Golgi defect as a major contributor to lysosomal dysfunction.

The Golgi apparatus plays a crucial role in lysosome biogenesis and the delivery of lysosomal enzymes, essential for maintaining cellular homeostasis and ensuring cell survival. Deficiencies in Golgi structure and function can profoundly impact lysosomal homeostasis, leading to various lysosomal storage diseases and neurodegenerative disorders. In this review, we highlight the role of the Golgi Reassembly Stacking Proteins (GRASPs) in the formation and function of the Golgi apparatus, emphasizing the current understanding of the association between the Golgi apparatus, lysosomes, and lysosomal storage diseases. Additionally, we discuss how Golgi dysfunction leads to the secretion of lysosomal enzymes. This review aims to serve as a concise resource, offering insights into Golgi structure, function, disease-related defects, and their consequential effects on lysosomal biogenesis and function. By highlighting Golgi defects as an underappreciated contributor to lysosomal dysfunction across various diseases, we aim to enhance comprehension of these intricate cellular processes.

Apoptotic Vesicular Metabolism Contributes to Organelle Assembly and Safeguards Liver Homeostasis and Regeneration.

BACKGROUND & AIMS: Apoptosis generates plenty of membrane-bound nanovesicles, the apoptotic vesicles (apoVs), which show promise for biomedical applications. The liver serves as a significant organ for apoptotic material removal. Whether and how the liver metabolizes apoptotic vesicular products and contributes to liver health and disease is unrecognized. METHODS: apoVs were labeled and traced after intravenous infusion. Apoptosis-deficient mice by Fas mutant (Fasmut) and Caspase-3 knockout (Casp3-/-) were used with apoV replenishment to evaluate the physiological apoV function. Combinations of morphologic, biochemical, cellular, and molecular assays were applied to assess the liver while hepatocyte analysis was performed. Partial hepatectomy and acetaminophen liver failure models were established to investigate liver regeneration and disease recovery. RESULTS: We discovered that the liver is a major metabolic organ of circulatory apoVs, in which apoVs undergo endocytosis by hepatocytes via a sugar recognition system. Moreover, apoVs play an indispensable role to counteract hepatocellular injury and liver impairment in apoptosis-deficient mice upon replenishment. Surprisingly, apoVs form a chimeric organelle complex with the hepatocyte Golgi apparatus through the soluble N-ethylmaleimide-sensitive factor attachment protein receptor machinery, which preserves Golgi integrity, promotes microtubule acetylation by regulating α-tubulin N-acetyltransferase 1, and consequently facilitates hepatocyte cytokinesis for liver recovery. The assembly of the apoV-Golgi complex is further revealed to contribute to liver homeostasis, regeneration, and protection against acute liver failure. CONCLUSIONS: These findings establish a previously unrecognized functional and mechanistic framework that apoptosis through vesicular metabolism safeguards liver homeostasis and regeneration, which holds promise for hepatic disease therapeutics.

Emerging roles of O-GlcNAcylation in protein trafficking and secretion.

The emerging roles of O-GlcNAcylation, a distinctive post-translational modification, are increasingly recognized for their involvement in the intricate processes of protein trafficking and secretion. This modification exerts its influence on both conventional and unconventional secretory pathways. Under healthy and stress conditions, such as during diseases, it orchestrates the transport of proteins within cells, ensuring timely delivery to their intended destinations. O-GlcNAcylation occurs on key factors, like coat protein complexes (COPI and COPII), clathrin, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), and GRASP55 (Golgi reassembly stacking protein of 55 kDa) that control vesicle budding and fusion in anterograde and retrograde trafficking and unconventional secretion. The understanding of O-GlcNAcylation offers valuable insights into its critical functions in cellular physiology and the progression of diseases, including neurodegeneration, cancer, and metabolic disorders. In this review, we summarize and discuss the latest findings elucidating the involvement of O-GlcNAc in protein trafficking and its significance in various human disorders.

Sulfoproteomics Workflow with Precursor Ion Accurate Mass Shift Analysis Reveals Novel Tyrosine Sulfoproteins in the Golgi.

Tyrosine sulfation in the Golgi of secreted and membrane proteins is an important post-translational modification (PTM). However, its labile nature has limited analysis by mass spectrometry (MS), a major reason why no sulfoproteome studies have been previously reported. Here, we show that a phosphoproteomics experimental workflow, which includes serial enrichment followed by high resolution, high mass accuracy MS, and tandem MS (MS/MS) analysis, enables sulfopeptide coenrichment and identification via accurate precursor ion mass shift open MSFragger database search. This approach, supported by manual validation, allows the confident identification of sulfotyrosine-containing peptides in the presence of high levels of phosphorylated peptides, thus enabling these two sterically and ionically similar isobaric PTMs to be distinguished and annotated in a single proteomic analysis. We applied this approach to isolated interphase and mitotic rat liver Golgi membranes and identified 67 tyrosine sulfopeptides, corresponding to 26 different proteins. This work discovered 23 new sulfoproteins with functions related to, for example, Ca2+-binding, glycan biosynthesis, and exocytosis. In addition, we report the first preliminary evidence for crosstalk between sulfation and phosphorylation in the Golgi, with implications for functional control.

Analysis of mannosidase I activity in interphase and mitotic cells by lectin staining and endoglycosidase H treatment.

N-Glycosylation is a common protein modification catalyzed by a series of glycosylation enzymes in the endoplasmic reticulum and Golgi apparatus. Here, based on a previously established Golgi α-mannosidase-I-deficient cell line, we present a protocol to investigate the enzymatic activity of exogenously expressed Golgi α-mannosidase IA in interphase and mitotic cells. We describe steps for cell surface lectin staining and subsequent live cell imaging. We also detail PNGase F and Endo H cleavage assays to analyze protein glycosylation. For complete details on the use and execution of this protocol, please refer to Huang et al.1.

Synchronized proinsulin trafficking reveals delayed Golgi export accompanies ß-cell secretory dysfunction in rodent models of hyperglycemia.

The pancreatic islet ß-cell's preference for release of newly synthesized insulin requires careful coordination of insulin exocytosis with sufficient insulin granule production to ensure that insulin stores exceed peripheral demands for glucose homeostasis. Thus, the cellular mechanisms regulating insulin granule production are critical to maintaining ß-cell function. In this report, we utilized the synchronous protein trafficking system, RUSH, in primary ß-cells to evaluate proinsulin transit through the secretory pathway leading to insulin granule formation. We demonstrate that the trafficking, processing, and secretion of the proinsulin RUSH reporter, proCpepRUSH, are consistent with current models of insulin maturation and release. Using both a rodent dietary and genetic model of hyperglycemia and ß-cell dysfunction, we show that proinsulin trafficking is impeded at the Golgi and coincides with the decreased appearance of nascent insulin granules at the plasma membrane. Ultrastructural analysis of ß-cells from diabetic leptin receptor deficient mice revealed gross morphological changes in Golgi structure, including shortened and swollen cisternae, and partial Golgi vesiculation, which are consistent with defects in secretory protein export. Collectively, this work highlights the utility of the proCpepRUSH reporter in studying proinsulin trafficking dynamics and suggests that altered Golgi export function contributes to ß-cell secretory defects in the pathogenesis of Type 2 diabetes.

Common Assays in Mammalian Golgi Studies.

The Golgi is a complex structure characterized by stacks of tightly aligned flat cisternae. In mammalian cells, Golgi stacks often concentrate in the perinuclear region and link together to form a ribbon. This structure is dynamic to accommodate continuous cargo flow in and out of the Golgi in both directions and undergoes morphological changes under physiological and pathological conditions. The fine, stacked Golgi structure makes it difficult to study by conventional light or even super-resolution microscopy. Furthermore, efforts to understand how Golgi structural dynamics impact cellular processes have been slow because of the knowledge gap in the protein machinery that maintains the complex and dynamic Golgi structure. In this method article, we list the common assays used in our research to help new and established researchers select the most appropriate method to properly address their questions.

Quantitative Proteomics Analysis of Purified Rat Liver Golgi.

The Golgi is the central organelle in the secretory pathway, essential for post-translational modifications, sorting and trafficking of secretory and membrane proteins and lipids in all eukaryotic cells. During mitosis, the mammalian Golgi membranes undergo continuous disassembly and reassembly processes which are critical for Golgi biogenesis during the cell division. To better understand the underlying molecular mechanism of this highly dynamic process, we analyzed the proteins that are in or associated with interphase and mitotic Golgi membranes using an in vitro Golgi assembly assay and quantitative proteomics. In this study, by combining an isobaric mass tag labeling strategy with OFFGEL peptide fractionation, LC-MS/MS analyses identified and quantified a total of 1193 Golgi-resident or -associated proteins. These proteins included Golgi structural proteins, Golgi-resident enzymes, Rab GTPases, and SNARE proteins. This systematic quantitative proteomic study revealed the comprehensive molecular machinery of the Golgi and the dynamic protein changes in its disassembly and reassembly processes. Here we describe the detailed procedures and protocols for this analysis.

Generation of Stable Cell Lines Expressing Golgi Reassembly Stacking Proteins (GRASPs) by Viral Transduction.

Stable cell lines that express a gene of specific interest provide an advantage over transient gene expression by reducing variations in transfection efficiency between experiments, sustaining expression for long-term studies, and controlling expression levels in particular if a clonal population is selected. Transient transfection requires introduction of an exogenous gene into host cells via typically harsh chemicals or conditions that permeabilize the cell membrane, which does not normally integrate into the target cell genome. Here, we describe the method of using retroviral transduction to stably express Golgi proteins fused to a promiscuous biotin ligase (TurboID) in HeLa cells, thus creating cell lines that can be leveraged in studies of the proximome/interactome. We also demonstrate a similar protocol for stable expression of a Golgi protein fused to a fluorescent tag via lentiviral transduction. These methods can be further adapted to establish other cell lines with different sub-cellular markers or fusion tags. Viral transduction is a convenient method to create stable cell lines in cell-based studies.

Common Markers and Small Molecule Inhibitors in Golgi Studies.

In this chapter, we provide a detailed guide for the application of commonly used small molecules to study Golgi structure and function in vitro. Furthermore, we have curated a concise, validated list of endomembrane markers typically used in downstream assays to examine the consequent effect on the Golgi via microscopy and western blot after drug treatment. This chapter will be useful for researchers beginning their foray into the field of intracellular trafficking and Golgi biology.

Mitotic phosphorylation inhibits the Golgi mannosidase MAN1A1.

N-glycans are processed mainly in the Golgi, and a well-organized Golgi structure is required for accurate glycosylation. However, during mitosis the Golgi undergoes severe fragmentation. The resulting trafficking block leads to an extended exposure of cargo molecules to Golgi enzymes. It is unclear how cells avoid glycosylation defects during mitosis. In this study, we report that Golgi α-1,2-mannosidase IA (MAN1A1), the first enzyme that cargo proteins encounter once arriving the Golgi, is phosphorylated at serine 12 by CDK1 in mitosis, which attenuates its activity, affects the production of glycan isomers, and reduces its interaction with the subsequent glycosyltransferase, MGAT1. Expression of wild-type MAN1A1, but not its phosphomimetic mutant, rescues the glycosylation defects in mannosidase I-deficient cells, whereas expression of its phosphorylation-deficient mutant in mitosis increases the formation of complex glycans. Our study reveals that glycosylation is regulated by cytosolic signaling during the cell cycle.

ER Redox Homeostasis Regulates Proinsulin Trafficking and Insulin Granule Formation in the Pancreatic Islet ß-Cell.

Defects in the pancreatic ß-cell's secretion system are well-described in type 2 diabetes (T2D) and include impaired proinsulin processing and a deficit in mature insulin-containing secretory granules; however, the cellular mechanisms underlying these defects remain poorly understood. To address this, we used an in situ fluorescent pulse-chase strategy to study proinsulin trafficking. We show that insulin granule formation and the appearance of nascent granules at the plasma membrane are decreased in rodent and cell culture models of prediabetes and hyperglycemia. Moreover, we link the defect in insulin granule formation to an early trafficking delay in endoplasmic reticulum (ER) export of proinsulin, which is independent of overt ER stress. Using a ratiometric redox sensor, we show that the ER becomes hyperoxidized in ß-cells from a dietary model of rodent prediabetes and that addition of reducing equivalents restores ER export of proinsulin and insulin granule formation and partially restores ß-cell function. Together, these data identify a critical role for the regulation of ER redox homeostasis in proinsulin trafficking and suggest that alterations in ER redox poise directly contribute to the decline in insulin granule production in T2D. This model highlights a critical link between alterations in ER redox and ER function with defects in proinsulin trafficking in T2D. Hyperoxidation of the ER lumen, shown as hydrogen peroxide, impairs proinsulin folding and disulfide bond formation that prevents efficient exit of proinsulin from the ER to the Golgi. This trafficking defect limits available proinsulin for the formation of insulin secretory granules during the development of T2D.

GCAF(TMEM251) regulates lysosome biogenesis by activating the mannose-6-phosphate pathway.

The mannose-6-phosphate (M6P) biosynthetic pathway for lysosome biogenesis has been studied for decades and is considered a well-understood topic. However, whether this pathway is regulated remains an open question. In a genome-wide CRISPR/Cas9 knockout screen, we discover TMEM251 as the first regulator of the M6P modification. Deleting TMEM251 causes mistargeting of most lysosomal enzymes due to their loss of M6P modification and accumulation of numerous undigested materials. We further demonstrate that TMEM251 localizes to the Golgi and is required for the cleavage and activity of GNPT, the enzyme that catalyzes M6P modification. In zebrafish, TMEM251 deletion leads to severe developmental defects including heart edema and skeletal dysplasia, which phenocopies Mucolipidosis Type II. Our discovery provides a mechanism for the newly discovered human disease caused by TMEM251 mutations. We name TMEM251 as GNPTAB cleavage and activity factor (GCAF) and its related disease as Mucolipidosis Type V.

Liquid-liquid phase separation facilitates the biogenesis of secretory storage granules.

Insulin is synthesized by pancreatic ß-cells and stored into secretory granules (SGs). SGs fuse with the plasma membrane in response to a stimulus and deliver insulin to the bloodstream. The mechanism of how proinsulin and its processing enzymes are sorted and targeted from the trans-Golgi network (TGN) to SGs remains mysterious. No cargo receptor for proinsulin has been identified. Here, we show that chromogranin (CG) proteins undergo liquid-liquid phase separation (LLPS) at a mildly acidic pH in the lumen of the TGN, and recruit clients like proinsulin to the condensates. Client selectivity is sequence-independent but based on the concentration of the client molecules in the TGN. We propose that the TGN provides the milieu for converting CGs into a "cargo sponge" leading to partitioning of client molecules, thus facilitating receptor-independent client sorting. These findings provide a new receptor-independent sorting model in ß-cells and many other cell types and therefore represent an innovation in the field of membrane trafficking.

GRASP55 regulates the unconventional secretion and aggregation of mutant huntingtin.

Recent studies demonstrated that the Golgi reassembly stacking proteins (GRASPs), especially GRASP55, regulate Golgi-independent unconventional secretion of certain cytosolic and transmembrane cargoes; however, the underlying mechanism remains unknown. Here, we surveyed several neurodegenerative disease-related proteins, including mutant huntingtin (Htt-Q74), superoxide dismutase 1 (SOD1), tau, and TAR DNA-binding protein 43 (TDP-43), for unconventional secretion; our results show that Htt-Q74 is most robustly secreted in a GRASP55-dependent manner. Using Htt-Q74 as a model system, we demonstrate that unconventional secretion of Htt is GRASP55 and autophagy dependent and is enhanced under stress conditions such as starvation and endoplasmic reticulum stress. Mechanistically, we show that GRASP55 facilitates Htt secretion by tethering autophagosomes to lysosomes to promote autophagosome maturation and subsequent lysosome secretion and by stabilizing p23/TMED10, a channel for translocation of cytoplasmic proteins into the lumen of the endoplasmic reticulum-Golgi intermediate compartment. Moreover, we found that GRASP55 levels are upregulated by various stresses to facilitate unconventional secretion, whereas inhibition of Htt-Q74 secretion by GRASP55 KO enhances Htt aggregation and toxicity. Finally, comprehensive secretomic analysis identified novel cytosolic cargoes secreted by the same unconventional pathway, including transgelin (TAGLN), multifunctional protein ADE2 (PAICS), and peroxiredoxin-1 (PRDX1). In conclusion, this study defines the pathway of GRASP55-mediated unconventional protein secretion and provides important insights into the progression of Huntington's disease.

Adaptor-Specific Antibody Fragment Inhibitors for the Intracellular Modulation of p97 (VCP) Protein-Protein Interactions.

Protein-protein interactions (PPIs) form complex networks to drive cellular signaling and cellular functions. Precise modulation of a target PPI helps explain the role of the PPI in cellular events and possesses therapeutic potential. For example, valosin-containing protein (VCP/p97) is a hub protein that interacts with more than 30 adaptor proteins involved in various cellular functions. However, the role of each p97 PPI during the relevant cellular event is underexplored. The development of small-molecule PPI modulators remains challenging due to a lack of grooves and pockets in the relatively large PPI interface and the fact that a common binding groove in p97 binds to multiple adaptors. Here, we report an antibody fragment-based modulator for the PPI between p97 and its adaptor protein NSFL1C (p47). We engineered these antibody modulators by phage display against the p97-interacting domain of p47 and minimizing binding to other p97 adaptors. The selected antibody fragment modulators specifically disrupt the intracellular p97/p47 interaction. The potential of this antibody platform to develop PPI inhibitors in therapeutic applications was demonstrated through the inhibition of Golgi reassembly, which requires the p97/p47 interaction. This study presents a unique approach to modulate specific intracellular PPIs using engineered antibody fragments, demonstrating a method to dissect the function of a PPI within a convoluted PPI network.

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.

SARS-CoV-2 triggers Golgi fragmentation via down-regulation of GRASP55 to facilitate viral trafficking.

The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an enveloped RNA virus. Despite the high economic and life losses caused by SARS-CoV-2, the detailed viral cycle, especially how it assembles and traffics in the secretory pathway, remains largely unknown. Here, we show that SARS-CoV-2 infection induces global alterations of the host endomembrane system, including dramatic Golgi fragmentation. Disrupting Golgi function with small molecules strongly inhibits viral infection. Furthermore, expression of several SARS-CoV-2 proteins individually is sufficient to trigger Golgi fragmentation. Significantly, SARS-CoV-2 infection down-regulates GRASP55 but up-regulates TGN46 expression, while expression of GRASP55 or knockdown of TGN46 reduces the infection rate of both USA-WA1 and Delta variants of SARS-CoV-2. Our study reveals that SARS-CoV-2 modulates Golgi structure and function via altering GRASP55 and TGN46 expression to facilitate viral trafficking, indicating the Golgi as a novel therapeutic target to block SARS-CoV-2 infection.