Abstract Immediate early response 3 interacting-protein 1 (IER3IP1) is an endoplasmic reticulum resident protein, highly expressed in pancreatic cells and the developing brain cortex. Homozygous mutations in IER3IP1 have been found in individuals with microcephaly and neonatal diabetes, yet the underlying mechanism causing beta cell failure remains unclear. Here, we utilized differentiation of genome edited-stem cells into pancreatic islet cells to elucidate the molecular basis of IER3IP1 neonatal diabetes. Using CRISPR-Cas9, we generated two distinct IER3IP1-mutant human embryonic stem cell lines: a homozygous knock-in model of a patient mutation (IER3IP1V21G), and a knockout model (IER3IP1-/-). While these mutant stem cell lines differentiated normally into definitive endoderm and pancreatic progenitors, we observed that IER3IP1-KO stem cell derived-islets (SC-islets) presented a significant decrease in beta cell numbers and elevated ER stress. Retention Using Selective Hooks (RUSH) assay revealed three-fold reduction in ER-to-Golgi trafficking of proinsulin in IER3IP1 mutant beta cells. Additionally, IER3IP1 mutant SC-islets implanted into immunocompromised mice displayed defective human insulin secretion, indicating the deleterious impact of IER3IP1 mutations on beta cell function. Our study provides valuable insights into the role of IER3IP1 in human beta cell biology and establishes a useful model to investigate ER-to-Golgi trafficking defects within beta cells. Article Highlights: · IER3IP1 mutations are linked to the development of microcephaly, epilepsy, and early-onset diabetes (MEDS1). However, the underlying molecular mechanisms of cell dysfunction are unknown. · Using targeted genome-editing, we generated specific IER3IP1 mutations in hESC lines which were differentiated into pancreatic islet lineages. · Loss of IER3IP1 resulted in a three-fold reduction in ER-to-Golgi trafficking of proinsulin in stem-cell derived beta cells, leading to beta cell dysfunction both in vitro and in vivo. · Loss of IER3IP1 also triggered increased markers of endoplasmic reticulum stress, indicating the pivotal role of the ER-to-Golgi trafficking pathway for beta cell homeostasis and function.

Our understanding of the ways in which peptides are used for communication in the nervous and endocrine systems began with the identification of oxytocin, vasopressin, and insulin, each of which is stored in electron-dense granules, ready for release in response to an appropriate stimulus. For each of these peptides, entry of its newly synthesized precursor into the ER lumen is followed by transport through the secretory pathway, exposing the precursor to a sequence of environments and enzymes that produce the bioactive products stored in mature granules. A final step in the biosynthesis of many peptides is C-terminal amidation by peptidylglycine α-amidating monooxygenase (PAM), an ascorbate- and copper-dependent membrane enzyme that enters secretory granules along with its soluble substrates. Biochemical and cell biological studies elucidated the highly conserved mechanism for amidated peptide production and raised many questions about PAM trafficking and the effects of PAM on cytoskeletal organization and gene expression. Phylogenetic studies and the discovery of active PAM in the ciliary membranes of Chlamydomonas reinhardtii, a green alga lacking secretory granules, suggested that a PAM-like enzyme was present in the last eukaryotic common ancestor. While the catalytic features of human and C. reinhardtii PAM are strikingly similar, the trafficking of PAM in C. reinhardtii and neuroendocrine cells and secretion of its amidated products differ. A comparison of PAM function in neuroendocrine cells, atrial myocytes, and C. reinhardtii reveals multiple ways in which altered trafficking allows PAM to accomplish different tasks in different species and cell types.

Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is a four-membrane spanning ceramide interacting protein that regulates mTORC1 signaling. Here, we show that LAPTM4B is sorted into intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs) and released in small extracellular vesicles (sEVs) into conditioned cell culture medium and human urine. Efficient sorting of LAPTM4B into ILV membranes depends on its third transmembrane domain containing a sphingolipid interaction motif (SLim). Unbiased lipidomic analysis reveals a strong enrichment of glycosphingolipids in sEVs secreted from LAPTM4B knockout cells and from cells expressing a SLim-deficient LAPTM4B mutant. The altered sphingolipid profile is accompanied by a distinct SLim-dependent co-modulation of ether lipid species. The changes in the lipid composition of sEVs derived from LAPTM4B knockout cells is reflected by an increased stability of membrane nanodomains of sEVs. These results identify LAPTM4B as a determinant of the glycosphingolipid profile and membrane properties of sEVs.

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is Peptidylglycine α-Amidating Monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM ( Pam Myh6-cKO/cKO ) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult Pam Myh6-cKO/cKO atria revealed a 20-fold drop in the volume fraction of secretory granules and a decrease in peripherally localized Golgi complexes. When primary cultures of Pam 0-Cre -cKO/cKO atrial myocytes (PAM floxed, no Cre recombinase) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in proANP levels. Expression of exogenous PAM in Pam Myh6-cKO/cKO atrial myocytes produced a dose-dependent increase in proANP content. Strikingly, rescue of proANP content did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact and its basal secretion is stimulated by drugs that inhibit Golgi-localized Arf activators. Increased basal secretion of proANP was a major contributor to its reduced levels in Pam Myh6-cKO/cKO myocytes; the inability of these drugs to inhibit basal proANP secretion by Pam Myh6-cKO/cKO myocytes revealed a role for COPI-mediated recycling of PAM to the endoplasmic reticulum. Analysis of atrial coated vesicles and the ability PAM to make fluorescently-tagged proANP accumulate in the cis -Golgi region of cells lacking secretory granules revealed a non-catalytic role for PAM in soluble cargo trafficking early in the secretory pathway. Significance Transmission electron microscopy of atrial cardiomyocytes revealed dense granules resembling those in endocrine cells and neurons, leading to the discovery of the natriuretic peptides stored in these granules. Subsequent studies revealed features unique to atrial granules, including high level expression of Peptidylglycine α-Amidating Monooxygenase (PAM), an enzyme required for the synthesis of many neuropeptides, but not for the synthesis of natriuretic peptides. The discovery that atrial myocytes lacking PAM are unable to produce granules and that PAM lacking its monooxygenase activity can rescue granule formation provides new information about the proANP secretory pathway. A better understanding of the unique features of atrial cell biology should provide insight into atrial fibrillation, the most common cardiac arrhythmia, atrial amyloidosis and heart failure.

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (PamMyh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult PamMyh6-cKO/cKO atria revealed a 13-fold drop in the number of secretory granules. When primary cultures of Pam0-Cre-cKO/cKO atrial myocytes (no Cre recombinase, PAM floxed) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in ANP precursor (proANP) levels. Expression of exogenous PAM in PamMyh6-cKO/cKO atrial myocytes produced a dose-dependent rescue of proANP content; strikingly, this response did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact. A threefold increase in the basal rate of proANP secretion by PamMyh6-cKO/cKO myocytes was a major contributor to its reduced levels. While proANP secretion was increased following treatment of control cultures with drugs that block the activation of Golgi-localized Arf proteins and COPI vesicle formation, proANP secretion by PamMyh6-cKO/cKO myocytes was unaffected. In cells lacking secretory granules, expression of exogenous PAM led to the accumulation of fluorescently tagged proANP in the cis-Golgi region. Our data indicate that COPI vesicle-mediated recycling of PAM from the cis-Golgi to the endoplasmic reticulum plays an essential role in the biogenesis of proANP containing atrial granules.

Sphingosine kinase 1 (SK1) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). S1P binds to G-protein-coupled receptors (S1PR1–5) to regulate cellular events, including Ca2+ signaling. The SK1/S1P axis and Ca2+ signaling both play important roles in health and disease. In this respect, Ca2+ microdomains at the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis. Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with malignancies. We show that overexpression of SK1 augments agonist-induced Ca2+ release from the ER resulting in increased mitochondrial matrix Ca2+. Also, overexpression of SK1 induces MFN2 fragmentation, likely through increased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments increases mitochondrial matrix Ca2+ during agonist stimulation, mimicking the SK1 overexpression in cells. Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2 fragmentation resulting in increased mitochondrial Ca2+ and downstream cellular effects.

Key features for progression to pancreatic β-cell failure and disease are loss of glucose responsiveness and an increased ratio of secreted proinsulin to insulin. Proinsulin and insulin are stored in secretory granules (SGs) and the fine-tuning of hormone output requires signal-mediated recruitment of select SG populations according to intracellular location and age. The GTPase Rac1 coordinates multiple signaling pathways that specify SG release, and Rac1 activity is controlled in part by GDP/GTP exchange factors (GEFs). To explore the function of two large multidomain GEFs, Kalirin and Trio in β-cells, we manipulated their Rac1-specific GEF1 domain activity by using small-molecule inhibitors and by genetically ablating Kalirin. We examined age-related SG behavior employing radiolabeling protocols. Loss of Kalirin/Trio function attenuated radioactive proinsulin release by reducing constitutive-like secretion and exocytosis of 2-h-old granules. At later chase times or at steady state, Kalirin/Trio manipulations decreased glucose-stimulated insulin output. Finally, use of a Rac1 FRET biosensor with cultured β-cell lines demonstrated that Kalirin/Trio GEF1 activity was required for normal rearrangement of Rac1 to the plasma membrane in response to glucose. Rac1 activation can be evoked by both glucose metabolism and signaling through the incretin glucagon-like peptide 1 (GLP-1) receptor. GLP-1 addition restored Rac1 localization/activity and insulin secretion in the absence of Kalirin, thereby assigning Kalirin’s participation to stimulatory glucose signaling.

5 1Department of Medicine, Stony Brook University School of Medicine, Stony Brook, NY 6 2Department of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki, Finland 7 3Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 8 4Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 9 5Departments of Pathology, Medicine, Albert Einstein College of Medicine, Bronx, NY 10 6School of Pharmacy, University of Saint Joseph, Hartford, CT 11 7Department of Molecular Biology and Biophysics, University of Connecticut Health Center, 12 Farmington, CT 13 8Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 14 15 16 17 To whom correspondence should be addressed: Regina Kuliawat, PhD, Department of 18 Developmental and Molecular Biology, Albert Einstein College of Medicine,1300 Morris Park 19 Avenue, Bronx, NY 10461, Tel: 718-430-8686; E-mail: regina.kuliawat@einstein.yu.edu 20 21 Short title: GEFs Kalirin/Trio impact glucose stimulated insulin secretion 22

Peptidylglycine α-amidating monooxygenase (PAM) is highly expressed in neurons and endocrine cells, where it catalyzes one of the final steps in the biosynthesis of bioactive peptides. PAM is also expressed in unicellular organisms such as Chlamydomonas reinhardtii, which do not store peptides in secretory granules. As for other granule membrane proteins, PAM is retrieved from the cell surface and returned to the trans-Golgi network. This pathway involves regulated entry of PAM into multivesicular body intralumenal vesicles (ILVs). The aim of this study was defining the endocytic pathways utilized by PAM in cells that do not store secretory products in granules. Using stably transfected HEK293 cells, endocytic trafficking of PAM was compared to that of the mannose 6-phosphate (MPR) and EGF (EGFR) receptors, established markers for the endosome to trans-Golgi network and degradative pathways, respectively. As in neuroendocrine cells, PAM internalized by HEK293 cells accumulated in the trans-Golgi network. Based on surface biotinylation, >70% of the PAM on the cell surface was recovered intact after a 4h chase and soluble, bifunctional PAM was produced. Endosomes containing PAM generally contained both EGFR and MPR and ultrastructural analysis confirmed that all three cargos accumulated in ILVs. PAM containing multivesicular bodies made frequent dynamic tubular contacts with younger and older multivesicular bodies. Frequent dynamic contacts were observed between lysosomes and PAM containing early endosomes and multivesicular bodies. The ancient ability of PAM to localize to ciliary membranes, which release bioactive ectosomes, may be related to its ability to accumulate in ILVs and exosomes.

Peptidylglycine α-amidating monooxygenase (PAM) (EC 1.14.17.3) catalyzes peptide amidation, a crucial post-translational modification, through the sequential actions of its monooxygenase (peptidylglycine α-hydroxylating monooxygenase) and lyase (peptidyl-α-hydroxyglycine α-amidating lyase (PAL)) domains. Alternative splicing generates two different regions that connect the protease-resistant catalytic domains. Inclusion of exon 16 introduces a pair of Lys residues, providing a site for controlled endoproteolytic cleavage of PAM and the separation of soluble peptidylglycine α-hydroxylating monooxygenase from membrane-associated PAL. Exon 16 also includes two O-glycosylation sites. PAM-1 lacking both glycosylation sites (PAM-1/OSX; where OSX is O-glycan-depleted mutant of PAM-1) was stably expressed in AtT-20 corticotrope tumor cells. In PAM-1/OSX, a cleavage site for furin-like convertases was exposed, generating a shorter form of membrane-associated PAL. The endocytic trafficking of PAM-1/OSX differed dramatically from that of PAM-1. A soluble fragment of the cytosolic domain of PAM-1 was produced in the endocytic pathway and entered the nucleus; very little soluble fragment of the cytosolic domain was produced from PAM-1/OSX. Internalized PAM-1/OSX was rapidly degraded; unlike PAM-1, very little internalized PAM-1/OSX was detected in multivesicular bodies. Blue native PAGE analysis identified high molecular weight complexes containing PAM-1; the ability of PAM-1/OSX to form similar complexes was markedly diminished. By promoting the formation of high molecular weight complexes, O-glycans may facilitate the recycling of PAM-1 through the endocytic compartment.

Lysosome-associated protein transmembrane-4b (LAPTM4B) associates with poor prognosis in several cancers, but its physiological function is not well understood. Here we use novel ceramide probes to provide evidence that LAPTM4B interacts with ceramide and facilitates its removal from late endosomal organelles (LEs). This lowers LE ceramide in parallel with and independent of acid ceramidase-dependent catabolism. In LAPTM4B-silenced cells, LE sphingolipid accumulation is accompanied by lysosomal membrane destabilization. However, these cells resist ceramide-driven caspase-3 activation and apoptosis induced by chemotherapeutic agents or gene silencing. Conversely, LAPTM4B overexpression reduces LE ceramide and stabilizes lysosomes but sensitizes to drug-induced caspase-3 activation. Together, these data uncover a cellular ceramide export route from LEs and identify LAPTM4B as its regulator. By compartmentalizing ceramide, LAPTM4B controls key sphingolipid-mediated cell death mechanisms and emerges as a candidate for sphingolipid-targeting cancer therapies.

The adaptor protein-1 complex (AP-1), which transports cargo between the trans-Golgi network and endosomes, plays a role in the trafficking of Atp7a, a copper-transporting P-type ATPase, and peptidylglycine α-amidating monooxygenase (PAM), a copper-dependent membrane enzyme. Lack of any of the four AP-1 subunits impairs function, and patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of the σ1A subunit, exhibit clinical and biochemical signs of impaired copper homeostasis. To explore the role of AP-1 in copper homeostasis in neuroendocrine cells, we used corticotrope tumor cells in which AP-1 function was diminished by reducing expression of its μ1A subunit. Copper levels were unchanged when AP-1 function was impaired, but cellular levels of Atp7a declined slightly. The ability of PAM to function was assessed by monitoring 18-kDa fragment-NH2 production from proopiomelanocortin. Reduced AP-1 function made 18-kDa fragment amidation more sensitive to inhibition by bathocuproine disulfonate, a cell-impermeant Cu(I) chelator. The endocytic trafficking of PAM was altered, and PAM-1 accumulated on the cell surface when AP-1 levels were reduced. Reduced AP-1 function increased the Atp7a presence in early/recycling endosomes but did not alter the ability of copper to stimulate its appearance on the plasma membrane. Co-immunoprecipitation of a small fraction of PAM and Atp7a supports the suggestion that copper can be transferred directly from Atp7a to PAM, a process that can occur only when both proteins are present in the same subcellular compartment. Altered luminal cuproenzyme function may contribute to deficits observed when the AP-1 function is compromised.

Background: Secretory granule membrane proteins are retrieved and reused or degraded after exocytosis. Results: A pH-sensitive His cluster in the amidating enzyme is essential for its trafficking. Conclusion: Lacking this conserved His cluster, proteolytic maturation and generation of a soluble cytosolic fragment of amidating enzyme fail to occur, disrupting secretion and nuclear signaling. Significance: The storage and regulated secretion of peptides requires intraluminal signaling. Decreasing luminal pH is thought to play a role in the entry of newly synthesized and endocytosed membrane proteins into secretory granules. The two catalytic domains of peptidylglycine α-amidating monooxygenase (PAM), a type I integral membrane protein, catalyze the sequential reactions that convert peptidyl-Gly substrates into amidated products. We explored the hypothesis that a conserved His-rich cluster (His-Gly-His-His) in the linker region connecting its two catalytic domains senses pH and affects PAM trafficking by mutating these His residues to Ala (Ala-Gly-Ala-Ala; H3A). Purified recombinant wild-type and H3A linker peptides were examined using circular dichroism and tryptophan fluorescence; mutation of the His cluster largely eliminated its pH sensitivity. An enzymatically active PAM protein with the same mutations (PAM-1/H3A) was expressed in HEK293 cells and AtT-20 corticotrope tumor cells. Metabolic labeling followed by immunoprecipitation revealed more rapid loss of newly synthesized PAM-1/H3A than PAM-1; although release of newly synthesized monofunctional PHM/H3A was increased, release of soluble bifunctional PAM/H3A, a product of the endocytic pathway, was decreased. Surface biotinylation revealed rapid loss of PAM-1/H3A, with no detectable return of the mutant protein to secretory granules. Consistent with its altered endocytic trafficking, little PAM-1/H3A was subjected to regulated intramembrane proteolysis followed by release of a small nuclear-targeted cytosolic fragment. AtT-20 cells expressing PAM-1/H3A adopted the morphology of wild-type AtT-20 cells; secretory products no longer accumulated in the trans-Golgi network and secretory granule exocytosis was more responsive to secretagogue.

The adaptor protein 1A complex (AP‐1A) transports cargo between the trans ‐Golgi network (TGN) and endosomes. In professional secretory cells, AP‐1A also retrieves material from immature secretory granules (SGs). The role of AP‐1A in SG biogenesis was explored using AtT‐20 corticotrope tumor cells expressing reduced levels of the AP‐1A μ1A subunit. A twofold reduction in μ1A resulted in a decrease in TGN cisternae and immature SGs and the appearance of regulated secretory pathway components in non‐condensing SGs. Although basal secretion of endogenous SG proteins was unaffected, secretagogue‐stimulated release was halved. The reduced μ1A levels interfered with the normal trafficking of carboxypeptidase D (CPD) and peptidylglycine α‐amidating monooxygenase‐1 (PAM‐1), integral membrane enzymes that enter immature SGs. The non‐condensing SGs contained POMC products and PAM‐1, but not CPD. Based on metabolic labeling and secretion experiments, the cleavage of newly synthesized PAM‐1 into PHM was unaltered, but PHM basal secretion was increased in sh‐μ1A PAM‐1 cells. Despite lacking a canonical AP‐1A binding motif, yeast two‐hybrid studies demonstrated an interaction between the PAM‐1 cytosolic domain and AP‐1A. Coimmunoprecipitation experiments with PAM‐1 mutants revealed an influence of the luminal domains of PAM‐1 on this interaction. Thus, AP‐1A is crucial for normal SG biogenesis, function and composition.

N-myc downstream-regulated gene 1 (NDRG1) mutations cause Charcot-Marie-Tooth disease type 4D (CMT4D). However, the cellular function of NDRG1 and how it causes CMT4D are poorly understood. We report that NDRG1 silencing in epithelial cells results in decreased uptake of low-density lipoprotein (LDL) due to reduced LDL receptor (LDLR) abundance at the plasma membrane. This is accompanied by the accumulation of LDLR in enlarged EEA1-positive endosomes that contain numerous intraluminal vesicles and sequester ceramide. Concomitantly, LDLR ubiquitylation is increased but its degradation is reduced and ESCRT (endosomal sorting complex required for transport) proteins are downregulated. Co-depletion of IDOL (inducible degrader of the LDLR), which ubiquitylates the LDLR and promotes its degradation, rescues plasma membrane LDLR levels and LDL uptake. In murine oligodendrocytes, Ndrg1 silencing not only results in reduced LDL uptake but also in downregulation of the oligodendrocyte differentiation factor Olig2. Both phenotypes are rescued by co-silencing of Idol, suggesting that ligand uptake through LDLR family members controls oligodendrocyte differentiation. These findings identify NDRG1 as a novel regulator of multivesicular body formation and endosomal LDLR trafficking. The deficiency of functional NDRG1 in CMT4D might impair lipid processing and differentiation of myelinating cells.

The aim was to study laminin (LM) synthesis, integration, and deposition into the basement membrane (BM) during adipogenesis. Human bone marrow-derived mesenchymal stromal cells (MSCs) were induced along the adipogenic lineage. LM chain mRNA and protein levels were followed using quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF) staining, transmission electron microscopy (TEM), and immunoprecipitation. MSCs produced low levels of LM mRNAs but were not surrounded by BM in IF and TEM imaging. LM-α4, LM-β1, and LM-γ1 mRNAs increased during adipogenesis 3.9-, 5.8-, and 2.8-fold by day 28. LM-411 was immunoprecipitated from the ECM of the differentiated cells. Immunostaining suggested deposition of LM-411 and some LM-421. BM build-up was probably organized in part by integrin (Int) α6β1. At day 28, TEM images revealed BM-like structures around fat droplet-containing cells. The first signs of BM formation and Int α6β1 were seen using IF imaging at day 14. Laminin-411 and Int α6β1 were expressed in vivo in mature human subcutaneous fat tissue. Undifferentiated human MSCs did not organize LM subunits into BM, whereas LM-411 and some LM-421 are precipitated in the BM around adipocytes. This is the first demonstration of LM-411 precipitation during hMSC adipogenesis around adipocytes as a structural scaffold and Int-regulated signaling element.

Rationale : The synthetic sphingosine analog FTY720 is undergoing clinical trials as an immunomodulatory compound, acting primarily via sphingosine 1-phosphate receptor activation. Sphingolipid and cholesterol homeostasis are closely connected but whether FTY720 affects atherogenesis in humans is not known. Objective : We examined the effects of FTY720 on the processing of scavenged lipoprotein cholesterol in human primary monocyte-derived macrophages. Methods and Results : FTY720 did not affect cholesterol uptake but inhibited its delivery to the endoplasmic reticulum, reducing cellular free cholesterol cytotoxicity. This was accompanied by increased levels of Niemann–Pick C1 protein (NPC1) and ATP-binding cassette transporter (ABC)A1 proteins and increased efflux of endosomal cholesterol to apolipoprotein A-I. These effects were not dependent on sphingosine 1-phosphate receptor activation. Instead, FTY720 stimulated the production of 27-hydroxycholesterol, an endogenous ligand of the liver X receptor, leading to liver X receptor–induced upregulation of ABCA1. Fluorescently labeled FTY720 was targeted to late endosomes, and the FTY720-induced upregulation of ABCA1 was NPC1-dependent, but the endosomal exit of FTY720 itself was not. Conclusions : We conclude that FTY720 decreases cholesterol toxicity in primary human macrophages by reducing the delivery of scavenged lipoprotein cholesterol to the endoplasmic reticulum and facilitating its release to physiological extracellular acceptors. Furthermore, FTY720 stimulates 27-hydroxycholesterol production, providing an explanation for the atheroprotective effects and identifying a novel mechanism by which FTY720 modulates signaling.

Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.