remained intact when loaded with MS2 bacteriophage particles pseudotyped with multiple B19V PLA2 subunits, providing superior enzymatic potential compared to native B19V. In globoside knockout cells, incoming viruses are arrested in the endosomal compartment and the infection is blocked. Infection can be rescued by promoting endosomal leakage with polyethyleneimine (PEI), demonstrating the essential role of globoside in facilitating endosomal escape. Incoming virus colocalizes with Golgi markers and interfering with Golgi function blocks infection, suggesting that globoside-mediated entry involves the Golgi compartment, which provides conditions favorable for the lipolytic PLA2. Our study challenges the current model of B19V entry and identifies globoside as an essential intracellular receptor required for endosomal escape.

Botulinum toxin intoxication requires retrograde transport and membrane translocation at the ER in RenVM neurons.

Botulinum neurotoxin A (BoNT/A) is a highly potent proteolytic toxin specific for neurons with numerous clinical and cosmetic uses. After uptake at the synapse, the protein is proposed to translocate from synaptic vesicles to the cytosol through a self-formed channel. Surprisingly, we found that after intoxication proteolysis of a fluorescent reporter occurs in the neuron soma first and then centrifugally in neurites. To investigate the molecular mechanisms at play, we use a genome-wide siRNA screen in genetically engineered neurons and identify over three hundred genes. An organelle-specific split-mNG complementation indicates BoNT/A traffic from the synapse to the soma-localized Golgi in a retromer-dependent fashion. The toxin then moves to the ER and appears to require the Sec61 complex for retro-translocation to the cytosol. Our study identifies genes and trafficking processes hijacked by the toxin, revealing a new pathway mediating BoNT/A cellular toxicity.

Sphingosine 1-Phosphate Stimulates ER to Golgi Ceramide Traffic to Promote Survival in T98G Glioma Cells.

Glioblastoma multiforme is the most common and fatal brain tumor among human cancers. Ceramide (Cer) and Sphingosine 1-phosphate (S1P) have emerged as bioeffector molecules that control several biological processes involved in both cancer development and resistance. Cer acts as a tumor suppressor, inhibiting cancer progression, promoting apoptosis, enhancing immunotherapy and sensitizing cells to chemotherapy. In contrast, S1P functions as an onco-promoter molecule, increasing proliferation, survival, invasiveness, and resistance to drug-induced apoptosis. The pro-survival PI3K/Akt pathway is a recognized downstream target of S1P, and we have previously demonstrated that in glioma cells it also improves Cer transport and metabolism towards complex sphingolipids in glioma cells. Here, we first examined the possibility that, in T98G glioma cells, S1P may regulate Cer metabolism through PI3K/Akt signaling. Our research showed that exogenous S1P increases the rate of vesicular trafficking of Cer from the endoplasmic reticulum (ER) to the Golgi apparatus through S1P receptor-mediated activation of the PI3K/Akt pathway. Interestingly, the effect of S1P results in cell protection against toxicity arising from Cer accumulation in the ER, highlighting the role of S1P as a survival factor to escape from the Cer-generating cell death response.

Hyperglycemia induced cathepsin L maturation linked to diabetic comorbidities and COVID-19 mortality.

Diabetes, a prevalent chronic condition, significantly increases the risk of mortality from COVID-19, yet the underlying mechanisms remain elusive. Emerging evidence implicates Cathepsin L (CTSL) in diabetic complications, including nephropathy and retinopathy. Our previous research identified CTSL as a pivotal protease promoting SARS-CoV-2 infection. Here, we demonstrate elevated blood CTSL levels in individuals with diabetes, facilitating SARS-CoV-2 infection. Chronic hyperglycemia correlates positively with CTSL concentration and activity in diabetic patients, while acute hyperglycemia augments CTSL activity in healthy individuals. In vitro studies reveal high glucose, but not insulin, promotes SARS-CoV-2 infection in wild-type cells, with CTSL knockout cells displaying reduced susceptibility. Utilizing lung tissue samples from diabetic and non-diabetic patients, alongside Leprdb/dbmice and Leprdb/+mice, we illustrate increased CTSL activity in both humans and mice under diabetic conditions. Mechanistically, high glucose levels promote CTSL maturation and translocation from the endoplasmic reticulum (ER) to the lysosome via the ER-Golgi-lysosome axis. Our findings underscore the pivotal role of hyperglycemia-induced CTSL maturation in diabetic comorbidities and complications.

People with diabetes are at greater risk of developing severe COVID-19 and dying from the illness, which is caused by a virus known as SARS-CoV-2. The high blood sugar levels associated with diabetes appear to be a contributing factor to this heightened risk. However, diabetes is a complex condition encompassing a range of metabolic disorders, and it is therefore likely that other factors may contribute. Previous research identified a link between an enzyme called cathepsin L and more severe COVID-19 in people with diabetes. Elevated cathepsin L levels are known to contribute to diabetes complications, such as kidney damage and vision loss. It has also been shown that cathepsin L helps SARS-CoV-2 to enter and infect cells. This raised the question of whether elevated cathepsin L is responsible for the increased COVID-19 vulnerability in patients with diabetes. To investigate, He, Zhao et al. monitored disease severity and cathepsin L levels in patients with COVID-19. This confirmed that people with diabetes had more severe COVID-19 and that higher levels of cathepsin L are linked to more severe disease. Analysis also revealed that cathepsin L activity increases as blood glucose levels increase. In laboratory experiments, cells exposed to glucose or fluid from the blood of people with diabetes were more easily infected with SARS-CoV-2, with cells genetically modified to lack cathepsin L being more resistant to infection. Further experiments revealed this was due to glucose promoting maturation and migration of cathepsin L in the cells. The findings of He, Zhao et al. help to explain why people with diabetes are more likely to develop severe or fatal COVID-19. Therefore, controlling blood glucose levels in people with diabetes may help to prevent or reduce the severity of the disease. Additionally, therapies targeting cathepsin L could also potentially help to treat COVID-19, especially in patients with diabetes, although more research is needed to develop and test these treatments.

Anaplasma phagocytophilum effector EgeA facilitates infection by hijacking TANGO1 and SCFD1 from ER-Golgi exit sites to pathogen-occupied inclusions.

The obligatory intracellular bacterium Anaplasma phagocytophilum causes human granulocytic anaplasmosis, an emerging zoonosis. Anaplasma has limited biosynthetic and metabolic capacities, yet it effectively replicates inside of inclusions/vacuoles of eukaryotic host cells. Here, we describe a unique Type IV secretion system (T4SS) effector, ER-Golgi exit site protein of Anaplasma (EgeA). In cells infected by Anaplasma, secreted native EgeA, EgeA-GFP, and the C-terminal half of EgeA (EgeA-C)-GFP localized to Anaplasma-containing inclusions. In uninfected cells, EgeA-C-GFP localized to cis-Golgi, whereas the N-terminal half of EgeA-GFP localized to the ER. Pull-down assays identified EgeA-GFP binding to a transmembrane protein in the ER, Transport and Golgi organization protein 1 (TANGO1). By yeast two-hybrid analysis, EgeA-C directly bound Sec1 family domain-containing protein 1 (SCFD1), a host protein of the cis-Golgi network that binds TANGO1 at ER-Golgi exit sites (ERES). Both TANGO1 and SCFD1 localized to the Anaplasma inclusion surface. Furthermore, knockdown of Anaplasma EgeA or either host TANGO1 or SCFD1 significantly reduced Anaplasma infection. TANGO1 and SCFD1 prevent ER congestion and stress by facilitating transport of bulky or unfolded proteins at ERES. A bulky cargo collagen and the ER-resident chaperon BiP were transported into Anaplasma inclusions, and several ER stress marker genes were not up-regulated in Anaplasma-infected cells. Furthermore, EgeA transfection reduced collagen overexpression-induced BiP upregulation. These results suggest that by binding to the two ERES proteins, EgeA redirects the cargo-adapted ERES to pathogen-occupied inclusions and reduces ERES congestion, which facilitates Anaplasma nutrient acquisition and reduces ER stress for Anaplasma survival and proliferation.

Evidence that the SARS-CoV-2 S protein undergoes a conformational change at the Golgi complex that leads to the formation of virus neutralising antibody binding epitopes in the S1 protein subunit.

Recombinant SARS-CoV-2 S protein expression was examined in Vero cells by imaging using the human monoclonal antibody panel (PD4, PD5, sc23, and sc29). The PD4 and sc29 antibodies recognised conformational specific epitopes in the S2 protein subunit at the Endoplasmic reticulum and Golgi complex. While PD5 and sc23 detected conformationally specific epitopes in the S1 protein subunit at the Golgi complex, only PD5 recognised the receptor binding domain (RBD). A comparison of the staining patterns of PD5 with non-conformationally specific antibodies that recognises the S1 subunit and RBD suggested the PD5 recognised a conformational structure within the S1 protein subunit. Our data suggests the antibody binding epitopes recognised by the human monoclonal antibodies formed at different locations in the secretory pathway during S protein transport, but a conformational change in the S1 protein subunit at the Golgi complex formed antibody binding epitopes that are recognised by virus neutralising antibodies.

IER3IP1-mutations cause microcephaly by selective inhibition of ER-Golgi transport.

Mutations in the IER3IP1 (Immediate Early Response-3 Interacting Protein 1) gene can give rise to MEDS1 (Microcephaly with Simplified Gyral Pattern, Epilepsy, and Permanent Neonatal Diabetes Syndrome-1), a severe condition leading to early childhood mortality. The small endoplasmic reticulum (ER)-membrane protein IER3IP1 plays a non-essential role in ER-Golgi transport. Here, we employed secretome and cell-surface proteomics to demonstrate that the absence of IER3IP1 results in the mistrafficking of proteins crucial for neuronal development and survival, including FGFR3, UNC5B and SEMA4D. This phenomenon correlates with the distension of ER membranes and increased lysosomal activity. Notably, the trafficking of cargo receptor ERGIC53 and KDEL-receptor 2 are compromised, with the latter leading to the anomalous secretion of ER-localized chaperones. Our investigation extended to in-utero knock-down of Ier3ip1 in mouse embryo brains, revealing a morphological phenotype in newborn neurons. In summary, our findings provide insights into how the loss or mutation of a 10 kDa small ER-membrane protein can cause a fatal syndrome.

GM130-silencing may aggravate blood-brain barrier damage and affect microglia polarization by down-regulating PD-L1 expression after experimental intracerebral hemorrhage.

BACKGROUND: In addition to primary injury, secondary injuries related to BBB disruption and immune-inflammatory response also play an important role in intracerebral hemorrhage (ICH). And the Golgi apparatus play an important role in the state of ICH. METHODS: ICH model and GM130-silencing ICH model were established in SD rats. The Garcia score was used to score the neurological defects of the rats. Blood-brain barrier (BBB) integrity were assessed by amount of extravasated Evans blue, and tight junction proteins. The expression of PD-L1 and GM130were detected through Western-blot and the subtype of microglia was showing with Immunofluorescence staining. RESULTS: Compared with the ICH group, GM130-silencing ICH rats got a worsened neurological deficit and enlarged volume of the hematoma. Evan's blue extravasation aggravated as well. The expression of GM130 in peri-hematoma tissue was further decreased, and the morphology and structure of the Golgi apparatus were further damaged. Meanwhile, the GM130 deficit resulted in decreased expression of PD-L1 and more polarization of microglia to the M1 subtype. CONCLUSION: We demonstrate that GM130 could influence the integrity of BBB and plays a role in neuroinflammation via regulation of PD-L1 after ICH. The manipulation of GM130 might be a promising therapeutical target in ICH.

Intestinal Nogo-B reduces GLP1 levels by binding to proglucagon on the endoplasmic reticulum to inhibit PCSK1 cleavage.

Glucagon-like peptide 1 (GLP1), which is mainly processed and cleaved from proglucagon in enteroendocrine cells (EECs) of the intestinal tract, acts on the GLP1 receptor in pancreatic cells to stimulate insulin secretion and to inhibit glucagon secretion. However, GLP1 processing is not fully understood. Here, we show that reticulon 4B (Nogo-B), an endoplasmic reticulum (ER)-resident protein, interacts with the major proglucagon fragment of proglucagon to retain proglucagon on the ER, thereby inhibiting PCSK1-mediated cleavage of proglucagon in the Golgi. Intestinal Nogo-B knockout in male type 2 diabetes mellitus (T2DM) mice increases GLP1 and insulin levels and decreases glucagon levels, thereby alleviating pancreatic injury and insulin resistance. Finally, we identify aberrantly elevated Nogo-B expression and inhibited proglucagon cleavage in EECs from diabetic patients. Our study reveals the subcellular regulatory processes involving Nogo-B during GLP1 production and suggests intestinal Nogo-B as a potential therapeutic target for T2DM.

Prognosis and metabolism with a Golgi apparatus-related genes-based formula in breast cancer.

The Golgi apparatus (GA), an organelle that processes, sorts, and transports proteins synthesized by the endoplasmic reticulum, is also involved in many cellular processes associated with cancer, such as angiogenesis, the innate immune response, and tumor invasion and migration. We aimed to construct a breast cancer (BC) prognosis prediction model based on GA-related genetic information to evaluate the prognosis of patients with BC more accurately than existing models and to stratify patients for clinical therapy. In this study, The Cancer Genome Atlas-breast invasive carcinoma was used as the training cohort, and the Molecular Taxonomy of Breast Cancer International Consortium cohort was used as the validation cohort. Using bioinformatics methods, we constructed a GA-related gene risk score (GRS). The GRS was used to divide BC patients into a high-GRS group and a low-GRS group, and functional analysis, survival analysis, mutation analysis, immune landscape analysis, and metabolic analysis were performed to compare the 2 groups. Finally, a nomogram was constructed for clinical application. The genes in the GRS model were mainly related to the glucose metabolism pathway, and the main mutations in the 2 groups of patients were mutations in TP53 and CHD1. The mutation rate in the high-GRS group was greater than that in the low-GRS group. The high GRS group had higher tumor immune activity glycolysis; the pentose phosphate pathway tended to be the dominant metabolic pathways in this group, while fatty acid oxidation and glutamine catabolism tended to be dominant in the low-GRS group. GA-related genes were used to construct a prediction model for BC patients and had high accuracy in predicting prognosis. The mutations associated with the GRS are mainly TP53 and CDH1. Interestingly, the GRS is correlated with glucose metabolism in terms of gene expression and functional enrichment. In summary, the role of GRS-related genes in glucose metabolism is worthy of further study.

The endolysosomal system in conventional and unconventional protein secretion.

Most secreted proteins are transported through the "conventional" endoplasmic reticulum-Golgi apparatus exocytic route for their delivery to the cell surface and release into the extracellular space. Nonetheless, formative discoveries have underscored the existence of alternative or "unconventional" secretory routes, which play a crucial role in exporting a diverse array of cytosolic proteins outside the cell in response to intrinsic demands, external cues, and environmental changes. In this context, lysosomes emerge as dynamic organelles positioned at the crossroads of multiple intracellular trafficking pathways, endowed with the capacity to fuse with the plasma membrane and recognized for their key role in both conventional and unconventional protein secretion. The recent recognition of lysosomal transport and exocytosis in the unconventional secretion of cargo proteins provides new and promising insights into our understanding of numerous physiological processes.

Golgi pH homeostasis stabilizes the lysosomal membrane through N-glycosylation of membrane proteins.

Protein glycosylation plays a vital role in various cellular functions, many of which occur within the Golgi apparatus. The Golgi pH regulator (GPHR) is essential for the proper functioning of the Golgi apparatus. The lysosomal membrane contains highly glycosylated membrane proteins in abundance. This study investigated the role of the Golgi luminal pH in N-glycosylation of lysosomal membrane proteins and the effect of this protein modification on membrane stability using Gphr-deficient MEFs. We showed that Gphr deficiency causes an imbalance in the Golgi luminal pH, resulting in abnormal protein N-glycosylation, indicated by a reduction in sialylated glycans and markedly reduced molecular weight of glycoproteins. Further experiments using FRAP and PLA revealed that Gphr deficiency prevented the trafficking dynamics and proximity condition of glycosyltransferases in the Golgi apparatus. In addition, incomplete N-glycosylation of lysosomal membrane proteins affected lysosomal membrane stability, as demonstrated by the increased susceptibility to lysosomal damage. Thus, this study highlights the critical role of Golgi pH regulation in controlling protein glycosylation and the impact of Golgi dysfunction on lysosomal membrane stability.

SPRING licenses S1P-mediated cleavage of SREBP2 by displacing an inhibitory pro-domain.

Site-one protease (S1P) conducts the first of two cleavage events in the Golgi to activate Sterol regulatory element binding proteins (SREBPs) and upregulate lipogenic transcription. S1P is also required for a wide array of additional signaling pathways. A zymogen serine protease, S1P matures through autoproteolysis of two pro-domains, with one cleavage event in the endoplasmic reticulum (ER) and the other in the Golgi. We recently identified the SREBP regulating gene, (SPRING), which enhances S1P maturation and is necessary for SREBP signaling. Here, we report the cryo-EM structures of S1P and S1P-SPRING at sub-2.5 Å resolution. SPRING activates S1P by dislodging its inhibitory pro-domain and stabilizing intra-domain contacts. Functionally, SPRING licenses S1P to cleave its cognate substrate, SREBP2. Our findings reveal an activation mechanism for S1P and provide insights into how spatial control of S1P activity underpins cholesterol homeostasis.

Oxysterol binding protein (OSBP) contributes to hepatitis E virus replication.

Hepatitis E virus (HEV) is a positive-sense, single-stranded RNA virus and causes primarily acute self-limiting infections. The ORF1 of the HEV genome encodes a polyprotein around 190 kDa, which contains several putative domains, including helicase and RNA-dependent RNA polymerase. The HEV-encoded helicase is a member of the superfamily 1 helicase family and possesses multiple enzymatic functions, such as RNA 5'-triphosphatase, RNA unwinding, and NTPase, which are thought to contribute to viral RNA synthesis. However, the helicase interaction with cellular proteins remains less known. Oxysterol binding protein (OSBP) is a lipid regulator that shuffles between the Golgi apparatus and the endoplasmic reticulum for cholesterol and phosphatidylinositol-4-phosphate exchange and controls the efflux of cholesterol from cells. In this study, the RNAi-mediated silencing of OSBP significantly reduced HEV replication. Further studies indicate that the HEV helicase interacted with OSBP, shown by co-immunoprecipitation and co-localization in co-transfected cells. The presence of helicase blocked OSBP preferential translocation to the Golgi apparatus. These results demonstrate that OSBP contributes to HEV replication and enrich our understanding of the HEV-cell interactions.

RNA scaffolds the Golgi ribbon by forming condensates with GM130.

The mammalian Golgi is composed of stacks that are laterally connected into a continuous ribbon-like structure. The integrity and function of the ribbon is disrupted under stress conditions, but the molecular mechanisms remain unclear. Here we show that the ribbon is maintained by biomolecular condensates of RNA and the Golgi matrix protein GM130 (GOLGA2). We identify GM130 as a membrane-bound RNA-binding protein, which directly recruits RNA and associated RNA-binding proteins to the Golgi membrane. Acute degradation of RNA or GM130 in cells disrupts the ribbon. Under stress conditions, RNA dissociates from GM130 and the ribbon is disjointed, but after the cells recover from stress the ribbon is restored. When overexpressed in cells, GM130 forms RNA-dependent liquid-like condensates. GM130 contains an intrinsically disordered domain at its amino terminus, which binds RNA to induce liquid-liquid phase separation. These co-condensates are sufficient to link purified Golgi membranes, reconstructing lateral linking of stacks into a ribbon-like structure. Together, these studies show that RNA acts as a structural biopolymer that together with GM130 maintains the integrity of the Golgi ribbon.