Golgi-resident Protein Research Articles

Acute GARP depletion disrupts vesicle transport, leading to severe defects in sorting, secretion, and O-glycosylation.

The GARP complex is an evolutionarily conserved protein complex proposed to tether endosome-derived vesicles at the trans-Golgi network. While prolonged depletion of GARP leads to severe trafficking and glycosylation defects, the primary defects linked to GARP dysfunction remain unclear. In this study, we utilized the mAID degron strategy to achieve rapid degradation of VPS54 in human cells, acutely disrupting GARP function. This resulted in the partial mislocalization and degradation of a subset of Golgi-resident proteins, including TGN46, ATP7A, TMEM87A, CPD, C1GALT1, and GS15. Enzyme recycling defects led to the early onset of O-glycosylation abnormalities. Additionally, while the secretion of fibronectin and cathepsin D was altered, mannose-6-phosphate receptors were largely unaffected. Partial displacement of COPI, AP1, and GGA coats caused a significant accumulation of vesicle-like structures and large vacuoles. Electron microscopy detection of GARP-dependent vesicles, along with the identification of specific cargo proteins, provides direct experimental evidence of GARP's role as a vesicular tether. We conclude that the primary defects of GARP dysfunction involve vesicular coat mislocalization, accumulation of GARP-dependent vesicles, degradation and mislocalization of specific Golgi proteins, and O-glycosylation defects.

The FAM114A proteins are adaptors for the recycling of Golgi enzymes.

Golgi-resident enzymes remain in place while their substrates flow through from the endoplasmic reticulum to elsewhere in the cell. COPI-coated vesicles bud from the Golgi to recycle Golgi residents to earlier cisternae. Different enzymes are present in different parts of the stack, and one COPI adaptor protein, GOLPH3, acts to recruit enzymes into vesicles in part of the stack. Here, we used proximity biotinylation to identify further components of intra-Golgi vesicles and found FAM114A2, a cytosolic protein. Affinity chromatography with FAM114A2, and its paralogue FAM114A1, showed that they bind to Golgi-resident membrane proteins, with membrane-proximal basic residues in the cytoplasmic tail being sufficient for the interaction. Deletion of both proteins from U2OS cells did not cause substantial defects in Golgi function. However, a Drosophila orthologue of these proteins (CG9590/FAM114A) is also localised to the Golgi and binds directly to COPI. Drosophila mutants lacking FAM114A have defects in glycosylation of glue proteins in the salivary gland. Thus, the FAM114A proteins bind Golgi enzymes and are candidate adaptors to contribute specificity to COPI vesicle recycling in the Golgi stack.

Comparative analysis of new mScarlet-based red fluorescent tags in Caenorhabditis elegans.

One problem that has hampered the use of red fluorescent proteins in the fast-developing nematode Caenorhabditis elegans has been the substantial time delay in maturation of several generations of red fluorophores. The recently described mScarlet-I3 protein has properties that may overcome this limitation. We compare here the brightness and onset of expression of CRISPR/Cas9 genome-engineered mScarlet, mScarlet3, mScarlet-I3, and GFP reporter knock-ins. Comparing the onset and brightness of expression of reporter alleles of C. elegans golg-4, encoding a broadly expressed Golgi resident protein, we found that the onset of detection of mScarlet-I3 in the embryo is several hours earlier than older versions of mScarlet and comparable to GFP. These findings were further supported by comparing mScarlet-I3 and GFP reporter alleles for pks-1, a gene expressed in the CAN neuron and cells of the alimentary system, as well as reporter alleles for the pan-neuronal, nuclear marker unc-75. Hence, the relative properties of mScarlet-I3 and GFP do not depend on cellular or subcellular context. In all cases, mScarlet-I3 reporters also show improved signal-to-noise ratio compared to GFP.

Comparative analysis of new, mScarlet-based red fluorescent tags in Caenorhabditis elegans.

One problem that has hampered the use of red fluorescent proteins in the fast-developing nematode C. elegans has been the substantial time delay in maturation of several generations of red fluorophores. The recently described mScarlet-I3 protein has properties that may overcome this limitation. We compare here the brightness and maturation time of CRISPR/Cas9 genome-engineered mScarlet, mScarlet3, mScarlet-I3 and GFP reporter knock-ins. Comparing the onset and brightness of expression of reporter alleles of C. elegans golg-4, encoding a broadly expressed Golgi resident protein, we found that the onset of detection of mScarlet-I3 in the embryo is several hours earlier than older versions of mScarlet and comparable to GFP. These findings were further supported by comparing mScarlet-I3 and GFP reporter alleles for pks-1, a gene expressed in the CAN neuron and cells of the alimentary system, as well as reporter alleles for the panneuronal, nuclear marker unc-75. Hence, the relative properties of mScarlet-I3 and GFP do not depend on cellular or subcellular context. In all cases, mScarlet-I3 reporters also show improved signal-to-noise ratio compared to GFP.

YIPF3 and YIPF4 regulate autophagic turnover of the Golgi apparatus

The degradation of organelles by autophagy is essential for cellular homeostasis. The Golgi apparatus has recently been demonstrated to be degraded by autophagy, but little is known about how the Golgi is recognized by the forming autophagosome. Using quantitative proteomic analysis and two novel Golgiphagy reporter systems, we found that the five-pass transmembrane Golgi-resident proteins YIPF3 and YIPF4 constitute a Golgiphagy receptor. The interaction of this complex with LC3B, GABARAP, and GABARAPL1 is dependent on a LIR motif within YIPF3 and putative phosphorylation sites immediately upstream; the stability of the complex is governed by YIPF4. Expression of a YIPF3 protein containing a mutated LIR motif caused an elongated Golgi morphology, indicating the importance of Golgi turnover via selective autophagy. The reporter assays reported here may be readily adapted to different experimental contexts to help deepen our understanding of Golgiphagy.

Chromosomal 3q amplicon encodes essential regulators of secretory vesicles that drive secretory addiction in cancer.

Cancer cells exhibit heightened secretory states that drive tumor progression. Here, we identify a chromosome 3q amplicon that serves as a platform for secretory regulation in cancer. The 3q amplicon encodes multiple Golgi-resident proteins, including the scaffold Golgi integral membrane protein 4 (GOLIM4) and the ion channel ATPase Secretory Pathway Ca2+ Transporting 1 (ATP2C1). We show that GOLIM4 recruits ATP2C1 and Golgi phosphoprotein 3 (GOLPH3) to coordinate calcium-dependent cargo loading and Golgi membrane bending and vesicle scission. GOLIM4 depletion disrupts the protein complex, resulting in a secretory blockade that inhibits the progression of 3q-amplified malignancies. In addition to its role as a scaffold, GOLIM4 maintains intracellular manganese (Mn) homeostasis by binding excess Mn in the Golgi lumen, which initiates the routing of Mn-bound GOLIM4 to lysosomes for degradation. We show that Mn treatment inhibits the progression of multiple types of 3q-amplified malignancies by degrading GOLIM4, resulting in a secretory blockade that interrupts pro-survival autocrine loops and attenuates pro-metastatic processes in the tumor microenvironment. Potentially underlying the selective activity of Mn against 3q-amplified malignancies, ATP2C1 co-amplification increases Mn influx into the Golgi lumen, resulting in a more rapid degradation of GOLIM4. These findings show that functional cooperativity between co-amplified genes underlies heightened secretion and a targetable secretory addiction in 3q-amplified malignancies.

Abstract 6980: GOLGA7 is essential for NRAS trafficking from the Golgi to the plasma membrane but not for its palmitoylation

Abstract RAS GTPases play a crucial role as oncogenic drivers in multiple cancer types. Targeting their localization to the plasma membrane (PM) is a promising strategy for cancer therapy, as RAS signaling requires PM localization. While KRAS traffics through a Golgi-independent route, palmitoylated RAS isoforms are transported to PM from the Golgi via a secretory pathway that is not yet fully understood. GOLGA7 is a Golgi-resident protein known as an accessory protein stabilizing the palmitoyltransferase ZDHHC9 for the palmitoylation of NRAS and HRAS. In this study, we found that among all four RAS isoforms, NRAS uniquely requires GOLGA7 for its association with the PM. Interestingly, the mislocalization of NRAS is achieved without detectable changes in its palmitoylation level in GOLGA7 deficient cells. We further observed that loss of GOLGA7 inhibit the anterograde trafficking of GFP-NRASG12D proteins and result in the accumulation of NRAS in the cis compartment of the Golgi apparatus. Moreover, targeting GOLGA7 suppresses cell proliferation by downregulating RAS signaling in a variety of NRAS-mutant cancer cells. Finally, GOLGA7 depletion attenuated the oncogenic transformation induced by NRASG12D in mice. These findings reveal a specific intracellular trafficking route mediated by GOLGA7 and have identified GOLGA7 as a potential therapeutic target for NRAS-driven cancers. Citation Format: Chenxuan Liu, Bo Jiao, Peihong Wang, Baoyuan Zhang, Jiaming Gao, Donghe Li, Xi Xie, Yunying Yao, Lei Yan, Zhenghong Qin, Ping Liu, Ruibao Ren. GOLGA7 is essential for NRAS trafficking from the Golgi to the plasma membrane but not for its palmitoylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6980.

The activation of the adaptor protein STING depends on its interactions with the phospholipid PI4P.

Activation of the endoplasmic reticulum (ER)-resident adaptor protein STING, a component of a cytosolic DNA-sensing pathway, induces the transcription of genes encoding type I interferons (IFNs) and other proinflammatory factors. Because STING is activated at the Golgi apparatus, control of the localization and activation of STING is important in stimulating antiviral and antitumor immune responses. Through a genome-wide CRISPR interference screen, we found that STING activation required the Golgi-resident protein ACBD3, which promotes the generation of phosphatidylinositol 4-phosphate (PI4P) at the trans-Golgi network, as well as other PI4P-associated proteins. Appropriate localization and activation of STING at the Golgi apparatus required ACBD3 and the PI4P-generating kinase PI4KB. In contrast, STING activation was enhanced when the lipid-shuttling protein OSBP, which removes PI4P from the Golgi apparatus, was inhibited by the US Food and Drug Administration-approved antifungal itraconazole. The increase in the abundance of STING-activating phospholipids at the trans-Golgi network resulted in the increased production of IFN-β and other cytokines in THP-1 cells. Furthermore, a mutant STING that could not bind to PI4P failed to traffic from the ER to the Golgi apparatus in response to a STING agonist, whereas forced relocalization of STING to PI4P-enriched areas elicited STING activation in the absence of stimulation with a STING agonist. Thus, PI4P is critical for STING activation, and manipulating PI4P abundance may therapeutically modulate STING-dependent immune responses.

MMD collaborates with ACSL4 and MBOAT7 to promote polyunsaturated phosphatidylinositol remodeling and susceptibility to ferroptosis

Ferroptosis is a form of regulated cell death with roles in degenerative diseases and cancer. Excessive iron-catalyzed peroxidation of membrane phospholipids, especially those containing the polyunsaturated fatty acid arachidonic acid (AA), is central in driving ferroptosis. Here, we reveal that an understudied Golgi-resident scaffold protein, MMD, promotes susceptibility to ferroptosis in ovarian and renal carcinoma cells in an ACSL4- and MBOAT7-dependent manner. Mechanistically, MMD physically interacts with both ACSL4 and MBOAT7, two enzymes that catalyze sequential steps to incorporate AA in phosphatidylinositol (PI) lipids. Thus, MMD increases the flux of AA into PI, resulting in heightened cellular levels of AA-PI and other AA-containing phospholipid species. This molecular mechanism points to a pro-ferroptotic role for MBOAT7 and AA-PI, with potential therapeutic implications, and reveals that MMD is an important regulator of cellular lipid metabolism.

Organelle mapping in dendrites of human iPSC-derived neurons reveals dynamic functional dendritic Golgi structures

Secretory pathways within dendrites of neurons have been proposed for local transport of newly synthesized proteins. However, little is known about the dynamics of the local secretory system and whether the organelles are transient or stable structures. Here, we quantify the spatial and dynamic behavior of dendritic Golgi and endosomes during differentiation of human neurons generated from induced pluripotent stem cells (iPSCs). In early neuronal development, before and during migration, the entire Golgi apparatus transiently translocates from the soma into dendrites. In mature neurons, dynamic Golgi elements, containing cis and trans cisternae, are transported from the soma along dendrites, in an actin-dependent process. Dendritic Golgi outposts are dynamic and display bidirectional movement. Similar structures were observed in cerebral organoids. Using the retention using selective hooks (RUSH) system, Golgi resident proteins are transported efficiently into Golgi outposts from the endoplasmic reticulum. This study reveals dynamic, functional Golgi structures in dendrites and a spatial map for investigating dendrite trafficking in human neurons.

Golgi-IP, a tool for multimodal analysis of Golgi molecular content

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.

The factory, the antenna and the scaffold: the three-way interplay between the Golgi, cilium and extracellular matrix underlying tissue function.

The growth and development of healthy tissues is dependent on the construction of a highly specialised extracellular matrix (ECM) to provide support for cell growth and migration and to determine the biomechanical properties of the tissue. These scaffolds are composed of extensively glycosylated proteins which are secreted and assembled into well-ordered structures that can hydrate, mineralise, and store growth factors as required. The proteolytic processing and glycosylation of ECM components is vital to their function. These modifications are under the control of the Golgi apparatus, an intracellular factory hosting spatially organised, protein-modifying enzymes. Regulation also requires a cellular antenna, the cilium, which integrates extracellular growth signals and mechanical cues to inform ECM production. Consequently, mutations in either Golgi or ciliary genes frequently lead to connective tissue disorders. The individual importance of each of these organelles to ECM function is well-studied. However, emerging evidence points towards a more tightly linked system of interdependence between the Golgi, cilium and ECM. This review examines how the interplay between all three compartments underpins healthy tissue. As an example, it will look at several members of the golgin family of Golgi-resident proteins whose loss is detrimental to connective tissue function. This perspective will be important for many future studies looking to dissect the cause and effect of mutations impacting tissue integrity.

Impact of Hypermannosylation on the Structure and Functionality of the ER and the Golgi Complex.

Proteins of the secretory pathway undergo glycosylation in the endoplasmic reticulum (ER) and the Golgi apparatus. Altered protein glycosylation can manifest in serious, sometimes fatal malfunctions. We recently showed that mutations in GDP-mannose pyrophosphorylase A (GMPPA) can cause a syndrome characterized by alacrima, achalasia, mental retardation, and myopathic alterations (AAMR syndrome). GMPPA acts as a feedback inhibitor of GDP-mannose pyrophosphorylase B (GMPPB), which provides GDP-mannose as a substrate for protein glycosylation. Loss of GMPPA thus enhances the incorporation of mannose into glycochains of various proteins, including α-dystroglycan (α-DG), a protein that links the extracellular matrix with the cytoskeleton. Here, we further characterized the consequences of loss of GMPPA for the secretory pathway. This includes a fragmentation of the Golgi apparatus, which comes along with a regulation of the abundance of several ER- and Golgi-resident proteins. We further show that the activity of the Golgi-associated endoprotease furin is reduced. Moreover, the fraction of α-DG, which is retained in the ER, is increased. Notably, WT cells cultured at a high mannose concentration display similar changes with increased retention of α-DG, altered structure of the Golgi apparatus, and a decrease in furin activity. In summary, our data underline the importance of a balanced mannose homeostasis for the secretory pathway.

Cab45 deficiency leads to the mistargeting of progranulin and prosaposin and aberrant lysosomal positioning.

The trans-Golgi Network (TGN) sorts molecular "addresses" and sends newly synthesized proteins to their destination via vesicular transport carriers. Despite the functional significance of packaging processes at the TGN, the sorting of soluble proteins remains poorly understood. Recent research has shown that the Golgi resident protein Cab45 is a significant regulator of secretory cargo sorting at the TGN. Cab45 oligomerizes upon transient Ca2+ influx, recruits soluble cargo molecules (clients), and packs them in sphingomyelin-rich transport carriers. However, the identity of client molecules packed into Cab45 vesicles is scarce. Therefore, we used a precise and highly efficient secretome analysis technology called hiSPECs. Intriguingly, we observed that Cab45 deficient cells manifest hypersecretion of lysosomal hydrolases. Specifically, Cab45 deficient cells secrete the unprocessed precursors of prosaposin (PSAP) and progranulin (PGRN). In addition, lysosomes in these cells show an aberrant perinuclear accumulation suggesting a new role of Cab45 in lysosomal positioning. This work uncovers a yet unknown function of Cab45 in regulating lysosomal function.

Acute COG complex inactivation unveiled its immediate impact on Golgi and illuminated the nature of intra-Golgi recycling vesicles.

Conserved Oligomeric Golgi (COG) complex controls Golgi trafficking and glycosylation, but the precise COG mechanism is unknown. The auxin-inducible acute degradation system was employed to investigate initial defects resulting from COG dysfunction. We found that acute COG inactivation caused a massive accumulation of COG-dependent (CCD) vesicles that carry the bulk of Golgi enzymes and resident proteins. v-SNAREs (GS15, GS28) and v-tethers (giantin, golgin84, and TMF1) were relocalized into CCD vesicles, while t-SNAREs (STX5, YKT6), t-tethers (GM130, p115), and most of Rab proteins remained Golgi-associated. Airyscan microscopy and velocity gradient analysis revealed that different Golgi residents are segregated into different populations of CCD vesicles. Acute COG depletion significantly affected three Golgi-based vesicular coats-COPI, AP1, and GGA, suggesting that COG uniquely orchestrates tethering of multiple types of intra-Golgi CCD vesicles produced by different coat machineries. This study provided the first detailed view of primary cellular defects associated with COG dysfunction in human cells.

Modeling N-Glycosylation: A Systems Biology Approach for Evaluating Changes in the Steady-State Organization of Golgi-Resident Proteins.

The organization of Golgi-resident proteins is crucial for sorting molecules within the secretory pathway and regulating posttranslational modifications. However, evaluating changes to Golgi organization can be challenging, often requiring extensive experimental investigations. Here, we propose a systems biology approach in which changes to Golgi-resident protein sorting and localization can be deduced using cellular N-glycan profiles as the only experimental input.The approach detailed here utilizes the influence of Golgi organization on N-glycan biosynthesis to investigate the mechanisms involved in establishing and maintaining Golgi organization. While N-glycosylation is carried out in a non-template-driven manner, the distribution of N-glycan biosynthetic enzymes within the Golgi ensures this process is not completely random. Therefore, changes to N-glycan profiles provide clues into how altered cell phenotypes affect the sorting and localization of Golgi-resident proteins. Here, we generate a stochastic simulation of N-glycan biosynthesis to produce a simulated glycan profile similar to that obtained experimentally and then combine this with Bayesian fitting to enable inference of changes in enzyme amounts and localizations. Alterations to Golgi organization are evaluated by calculating how the fitted enzyme parameters shift when moving from simulating the glycan profile of one cellular state (e.g., a wild type) to an altered cellular state (e.g., a mutant). Our approach illustrates how an iterative combination of mathematical systems biology and minimal experimental cell biology can be utilized to maximally integrate biological knowledge to gain insightful knowledge of the underlying mechanisms in a manner inaccessible to either alone.

Quantification of Golgi Protein Mislocalization to the Budding Yeast Vacuole.

The localization of proteins to the Golgi complex is a dynamic process requiring sorting signals in the cytosolic domains of resident Golgi proteins and retrograde vesicular trafficking. Disruptions in these signals or in the retrograde pathways often lead to mislocalization of Golgi proteins to the vacuole in budding yeast. The extent of vacuolar mislocalization can be quantified through colocalization of GFP-tagged Golgi proteins with fluorescent dyes that mark either the vacuole limiting membrane or the vacuole lumen. Manders' colocalization coefficient (MCC) is a useful tool for quantifying the degree of colocalization. However, the dilution of fluorescence signal intensity that occurs when GFP-tagged Golgi proteins mislocalize to the much larger vacuole is problematic for thresholding the images prior to calculating the MCC. In this chapter, we describe the use of Multi-Otsu thresholding in ImageJ to quantify the degree of GFP-tagged protein mislocalization to the vacuole. Furthermore, these methods can be applied to other colocalization events within the cell.

GARP dysfunction results in COPI displacement, depletion of Golgi v-SNAREs and calcium homeostasis proteins.

Golgi-associated retrograde protein (GARP) is an evolutionary conserved heterotetrameric protein complex that tethers endosome-derived vesicles and is vital for Golgi glycosylation. Microscopy and proteomic approaches were employed to investigate defects in Golgi physiology in RPE1 cells depleted for the GARP complex. Both cis and trans-Golgi compartments were significantly enlarged in GARP-knock-out (KO) cells. Proteomic analysis of Golgi-enriched membranes revealed significant depletion of a subset of Golgi residents, including Ca2+ binding proteins, enzymes, and SNAREs. Validation of proteomics studies revealed that SDF4 and ATP2C1, related to Golgi calcium homeostasis, as well as intra-Golgi v-SNAREs GOSR1 and BET1L, were significantly depleted in GARP-KO cells. Finding that GARP-KO is more deleterious to Golgi physiology than deletion of GARP-sensitive v-SNAREs, prompted a detailed investigation of COPI trafficking machinery. We discovered that in GARP-KO cells COPI is significantly displaced from the Golgi and partially relocalized to the ER-Golgi intermediate compartment (ERGIC). Moreover, COPI accessory proteins GOLPH3, ARFGAP1, GBF1, and BIG1 are also relocated to off-Golgi compartments. We propose that the dysregulation of COPI machinery, along with the depletion of Golgi v-SNAREs and alteration of Golgi Ca2+ homeostasis, are the major driving factors for the depletion of Golgi resident proteins, structural alterations, and glycosylation defects in GARP deficient cells.

The Golgi-resident protein ACBD3 concentrates STING at ER-Golgi contact sites to drive export from the ER.

STING, an endoplasmic reticulum (ER)-resident receptor for cyclic di-nucleotides (CDNs), is essential for innate immune responses. Upon CDN binding, STING moves from the ER to the Golgi, where it activates downstream type-I interferon (IFN) signaling. General cargo proteins exit from the ER via concentration at ER exit sites. However, the mechanism of STING concentration is poorly understood. Here, we visualize the ER exit sites of STING by blocking its transport at low temperature or by live-cell imaging with the cell-permeable ligand bis-pivSATE-2'F-c-di-dAMP, which we have developed. After ligand binding, STING forms punctate foci at non-canonical ER exit sites. Unbiased proteomic screens and super-resolution microscopy show that the Golgi-resident protein ACBD3/GCP60 recognizes and concentrates ligand-bound STING at specialized ER-Golgi contact sites. Depletion of ACBD3 impairs STING ER-to-Golgi trafficking and type-I IFN responses. Our results identify the ACBD3-mediated non-canonical cargo concentration system that drives the ER exit of STING.

Sec14l4 SNP Mice Have Increased Susceptibility to Oxidative Hemolysis through Modulation of G6PD and the Pentose Phosphate Pathway

Red blood cell function and recovery following transfusion is influenced by a variety of factors including sex, race, sickle cell trait and single nucleotide polymorphisms (SNPs). In collaboration with the REDS III program, we performed a genome wide association study identifying SNPs that are associated with in vitro osmotic, oxidative and spontaneous storage hemolysis stress. Sec14L4, was one of 27 genes identified with enhanced hemolysis, specifically following oxidative insult and later confirmed by an independent study to be associated with decreased hemoglobin increments following transfusion. SEC14L4 is an understudied, highly conserved phosphatidylinositol transfer protein that is predicted to reside with the Golgi Apparatus. While the function of SEC14L4 is not understood, it is clear that polymorphisms in this gene have clinical impact and understanding its role in red cell biology and transfusion is critical. We generated a novel humanized Sec14L4 SNP and knockout mouse model to assess RBC metabolism, function and transfusion outcomes following storage. While Sec14l4 KO mice have complete loss of SEC14L4 protein expression, the SNP has an approximate 50% decrease in expression, suggesting the mutation decreases protein and/or mRNA stability. Interestingly, knockout mice experience anemia with decreased hemoglobin levels and lower hematocrit at baseline. Following 12 days of storage (equivalent to 42 days of human blood storage) Sec14l4 knockout RBCs have increased hemolysis and are more susceptible to lysis following treatment with the oxidant diamide. Tracing experiments with 1,2,313C3-glucose from Sec14l4 SNP mouse RBCs reveals decreased levels of glucose-6-phosaphate dehydrogenase (G6PD) and depression of metabolic fluxes through the pentose phosphate pathway with lower levels of 13C3-6-phosphogluconate and nicotinamide adenine dinucleotide phosphate (NADP+), and reduced ADP and ATP. Compensatory increases in glycolytic flux was also observed. G6PD is the rate liming enzyme of the pentose phosphate pathway which is the sole producer of NADPH in the red blood cell. NADPH is an essential cofactor for glutathione reductase and peroxidase, the primary erythrocyte antioxidant enzymes. Red blood cells deficient for G6PD undergo acute hemolysis if exposed to oxidative stress due to decreased antioxidant capacity. These data suggest that red blood cells deficient in SEC14L4 phenocopy G6PD deficient red cells, possibly explaining increased susceptibility to oxidative insult. Lipidomic evaluation also reveled increased accumulation of oxidized lipids during storage (12-HETE and 15-HETE). Oxilipins are markers of poor post-transfusion recovery in stored mouse and human red blood cells. Together these data indicate that SEC14L4 is an important player in red blood cell stability during storage, oxidative stress, and following transfusion. How a Golgi resident protein influences the adult red cell is not yet understood but suggests a role for SEC14L4 in early erythrocyte development which has an effect over the lifetime of the cell, with the result of increased stability and antioxidant capacity. In support of this hypothesis, Sec14l4 is highly expressed in the bone marrow with decreasing expression as the red cell terminally differentiates. Future work will confirm proteomic, metabolomic and lipidomic findings and focus on how SEC14L4 may be regulating the G6PD pathway in mature red cells.