ABSTRACT
OBJECTIVE: Precursors of peptide hormones undergo posttranslational modifications within the trans-Golgi network (TGN). Dysfunction of proteins involved at different steps of this process cause several complex syndromes affecting the central nervous system (CNS). We aimed to clarify the genetic cause in a group of patients characterized by hypopituitarism in combination with brain atrophy, thin corpus callosum, severe developmental delay, visual impairment, and epilepsy. METHODS: Whole exome sequencing was performed in seven individuals of six unrelated families with these features. Postmortem histopathological and HID1 expression analysis of brain tissue and pituitary gland were conducted in one patient. Functional consequences of the homozygous HID1 variant p.R433W were investigated by Seahorse XF Assay in fibroblasts of two patients. RESULTS: Bi-allelic variants in the gene HID1 domain-containing protein 1 (HID1) were identified in all patients. Postmortem examination confirmed cerebral atrophy with enlarged lateral ventricles. Markedly reduced expression of pituitary hormones was found in pituitary gland tissue. Colocalization of HID1 protein with the TGN was not altered in fibroblasts of patients compared to controls, while the extracellular acidification rate upon stimulation with potassium chloride was significantly reduced in patient fibroblasts compared to controls. INTERPRETATION: Our findings indicate that mutations in HID1 cause an early infantile encephalopathy with hypopituitarism as the leading presentation, and expand the list of syndromic CNS diseases caused by interference of TGN function. ANN NEUROL 2021;90:149-164.
Subject(s)
Brain Diseases/genetics , Epilepsy/genetics , Hypopituitarism/genetics , Alleles , Brain Diseases/pathology , Child, Preschool , Epilepsy/pathology , Female , Humans , Hypopituitarism/pathology , Infant , Male , Pituitary Gland/pathology , Exome Sequencing , Young AdultABSTRACT
Astrocytes orchestrate neural development by powerfully coordinating synapse formation and function and, as such, may be critically involved in the pathogenesis of neurodevelopmental abnormalities and cognitive deficits commonly observed in psychiatric disorders. Here, we report the identification of a subset of cortical astrocytes that are competent for regulating dopamine (DA) homeostasis during postnatal development of the prefrontal cortex (PFC), allowing for optimal DA-mediated maturation of excitatory circuits. Such control of DA homeostasis occurs through the coordinated activity of astroglial vesicular monoamine transporter 2 (VMAT2) together with organic cation transporter 3 and monoamine oxidase type B, two key proteins for DA uptake and metabolism. Conditional deletion of VMAT2 in astrocytes postnatally produces loss of PFC DA homeostasis, leading to defective synaptic transmission and plasticity as well as impaired executive functions. Our findings show a novel role for PFC astrocytes in the DA modulation of cognitive performances with relevance to psychiatric disorders.
Subject(s)
Astrocytes/metabolism , Cognitive Dysfunction/metabolism , Dopamine/metabolism , Animals , Astrocytes/drug effects , Brain/metabolism , Cognitive Dysfunction/physiopathology , Dopamine/pharmacology , Homeostasis , Male , Mice , Mice, Knockout , Neurons/metabolism , Prefrontal Cortex/drug effects , Prefrontal Cortex/metabolism , Rats , Rats, Sprague-Dawley , Synaptic Transmission/physiologyABSTRACT
The regulated secretion of peptide hormones, neural peptides and many growth factors depends on their sorting into large dense core vesicles (LDCVs) capable of regulated exocytosis. LDCVs form at the trans-Golgi network, but the mechanisms that sort proteins to this regulated secretory pathway and the cytosolic machinery that produces LDCVs remain poorly understood. Recently, we used an RNAi screen to identify a role for heterotetrameric adaptor protein AP-3 in regulated secretion and in particular, LDCV formation. Indeed, mocha mice lacking AP-3 have a severe neurological and behavioral phenotype, but this has been attributed to a role for AP-3 in the endolysosomal rather than biosynthetic pathway. We therefore used mocha mice to determine whether loss of AP-3 also dysregulates peptide release in vivo. We find that adrenal chromaffin cells from mocha animals show increased constitutive exocytosis of both soluble cargo and LDCV membrane proteins, reducing the response to stimulation. We also observe increased basal release of both insulin and glucagon from pancreatic islet cells of mocha mice, suggesting a global disturbance in the release of peptide hormones. AP-3 exists as both ubiquitous and neuronal isoforms, but the analysis of mice lacking each of these isoforms individually and together shows that loss of both is required to reproduce the effect of the mocha mutation on the regulated pathway. In addition, we show that loss of the related adaptor protein AP-1 has a similar effect on regulated secretion but exacerbates the effect of AP-3 RNAi, suggesting distinct roles for the two adaptors in the regulated secretory pathway.
Subject(s)
Adaptor Protein Complex 3/genetics , Adaptor Protein Complex beta Subunits/genetics , Cytosol/metabolism , Exocytosis/genetics , Peptide Hormones/metabolism , Adaptor Protein Complex 3/metabolism , Adaptor Protein Complex beta Subunits/metabolism , Animals , Glucagon/genetics , Glucagon/metabolism , Insulin/genetics , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Lysosomes , Metabolic Networks and Pathways , Mice , Neurons/metabolism , RNA Interference , Transcription Factor AP-1/geneticsABSTRACT
Large dense core vesicles (LDCVs) mediate the regulated release of neuropeptides and peptide hormones. HID-1 is a trans-Golgi network (TGN) localized peripheral membrane protein contributing to LDCV formation. There is no information about HID-1 structure or domain architecture, and thus it remains unknown how HID-1 binds to the TGN and performs its function. We report that the N-terminus of HID-1 mediates membrane binding through a myristoyl group with a polybasic amino acid patch but lacks specificity for the TGN. In addition, we show that the C-terminus serves as the functional domain. Indeed, this isolated domain, when tethered to the TGN, can rescue the neuroendocrine secretion and sorting defects observed in HID-1 KO cells. Finally, we report that a point mutation within that domain, identified in patients with endocrine and neurological deficits, leads to loss of function.
Subject(s)
Dense Core Vesicles , Peptide Hormones , Humans , Amino Acids , Cell Movement , Neurosecretory SystemsABSTRACT
The retention using selective hook (RUSH) system enables us to synchronize and visualize the movement of cargoes along the secretory pathway. A fluorescently tagged cargo of interest is retained in the endoplasmic reticulum and released in a biotin-dependent manner. Here, we report a detailed protocol describing the steps necessary to perform RUSH experiments to study secretory granule biogenesis at the trans-Golgi network (TGN).
Subject(s)
Golgi Apparatus , trans-Golgi Network , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Secretory Pathway , Secretory Vesicles/metabolism , trans-Golgi Network/metabolismABSTRACT
Insulin secretory granules (SGs) mediate the regulated secretion of insulin, which is essential for glucose homeostasis. The basic machinery responsible for this regulated exocytosis consists of specific proteins present both at the plasma membrane and on insulin SGs. The protein composition of insulin SGs thus dictates their release properties, yet the mechanisms controlling insulin SG formation, which determine this molecular composition, remain poorly understood. VPS41, a component of the endolysosomal tethering homotypic fusion and vacuole protein sorting (HOPS) complex, was recently identified as a cytosolic factor involved in the formation of neuroendocrine and neuronal granules. We now find that VPS41 is required for insulin SG biogenesis and regulated insulin secretion. Loss of VPS41 in pancreatic ß-cells leads to a reduction in insulin SG number, changes in their transmembrane protein composition, and defects in granule-regulated exocytosis. Exploring a human point mutation, identified in patients with neurological but no endocrine defects, we show that the effect on SG formation is independent of HOPS complex formation. Finally, we report that mice with a deletion of VPS41 specifically in ß-cells develop diabetes due to severe depletion of insulin SG content and a defect in insulin secretion. In sum, our data demonstrate that VPS41 contributes to glucose homeostasis and metabolism.
Subject(s)
Diabetes Mellitus/metabolism , Insulin Secretion/genetics , Insulin-Secreting Cells/metabolism , Secretory Vesicles/metabolism , Vesicular Transport Proteins/metabolism , Animals , Cell Line , Diabetes Mellitus/genetics , Exocytosis/physiology , Glucose Tolerance Test , Mice , Mice, Knockout , Rats , Vesicular Transport Proteins/geneticsABSTRACT
Vacuolar protein sorting 41 (VPS41) is as part of the Homotypic fusion and Protein Sorting (HOPS) complex required for lysosomal fusion events and, independent of HOPS, for regulated secretion. Here, we report three patients with compound heterozygous mutations in VPS41 (VPS41S285P and VPS41R662* ; VPS41c.1423-2A>G and VPS41R662* ) displaying neurodegeneration with ataxia and dystonia. Cellular consequences were investigated in patient fibroblasts and VPS41-depleted HeLa cells. All mutants prevented formation of a functional HOPS complex, causing delayed lysosomal delivery of endocytic and autophagic cargo. By contrast, VPS41S285P enabled regulated secretion. Strikingly, loss of VPS41 function caused a cytosolic redistribution of mTORC1, continuous nuclear localization of Transcription Factor E3 (TFE3), enhanced levels of LC3II, and a reduced autophagic response to nutrient starvation. Phosphorylation of mTORC1 substrates S6K1 and 4EBP1 was not affected. In a C. elegans model of Parkinson's disease, co-expression of VPS41S285P /VPS41R662* abolished the neuroprotective function of VPS41 against α-synuclein aggregates. We conclude that the VPS41 variants specifically abrogate HOPS function, which interferes with the TFEB/TFE3 axis of mTORC1 signaling, and cause a neurodegenerative disease.
Subject(s)
Neurodegenerative Diseases , Animals , Autophagy , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Caenorhabditis elegans/genetics , HeLa Cells , Humans , Lysosomes/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Neurodegenerative Diseases/genetics , Protein Transport , Vesicular Transport Proteins/metabolismSubject(s)
CRISPR-Cas Systems/physiology , Synapses/physiology , Synaptic Transmission/physiology , Animals , Biolistics , CRISPR-Cas Systems/genetics , Cells, Cultured , Gene Knockdown Techniques , Genes, Reporter , Hippocampus , Nerve Tissue Proteins/physiology , Organ Culture Techniques , Phenotype , Protein Subunits , Pyramidal Cells/physiology , Rats , Receptors, AMPA/physiology , Receptors, N-Methyl-D-Aspartate/physiology , Recombinant Fusion Proteins/metabolism , TransfectionABSTRACT
Dense-core vesicles (DCVs) are secretory vesicles found in neurons and endocrine cells. DCVs package and release cargoes including neuropeptides, biogenic amines, and peptide hormones. We recently identified the endosome-associated recycling protein (EARP) complex and the EARP-interacting-protein EIPR-1 as proteins important for controlling levels of DCV cargoes in Caenorhabditis elegans neurons. Here we determine the role of mammalian EIPR1 in insulinoma cells. We find that in Eipr1 KO cells, there is reduced insulin secretion, and mature DCV cargoes such as insulin and carboxypeptidase E (CPE) accumulate near the trans-Golgi network and are not retained in mature DCVs in the cell periphery. In addition, we find that EIPR1 is required for the stability of the EARP complex subunits and for the localization of EARP and its association with membranes, but EIPR1 does not affect localization or function of the related Golgi-associated retrograde protein (GARP) complex. EARP is localized to two distinct compartments related to its function: an endosomal compartment and a DCV biogenesis-related compartment. We propose that EIPR1 functions with EARP to control both endocytic recycling and DCV maturation.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Carrier Proteins/metabolism , Insulin-Secreting Cells/metabolism , Secretory Vesicles/physiology , Animals , Biophysical Phenomena , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/physiology , Carrier Proteins/physiology , Cell Line , Endosomes/metabolism , Golgi Apparatus/metabolism , Neurons/metabolism , Neuropeptides/metabolism , Protein Transport , Secretory Vesicles/metabolism , Vesicular Transport Proteins/metabolism , trans-Golgi Network/metabolismABSTRACT
Regulated secretion of neuropeptides and peptide hormones by secretory granules (SGs) is central to physiology. Formation of SGs occurs at the trans-Golgi network (TGN) where their soluble cargo aggregates to form a dense core, but the mechanisms controlling the sorting of regulated secretory cargoes (soluble and transmembrane) away from constitutively secreted proteins remain unclear. Optimizing the use of the retention using selective hooks method in (neuro-)endocrine cells, we now quantify TGN budding kinetics of constitutive and regulated secretory cargoes. We further show that, by monitoring two cargoes simultaneously, it becomes possible to visualize sorting to the constitutive and regulated secretory pathways in real time. Further analysis of the localization of SG cargoes immediately after budding from the TGN revealed that, surprisingly, the bulk of two studied transmembrane SG cargoes (phogrin and VMAT2) does not sort directly onto SGs during budding, but rather exit the TGN into nonregulated vesicles to get incorporated to SGs at a later step. This differential behavior of soluble and transmembrane cargoes suggests a more complex model of SG biogenesis than anticipated.
Subject(s)
Endocrine Cells/metabolism , Secretory Vesicles/metabolism , trans-Golgi Network/metabolism , Animals , Biological Transport , Cell Line , Cytoplasmic Granules/metabolism , Exocytosis , Golgi Apparatus/metabolism , Neuropeptides/metabolism , PC12 Cells , Protein Transport/physiology , Rats , trans-Golgi Network/physiologyABSTRACT
In contrast to temporal coding by synaptically acting neurotransmitters such as glutamate, neuromodulators such as monoamines signal changes in firing rate. The two modes of signaling have been thought to reflect differences in release by different cells. We now find that midbrain dopamine neurons release glutamate and dopamine with different properties that reflect storage in different synaptic vesicles. The vesicles differ in release probability, coupling to presynaptic Ca2+ channels and frequency dependence. Although previous work has attributed variation in these properties to differences in location or cytoskeletal association of synaptic vesicles, the release of different transmitters shows that intrinsic differences in vesicle identity drive different modes of release. Indeed, dopamine but not glutamate vesicles depend on the adaptor protein AP-3, revealing an unrecognized linkage between the pathway of synaptic vesicle recycling and the properties of exocytosis. Storage of the two transmitters in different vesicles enables the transmission of distinct signals.
Subject(s)
Adaptor Protein Complex 3/metabolism , Calcium Channels/metabolism , Dopamine/metabolism , Dopaminergic Neurons/metabolism , Exocytosis , Glutamic Acid/metabolism , Synaptic Vesicles/metabolism , Animals , Mesencephalon/cytology , Mice , Neurons/metabolism , Neurotransmitter Agents/metabolismABSTRACT
CaMKII is one of the most studied synaptic proteins, but many critical issues regarding its role in synaptic function remain unresolved. Using a CRISPR-based system to delete CaMKII and replace it with mutated forms in single neurons, we have rigorously addressed its various synaptic roles. In brief, basal AMPAR and NMDAR synaptic transmission both require CaMKIIα, but not CaMKIIß, indicating that, even in the adult, synaptic transmission is determined by the ongoing action of CaMKIIα. While AMPAR transmission requires kinase activity, NMDAR transmission does not, implying a scaffolding role for the CaMKII protein instead. LTP is abolished in the absence of CaMKIIα and/or CaMKIIß and with an autophosphorylation impaired CaMKIIα (T286A). With the exception of NMDAR synaptic currents, all aspects of CaMKIIα signaling examined require binding to the NMDAR, emphasizing the essential role of this receptor as a master synaptic signaling hub.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Hippocampus/physiology , Receptors, N-Methyl-D-Aspartate/metabolism , Synaptic Transmission/physiology , Animals , CRISPR-Cas Systems , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Cell Membrane/metabolism , Female , HEK293 Cells , Hippocampus/cytology , Humans , Long-Term Potentiation/physiology , Mice , Mice, Knockout , Neurons/cytology , Neurons/physiology , Phosphorylation , Rats , Receptors, Glutamate/metabolism , Signal Transduction/physiology , Synapses/physiologyABSTRACT
The originally published version of this Article contained errors in Figure 5, for which we apologise. In panel c, the scatter graph was inadvertently replaced with a scatter graph comprising a subset of data points from panel d. Furthermore, the legends to Figures 5c and 5d were inverted. These errors have now been corrected in both the PDF and HTML versions of the Article, and the incorrect version of Fig. 5c is presented in the Author Correction associated with this Article.
ABSTRACT
Large dense core vesicles (LDCVs) mediate the regulated release of neuropeptides and peptide hormones. They form at the trans-Golgi network (TGN), where their soluble content aggregates to form a dense core, but the mechanisms controlling biogenesis are still not completely understood. Recent studies have implicated the peripheral membrane protein HID-1 in neuropeptide sorting and insulin secretion. Using CRISPR/Cas9, we generated HID-1 KO rat neuroendocrine cells, and we show that the absence of HID-1 results in specific defects in peptide hormone and monoamine storage and regulated secretion. Loss of HID-1 causes a reduction in the number of LDCVs and affects their morphology and biochemical properties, due to impaired cargo sorting and dense core formation. HID-1 KO cells also exhibit defects in TGN acidification together with mislocalization of the Golgi-enriched vacuolar H+-ATPase subunit isoform a2. We propose that HID-1 influences early steps in LDCV formation by controlling dense core formation at the TGN.
Subject(s)
Homeodomain Proteins/metabolism , Membrane Proteins/metabolism , Secretory Vesicles/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , trans-Golgi Network/metabolism , Animals , Exocytosis , Gene Knockout Techniques , Golgi Apparatus/metabolism , HEK293 Cells , Humans , Neuropeptides/metabolism , PC12 Cells , Protein Transport , RatsABSTRACT
We and others have previously shown that triple knockout mice lacking the beta1/beta2/beta3-adrenoceptors (beta-less mice) developed a progressive obesity at adulthood. Here, we studied the glucose homeostasis in beta-less mice before the onset of obesity. We show that beta-less mice have increased fat mass and are glucose intolerant. In addition, we observed that beta-less mice have impaired glucose-induced insulin secretion and exhibit an increase in liver PEPCK gene expression in the fed state, suggesting that they have increased gluconeogenesis. Although these characteristics are usually associated with insulin resistance, beta-less mice exhibit enhanced insulin sensitivity during insulin tolerance tests. This is keeping with the results obtained during euglycemic-hyperinsulinemic clamps showing that beta-less mice display increased insulin responsiveness with normal suppression of hepatic glucose production. Altogether, our results suggest that an intact beta-adrenergic system is required to regulate overall glucose homeostasis and, in particular, insulin-mediated glucose uptake, most likely at the level of muscles and adipose tissue.
Subject(s)
Glucose Intolerance/physiopathology , Insulin/physiology , Receptors, Adrenergic, beta/deficiency , Weight Gain , Animals , Blood Glucose/metabolism , Body Composition , Eating , Gene Expression Regulation, Enzymologic , Gluconeogenesis , Glucose/metabolism , Glucose Clamp Technique , Homeostasis , Insulin/blood , Insulin/pharmacology , Kinetics , Mice , Mice, Knockout , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolismABSTRACT
In rodent brown adipose tissue, the beta-adrenergic signaling is believed, by an action on PGC-1alpha, to control UCP1 expression and mitochondriogenesis. We addressed this hypothesis using beta(1)/beta(2)/beta(3)-adrenoceptor knockout (beta-less) brown adipocytes in primary culture. In these cells: (a) proliferation and differentiation into multilocular cells were normal; (b) UCP1 mRNA expression was dramatically decreased (by 93%), whereas PGC-1alpha and mtTFA mRNA expressions were not; (c) UCP1, PGC-1alpha and COX IV protein expressions were decreased by 97%, 62% and 22%, respectively. Altogether the data show a dissociation between the control of UCP1, which is mostly beta-adrenoceptor-dependent and that of PGC-1alpha and of mitochondriogenesis which are not.
Subject(s)
Adipocytes/physiology , Adipose Tissue, Brown/physiology , Carrier Proteins/physiology , Membrane Proteins/physiology , Receptors, Adrenergic, beta/physiology , Trans-Activators/physiology , Adipocytes/cytology , Adipose Tissue, Brown/cytology , Animals , Base Sequence , Blotting, Western , Carrier Proteins/genetics , DNA Primers , Ion Channels , Membrane Proteins/genetics , Mice , Mitochondrial Proteins , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Polymerase Chain Reaction , RNA, Messenger/genetics , Receptors, Adrenergic, beta/genetics , Transcription Factors , Uncoupling Protein 1ABSTRACT
Resistin is an adipose-derived hormone that has been proposed as a link among obesity, insulin resistance, and diabetes. In agreement with a role of resistin in insulin resistance, the administration of recombinant resistin led to glucose intolerance in mice and impaired insulin action in rat liver. However, the regulation of resistin expression by physiological conditions, hormones, or agents known to modulate insulin sensitivity does not always support the association between resistin and obesity-induced insulin resistance. In the present study we investigated the effects of leptin administration on adipose resistin expression in insulin-resistant and obese ob/ob mice. We show that the expression of resistin mRNA and protein in adipose tissue is lower in ob/ob than in wild-type control mice, in agreement with the reduced adipocyte resistin mRNA level reported in several models of obesity. Leptin administration in ob/ob mice resulted in improvement of insulin sensitivity concomitant with a decrease in resistin gene expression. The lack of effect of leptin on resistin in db/db mice indicated that the leptin inhibitory action on resistin expression requires the long leptin receptor isoform. In addition, we demonstrated that the effect of leptin on resistin expression was centrally mediated. High-fat feeding in C57BL/6J wild-type mice, which is known to induce the development of obesity and insulin resistance, produced an increase in resistin expression. Interestingly, in both ob/ob and high fat-fed mice we obtained a striking positive correlation between glycemia and resistin gene expression. In conclusion, our results demonstrate that leptin decreases resistin expression and suggest that resistin may influence glucose homeostasis.
Subject(s)
Blood Glucose/analysis , Diabetes Mellitus, Type 2/blood , Dietary Fats/administration & dosage , Hormones, Ectopic/genetics , Leptin/administration & dosage , Nerve Tissue Proteins , Obesity/blood , 11-beta-Hydroxysteroid Dehydrogenases/genetics , Adipose Tissue/chemistry , Animals , Carrier Proteins/genetics , Diabetes Mellitus/blood , Disease Models, Animal , Fatty Acid-Binding Protein 7 , Fatty Acid-Binding Proteins , Gene Expression/drug effects , Homeostasis , Hormones, Ectopic/analysis , Insulin Resistance , Male , Mice , Mice, Inbred C57BL , Mice, Obese , RNA, Messenger/analysis , ResistinABSTRACT
One of the most powerful ways to test the function of a protein is to characterize the consequences of its deletion. In the past, this has involved inactivation of the gene by homologous recombination either in the germline or later through conditional deletion. RNA interference (RNAi) provides an alternative way to knock down proteins, but both of these approaches have their limitations. Recently, the CRISPR/Cas9 system has suggested another way to selectively inactivate genes. We have now tested this system in postmitotic neurons by targeting two well-characterized synaptic proteins, the obligatory GluN1 subunit of the NMDA receptor and the GluA2 subunit of the AMPA receptor. Expression of CRISPR/Cas9 in hippocampal slice cultures completely eliminated NMDA receptor and GluA2 function. CRISPR/Cas9 thus provides a powerful tool to study the function of synaptic proteins.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Receptors, AMPA/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Synapses/metabolism , Animals , Animals, Newborn , Cells, Cultured , Excitatory Amino Acid Agents/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hippocampus/cytology , Humans , In Vitro Techniques , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , N-Methylaspartate/pharmacology , Nerve Tissue Proteins/genetics , Quinoxalines/pharmacology , RNA/genetics , RNA/metabolism , Rats , Receptors, AMPA/genetics , Receptors, N-Methyl-D-Aspartate/genetics , Synapses/genetics , Synaptophysin/metabolism , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/pharmacology , Red Fluorescent ProteinABSTRACT
The regulated release of polypeptides has a central role in physiology, behavior, and development, but the mechanisms responsible for production of the large dense core vesicles (LDCVs) capable of regulated release have remained poorly understood. Recent work has implicated cytosolic adaptor protein AP-3 in the recruitment of LDCV membrane proteins that confer regulated release. However, AP-3 in mammals has been considered to function in the endolysosomal pathway and in the biosynthetic pathway only in yeast. We now find that the mammalian homolog of yeast VPS41, a member of the homotypic fusion and vacuole protein sorting (HOPS) complex that delivers biosynthetic cargo to the endocytic pathway in yeast, promotes LDCV formation through a common mechanism with AP-3, indicating a conserved role for these proteins in the biosynthetic pathway. VPS41 also self-assembles into a lattice, suggesting that it acts as a coat protein for AP-3 in formation of the regulated secretory pathway.
Subject(s)
DNA-Binding Proteins/metabolism , Drosophila melanogaster/metabolism , Exocytosis/physiology , Organelle Biogenesis , Secretory Pathway/physiology , Transcription Factors/metabolism , Vesicular Transport Proteins/metabolism , Animals , COS Cells , Chlorocebus aethiops , DNA-(Apurinic or Apyrimidinic Site) Lyase , DNA-Binding Proteins/genetics , Drosophila Proteins , Drosophila melanogaster/genetics , Endosomes/metabolism , Humans , Membrane Fusion , Membrane Proteins/metabolism , PC12 Cells , Protein Transport , Rats , Secretory Vesicles/metabolism , Transcription Factors/genetics , Vesicular Monoamine Transport Proteins/metabolism , Vesicular Transport Proteins/geneticsABSTRACT
The regulated release of proteins depends on their inclusion within large dense-core vesicles (LDCVs) capable of regulated exocytosis. LDCVs form at the trans-Golgi network (TGN), but the mechanism for protein sorting to this regulated secretory pathway (RSP) and the cytosolic machinery involved in this process have remained poorly understood. Using an RNA interference screen in Drosophila melanogaster S2 cells, we now identify a small number of genes, including several subunits of the heterotetrameric adaptor protein AP-3, which are required for sorting to the RSP. In mammalian neuroendocrine cells, loss of AP-3 dysregulates exocytosis due to a primary defect in LDCV formation. Previous work implicated AP-3 in the endocytic pathway, but we find that AP-3 promotes sorting to the RSP within the biosynthetic pathway at the level of the TGN. Although vesicles with a dense core still form in the absence of AP-3, they contain substantially less synaptotagmin 1, indicating that AP-3 concentrates the proteins required for regulated exocytosis.