Phosphatidylinositol Transfer Protein Research Articles

Overexpression of PDR16 Confers Amphotericin B Resistance in a PMP3-Dependent Manner in Yeast Saccharomyces cerevisiae.

Invasive fungal infections in humans with compromised immune systems are the primary cause of morbidity and mortality, which is becoming more widely acknowledged. Amphotericin B (AmB) is one of the antifungal drugs used to treat such infections. AmB binds with plasma membrane ergosterol, inducing cellular ions to leak and causing cell death. Reduction in ergosterol content and modification of cell walls have been described as AmB resistance mechanisms. In addition, when the sphingolipid level is decreased, the cell becomes more susceptible to AmB. Previously, PDR16, a gene that encodes phosphatidylinositol transfer protein in Saccharomyces cerevisiae, was shown to enhance AmB resistance upon overexpression. However, the mechanism of PDR16-mediated AmB resistance is not clear. Here, in this study, it was discovered that a plasma membrane proteolipid 3 protein encoded by PMP3 is essential for PDR16-mediated AmB resistance. PDR16-mediated AmB resistance does not depend on ergosterol, but a functional sphingolipid biosynthetic pathway is required. Additionally, PMP3-mediated alteration in membrane integrity abolishes PDR16 mediated AmB resistance, confirming the importance of PMP3 in the PDR16 mediated AmB resistance.

Non-vesicular phosphatidylinositol transfer plays critical roles in defining organelle lipid composition

Phosphatidylinositol (PI) is the precursor lipid for the minor phosphoinositides (PPIns), which are critical for multiple functions in all eukaryotic cells. It is poorly understood how phosphatidylinositol, which is synthesized in the ER, reaches those membranes where PPIns are formed. Here, we used VT01454, a recently identified inhibitor of class I PI transfer proteins (PITPs), to unravel their roles in lipid metabolism, and solved the structure of inhibitor-bound PITPNA to gain insight into the mode of inhibition. We found that class I PITPs not only distribute PI for PPIns production in various organelles such as the plasma membrane (PM) and late endosomes/lysosomes, but that their inhibition also significantly reduced the levels of phosphatidylserine, di- and triacylglycerols, and other lipids, and caused prominent increases in phosphatidic acid. While VT01454 did not inhibit Golgi PI4P formation nor reduce resting PM PI(4,5)P2 levels, the recovery of the PM pool of PI(4,5)P2 after receptor-mediated hydrolysis required both class I and class II PITPs. Overall, these studies show that class I PITPs differentially regulate phosphoinositide pools and affect the overall cellular lipid landscape.

A genetic screen to uncover mechanisms underlying lipid transfer protein function at membrane contact sites.

Lipid transfer proteins mediate the transfer of lipids between organelle membranes, and the loss of function of these proteins has been linked to neurodegeneration. However, the mechanism by which loss of lipid transfer activity leads to neurodegeneration is not understood. In Drosophila photoreceptors, depletion of retinal degeneration B (RDGB), a phosphatidylinositol transfer protein, leads to defective phototransduction and retinal degeneration, but the mechanism by which loss of this activity leads to retinal degeneration is not understood. RDGB is localized to membrane contact sites through the interaction of its FFAT motif with the ER integral protein VAP. To identify regulators of RDGB function in vivo, we depleted more than 300 VAP-interacting proteins and identified a set of 52 suppressors of rdgB The molecular identity of these suppressors indicates a role of novel lipids in regulating RDGB function and of transcriptional and ubiquitination processes in mediating retinal degeneration in rdgB9 The human homologs of several of these molecules have been implicated in neurodevelopmental diseases underscoring the importance of VAP-mediated processes in these disorders.

Abstract 2365 Class I phosphatidylinositol transfer proteins control organelle membrane lipid composition beyond supporting phosphoinositides synthesis

Abstract 2365 Class I phosphatidylinositol transfer proteins control organelle membrane lipid composition beyond supporting phosphoinositides synthesis

A metabolically controlled contact site between vacuoles and lipid droplets in yeast

The lipid droplet (LD) organization proteins Ldo16 and Ldo45 affect multiple aspects of LD biology in yeast. They are linked to the LD biogenesis machinery seipin, and their loss causes defects in LD positioning, protein targeting, and breakdown. However, their molecular roles remained enigmatic. Here, we report that Ldo16/45 form a tether complex with Vac8 to create vacuole lipid droplet (vCLIP) contact sites, which can form in the absence of seipin. The phosphatidylinositol transfer protein (PITP) Pdr16 is a further vCLIP-resident recruited specifically by Ldo45. While only an LD subpopulation is engaged in vCLIPs at glucose-replete conditions, nutrient deprivation results in vCLIP expansion, and vCLIP defects impair lipophagy upon prolonged starvation. In summary, Ldo16/45 are multifunctional proteins that control the formation of a metabolically regulated contact site. Our studies suggest a link between LD biogenesis and breakdown and contribute to a deeper understanding of how lipid homeostasis is maintained during metabolic challenges.

PITPNC1 Suppress CD8+ T cell immune function and promote radioresistance in rectal cancer by modulating FASN/CD155

BackgroundRadioresistance is a primary factor contributing to the failure of rectal cancer treatment. Immune suppression plays a significant role in the development of radioresistance. We have investigated the potential role of phosphatidylinositol transfer protein cytoplasmic 1 (PITPNC1) in regulating immune suppression associated with radioresistance.MethodsTo elucidate the mechanisms by which PITPNC1 influences radioresistance, we established HT29, SW480, and MC38 radioresistant cell lines. The relationship between radioresistance and changes in the proportion of immune cells was verified through subcutaneous tumor models and flow cytometry. Changes in the expression levels of PITPNC1, FASN, and CD155 were determined using immunohistochemistry and western blotting techniques. The interplay between these proteins was investigated using immunofluorescence co-localization and immunoprecipitation assays. Additionally, siRNA and lentivirus-mediated gene knockdown or overexpression, as well as co-culture of tumor cells with PBMCs or CD8+ T cells and establishment of stable transgenic cell lines in vivo, were employed to validate the impact of the PITPNC1/FASN/CD155 pathway on CD8+ T cell immune function.ResultsUnder irradiation, the apoptosis rate and expression of apoptosis-related proteins in radioresistant colorectal cancer cell lines were significantly decreased, while the cell proliferation rate increased. In radioresistant tumor-bearing mice, the proportion of CD8+ T cells and IFN-γ production within immune cells decreased. Immunohistochemical analysis of human and animal tissue specimens resistant to radiotherapy showed a significant increase in the expression levels of PITPNC1, FASN, and CD155. Gene knockdown and rescue experiments demonstrated that PITPNC1 can regulate the expression of CD155 on the surface of tumor cells through FASN. In addition, co-culture experiments and in vivo tumor-bearing experiments have shown that silencing PITPNC1 can inhibit FASN/CD155, enhance CD8+ T cell immune function, promote colorectal cancer cell death, and ultimately reduce radioresistance in tumor-bearing models.ConclusionsPITPNC1 regulates the expression of CD155 through FASN, inhibits CD8+ T cell immune function, and promotes radioresistance in rectal cancer.

Microcolin H, a novel autophagy inducer, exerts potent antitumour activity by targeting PITPα/β

The identification of effective drug targets and the development of bioactive molecules are areas of high need in cancer therapy. The phosphatidylinositol transfer protein alpha/beta isoform (PITPα/β) has been reported to play an essential role in integrating phosphoinositide trafficking and lipid metabolism in diverse cellular processes but remains unexplored as a potential target for cancer treatment. Herein, data analysis of clinical cancer samples revealed that PITPα/β expression is closely correlated with the poor prognosis. Target identification by chemical proteomic methods revealed that microcolin H, a naturally occurring marine lipopeptide, directly binds PITPα/β and displays antiproliferative activity on different types of tumour cell lines. Furthermore, we identified that microcolin H treatment increased the conversion of LC3I to LC3II, accompanied by a reduction of the level of p62 in cancer cells, leading to autophagic cell death. Moreover, microcolin H showed preeminent antitumour efficacy in nude mouse subcutaneous tumour models with low toxicity. Our discoveries revealed that by targeting PITPα/β, microcolin H induced autophagic cell death in tumours with efficient anti-proliferating activity, which sheds light on PITPα/β as a promising therapeutic target for cancer treatment.

The expanding roles of PI4P and PI(4,5)P2 at the plasma membrane: Role of phosphatidylinositol transfer proteins

Phosphoinositides are phosphorylated derivatives of phosphatidylinositol, a phospholipid that is synthesised at the endoplasmic reticulum. The plasma membrane contains the enzymes to phosphorylate phosphatidylinositol and is therefore rich in the phosphorylated derivatives, PI4P and PI(4,5)P2. PI(4,5)P2 is a substrate for phospholipase C and during cell signaling, PI(4,5)P2 levels are reduced. Here I discuss a family of proteins, phosphatidylinositol transfer proteins (PITPs) that can restore PI(4,5)P2 levels.

Synthesis of a photocleavable bola-phosphatidylcholine

Phosphatidylinositol transfer proteins (PITPs) are ubiquitous in eukaryotes and are involved in the regulation of phospholipid metabolism, membrane trafficking, and signal transduction. Sec14 is a yeast PITP that has been shown to transfer phosphatidylinositol (PI) or phosphatidylcholine (PC) from the endoplasmic reticulum to the Golgi. It is now believed that Sec14 may play a greater role than just shuttling PI and PC throughout the cell. Genetic evidence suggests that retrieval of membrane-bound PI by Sec14 also manages to present PI to the phosphatidylinositol-4-kinase, Pik1, to generate phosphatidylinositol-4-phosphate, PI(4)P. To test this hypothetical model, we designed a photocleavable bolalipid to span the entire membrane, having one phosphatidylcholine or phosphatidylinositol headgroup on each leaflet connected by a photocleavable diacid. Sec14 should not be able to present the bola-PI to Pik1 for phosphorylation as the head group will be difficult to lift from the bilayer as it is tethered on the opposite leaflet. After photocleavage the two halves would behave as a normal phospholipid, thus phosphorylation by Pik1 would resume. We report here the synthesis of a photocleavable bola-PC, a precursor to the desired bola-PI. The mono-photocleavable bola-PC lipid was designed to contain two glycerol molecules with choline head groups connected through a phosphodiester bond at the sn3 position. Each glycerol was acylated with palmitic acid at the sn1 position. These two glycerol moieties were then connected through their respective sn2 hydroxyls via a photocleavable dicarboxylic acid containing a nitrophenyl ethyl photolabile protecting group. The bola-PC and its precursors were found to undergo efficient photocleavage when irradiated in solution or in vesicles with 365 nm light for two minutes. Treatment of the bola-PC with a mutant phospholipase D and myo-inositol produced a mono-inositol bola-PC-PI.

Rescuing β cells.

Loss of phosphatidylinositol transfer protein alpha underlies pancreatic β cell failure.

Restoration of PITPNA in Type 2 diabetic human islets reverses pancreatic beta-cell dysfunction

Defects in insulin processing and granule maturation are linked to pancreatic beta-cell failure during type 2 diabetes (T2D). Phosphatidylinositol transfer protein alpha (PITPNA) stimulates activity of phosphatidylinositol (PtdIns) 4-OH kinase to produce sufficient PtdIns-4-phosphate (PtdIns-4-P) in the trans-Golgi network to promote insulin granule maturation. PITPNA in beta-cells of T2D human subjects is markedly reduced suggesting its depletion accompanies beta-cell dysfunction. Conditional deletion of Pitpna in the beta-cells of Ins-Cre, Pitpnaflox/flox mice leads to hyperglycemia resulting from decreasing glucose-stimulated insulin secretion (GSIS) and reducing pancreatic beta-cell mass. Furthermore, PITPNA silencing in human islets confirms its role in PtdIns-4-P synthesis and leads to impaired insulin granule maturation and docking, GSIS, and proinsulin processing with evidence of ER stress. Restoration of PITPNA in islets of T2D human subjects reverses these beta-cell defects and identify PITPNA as a critical target linked to beta-cell failure in T2D.

The lipid transfer proteins Nir2 and Nir3 sustain phosphoinositide signaling and actin dynamics during phagocytosis.

Changes in membrane phosphoinositides and local Ca2+ elevations at sites of particle capture coordinate the dynamic remodeling of the actin cytoskeleton during phagocytosis. Here, we show that the phosphatidylinositol (PI) transfer proteins PITPNM1 (Nir2) and PITPNM2 (Nir3) maintain phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] homeostasis at phagocytic cups, thereby promoting actin contractility and the sealing of phagosomes. Nir3 and to a lesser extent Nir2 accumulated on endoplasmic reticulum (ER) cisternae juxtaposed to phagocytic cups when expressed in phagocytic COS-7 cells. CRISPR-Cas9 editing of Nir2 and Nir3 genes decreased plasma membrane PI(4,5)P2 levels, store-operated Ca2+ entry (SOCE) and receptor-mediated phagocytosis, stalling particle capture at the cup stage. Re-expression of either Nir2 or Nir3 restored phagocytosis, but not SOCE, proportionally to the PM PI(4,5)P2 levels. Phagosomes forming in Nir2 and Nir3 (Nir2/3) double-knockout cells had decreased overall PI(4,5)P2 levels but normal periphagosomal Ca2+ signals. Nir2/3 depletion reduced the density of contractile actin rings at sites of particle capture, causing repetitive low-intensity contractile events indicative of abortive phagosome closure. We conclude that Nir proteins maintain phosphoinositide homeostasis at phagocytic cups, thereby sustaining the signals that initiate the remodeling of the actin cytoskeleton during phagocytosis.

Role of SEC14-like phosphatidylinositol transfer proteins in membrane identity and dynamics.

Membrane identity and dynamic processes, that act at membrane sites, provide important cues for regulating transport, signal transduction and communication across membranes. There are still numerous open questions as to how membrane identity changes and the dynamic processes acting at the surface of membranes are regulated in diverse eukaryotes in particular plants and which roles are being played by protein interaction complexes composed of peripheral and integral membrane proteins. One class of peripheral membrane proteins conserved across eukaryotes comprises the SEC14-like phosphatidylinositol transfer proteins (SEC14L-PITPs). These proteins share a SEC14 domain that contributes to membrane identity and fulfills regulatory functions in membrane trafficking by its ability to sense, bind, transport and exchange lipophilic substances between membranes, such as phosphoinositides and diverse other lipophilic substances. SEC14L-PITPs can occur as single-domain SEC14-only proteins in all investigated organisms or with a modular domain structure as multi-domain proteins in animals and streptophytes (comprising charales and land plants). Here, we present an overview on the functional roles of SEC14L-PITPs, with a special focus on the multi-domain SEC14L-PITPs of the SEC14-nodulin and SEC14-GOLD group (PATELLINs, PATLs in plants). This indicates that SEC14L-PITPs play diverse roles from membrane trafficking to organism fitness in plants. We concentrate on the structure of SEC14L-PITPs, their ability to not only bind phospholipids but also other lipophilic ligands, and their ability to regulate complex cellular responses through interacting with proteins at membrane sites.

Individual phosphatidylinositol transfer proteins have distinct functions that do not involve lipid transfer activity

AbstractPlatelets use signal transduction pathways facilitated by class I phosphatidylinositol transfer proteins (PITPs). The 2 mammalian class I PITPs, PITPα and PITPβ, are single PITP domain soluble proteins that are encoded by different genes and share 77% sequence identity, although their individual roles in mammalian biology remain uncharacterized. These proteins are believed to shuttle phosphatidylinositol and phosphatidylcholine between separate intracellular membrane compartments, thereby regulating phosphoinositide synthesis and second messenger formation. Previously, we observed that platelet-specific deletion of PITPα, the predominantly expressed murine PITP isoform, had no effect on hemostasis but impaired tumor metastasis formation and disrupted phosphoinositide signaling. Here, we found that mice lacking the less expressed PITPβ in their platelets exhibited a similar phenotype. However, in contrast to PITPα-null platelet lysates, which have impaired lipid transfer activity, PITPβ-null platelet lysates have essentially normal lipid transfer activity, although both isoforms contribute to phosphoinositide synthesis in vitro. Moreover, we found that platelet-specific deletion of both PITPs led to ex vivo platelet aggregation/secretion and spreading defects, impaired tail bleeding, and profound tumor dissemination. Our study also demonstrated that PITP isoforms are required to maintain endogenous phosphoinositide PtdInsP2 levels and agonist-stimulated second messenger formation. The data shown here demonstrate that the 2 isoforms are functionally overlapping and that a single isoform is able to maintain the homeostasis of platelets. However, both class I PITP isoforms contribute to phosphoinositide signaling in platelets through distinct biochemical mechanisms or different subcellular domains.

Golgi signalling proteins GOLPH3, MYO18A, PITPNC1 and RAB1B: implications in prognosis and survival outcomes of AML patients

Background The role of different Golgi signalling proteins remains unexplored in the progression and spread of acute myeloid leukaemia (AML), whom all interact together in a way that facilitates proliferation and differentiation of myeloid lineage cells. Objective Since Golgi apparatus acts as master brain in membrane trafficking and signalling events that affect cell polarity necessary for migration, division, or differentiation; this study aims to explore the association between signalling proteins and the diagnosis, prognosis, and survival of AML patients. Material and methods This study comprised 70 newly diagnosed AML patients and 20 healthy controls to investigate the serum levels of signalling proteins; Golgi Phosphoprotein 3 (GOLPH3), Myosin 18A (MYO18A), Cytoplasmic Phosphatidylinositol Transfer Protein 1 (PITPNC1) and Ras-Associated Binding Protein 1B (RAB1B). Results AML patients showed higher serum levels of GOLPH3, MYO18A, PITPNC1 and RAB1B when compared to control (p < 0.001). A significant negative correlation was found between the patients’ overall survival and GOLPH3 (p = 0.001), MYO18A (p = 0.011), PITPNC1 (p = 0.001) and RAB1B (p = 0.042). Results were confirmed by Kaplen–Meier survival analysis showing lower survival estimates in patients with higher GOLPH3 (p = 0.014), MYO18A (p = 0.047), PITPNC1 (p = 0.008) and RAB1B (p = 0.033) serum levels. Conclusion GOLPH3, MYO18A, PITPNC1 and RAB1B maybe promising diagnostic and prognostic biomarkers in AML patients.

Mechanisms by which small molecules of diverse chemotypes arrest Sec14 lipid transfer activity

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) enhance the activities of PtdIns 4-OH kinases that generate signaling pools of PtdIns-4-phosphate. In that capacity, PITPs serve as key regulators of lipid signaling in eukaryotic cells. Although the PITP phospholipid exchange cycle is the engine that stimulates PtdIns 4-OH kinase activities, the underlying mechanism is not understood. Herein, we apply an integrative structural biology approach to investigate interactions of the yeast PITP Sec14 with small-molecule inhibitors (SMIs) of its phospholipid exchange cycle. Using a combination of X-ray crystallography, solution NMR spectroscopy, and atomistic MD simulations, we dissect how SMIs compete with native Sec14 phospholipid ligands and arrest phospholipid exchange. Moreover, as Sec14 PITPs represent new targets for the development of next-generation antifungal drugs, the structures of Sec14 bound to SMIs of diverse chemotypes reported in this study will provide critical information required for future structure-based design of next-generation lead compounds directed against Sec14 PITPs of virulent fungi.

The phosphatidylinositol-transfer protein Nir3 promotes PI(4,5)P2 replenishment in response to TCR signaling during T cell development and survival

Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C-γ (PLCγ1) represents a critical step in T cell antigen receptor (TCR) signaling and subsequent thymocyte and T cell responses. PIP2 replenishment following its depletion in the plasma membrane (PM) is dependent on delivery of its precursor phosphatidylinositol (PI) from the endoplasmic reticulum (ER) to the PM. We show that a PI transfer protein (PITP), Nir3 (Pitpnm2), promotes PIP2 replenishment following TCR stimulation and is important for T cell development. In Nir3–/– T lineage cells, the PIP2 replenishment following TCR stimulation is slower. Nir3 deficiency attenuates calcium mobilization in double-positive (DP) thymocytes in response to weak TCR stimulation. This impaired TCR signaling leads to attenuated thymocyte development at TCRβ selection and positive selection as well as diminished mature T cell fitness in Nir3–/– mice. This study highlights the importance of PIP2 replenishment mediated by PITPs at ER-PM junctions during TCR signaling.

Yeast Sec14-like lipid transfer proteins Pdr16 and Pdr17 bind and transfer the ergosterol precursor lanosterol in addition to phosphatidylinositol.

Yeast Sec14-like phosphatidylinositol transfer proteins (PITPs) contain a hydrophobic cavity capable of accepting a single molecule of phosphatidylinositol (PI) or another molecule in a mutually exclusive manner. We report here that two yeast Sec14 family PITPs, Pdr16p (Sfh3p) and Pdr17p (Sfh4p), possess high-affinity binding and transfer towards lanosterol. To our knowledge, this is the first identification of lanosterol transfer proteins. In addition, a pdr16Δpdr17Δ double mutant had a significantly increased level of cellular lanosterol compared with the corresponding wild-type. Based on the lipid profiles of wild-type and pdr16Δpdr17Δ cells grown in aerobic and anaerobic conditions, we suggest that PI-lanosterol transfer proteins are important predominantly for the optimal functioning of the post-lanosterol part of sterol biosynthesis.

RdgB knockdown in neurons reduced nocturnal sleep in Drosophila melanogaster

Recent studies revealed behaviorally defined sleep is conserved across broad species from insect to human. For evolutional analysis, it is critical to determine how homologous genes regulate the homologous function among species. Drosophila melanogaster shares numerous sleep related genes with mammals including Sik3, salt-inducible kinase 3, whose mutation caused long sleep both in mouse and fruit fly. The Drosophila rdgB (retinal degeneration B) encodes a membrane-associated phosphatidylinositol transfer protein and its mutation caused light-induced degeneration of photoreceptor cells. rdgB mutation also impaired phototransduction and olfactory behavior, indicating rdgB is involved in the normal neural transmission. Mammalian rdgB homologue, Pitpnm2 (phosphatidylinositol transfer protein membrane-associated 2) was discovered as one of SNIPPs (sleep-need index phosphoproteins), suggesting its role in sleep. Here, we show that rdgB is involved in sleep regulation in Drosophila. Pan-neuronal and mushroom body (MB) specific rdgB knockdown decreased nocturnal sleep. MB neurons play a dominant role, since the rescue of rdgB expression only in MB neurons in pan-neuronal knockdown reversed the sleep reducing effect of rdgB knockdown. These results revealed the sleep-related function of rdgB in Drosophila which may be conserved across species.

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.