RESUMEN
The life of eukaryotic cells requires the transport of lipids between membranes, which are separated by the aqueous environment of the cytosol. Vesicle-mediated traffic along the secretory and endocytic pathways and lipid transfer proteins (LTPs) cooperate in this transport. Until recently, known LTPs were shown to carry one or a few lipids at a time and were thought to mediate transport by shuttle-like mechanisms. Over the last few years, a new family of LTPs has been discovered that is defined by a repeating ß-groove (RBG) rod-like structure with a hydrophobic channel running along their entire length. This structure and the localization of these proteins at membrane contact sites suggest a bridge-like mechanism of lipid transport. Mutations in some of these proteins result in neurodegenerative and developmental disorders. Here we review the known properties and well-established or putative physiological roles of these proteins, and we highlight the many questions that remain open about their functions.
Asunto(s)
Proteínas Portadoras , Proteínas , Proteínas Portadoras/química , Proteínas/metabolismo , Transporte Biológico/genética , Membrana Celular/metabolismo , Lípidos/químicaRESUMEN
The endoplasmic reticulum (ER) has a broad localization throughout the cell and forms direct physical contacts with all other classes of membranous organelles, including the plasma membrane (PM). A number of protein tethers that mediate these contacts have been identified, and study of these protein tethers has revealed a multiplicity of roles in cell physiology, including regulation of intracellular Ca2+ dynamics and signaling as well as control of lipid traffic and homeostasis. In this review, we discuss the cross talk between the ER and the PM mediated by direct contacts. We review factors that tether the two membranes, their properties, and their dynamics in response to the functional state of the cell. We focus in particular on the role of ER-PM contacts in nonvesicular lipid transport between the two bilayers mediated by lipid transfer proteins.
Asunto(s)
Calcio/metabolismo , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteína ORAI1/metabolismo , Transporte Biológico , Señalización del Calcio , Proteínas Portadoras/genética , Membrana Celular/ultraestructura , Retículo Endoplásmico/ultraestructura , Células Eucariotas/metabolismo , Células Eucariotas/ultraestructura , Expresión Génica , Homeostasis , Humanos , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Sinaptotagminas/genética , Sinaptotagminas/metabolismoRESUMEN
VAP (VAPA and VAPB) is an evolutionarily conserved endoplasmic reticulum (ER)-anchored protein that helps generate tethers between the ER and other membranes through which lipids are exchanged across adjacent bilayers. Here, we report that by regulating PI4P levels on endosomes, VAP affects WASH-dependent actin nucleation on these organelles and the function of the retromer, a protein coat responsible for endosome-to-Golgi traffic. VAP is recruited to retromer budding sites on endosomes via an interaction with the retromer SNX2 subunit. Cells lacking VAP accumulate high levels of PI4P, actin comets, and trans-Golgi proteins on endosomes. Such defects are mimicked by downregulation of OSBP, a VAP interactor and PI4P transporter that participates in VAP-dependent ER-endosomes tethers. These results reveal a role of PI4P in retromer-/WASH-dependent budding from endosomes. Collectively, our data show how the ER can control budding dynamics and association with the cytoskeleton of another membrane by direct contacts leading to bilayer lipid modifications.
Asunto(s)
Endosomas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Actinas/metabolismo , Retículo Endoplásmico/metabolismo , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Proteínas de Microfilamentos/metabolismo , Receptores de Esteroides/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Proteínas de Transporte Vesicular/genéticaRESUMEN
Protein interactions form a network whose structure drives cellular function and whose organization informs biological inquiry. Using high-throughput affinity-purification mass spectrometry, we identify interacting partners for 2,594 human proteins in HEK293T cells. The resulting network (BioPlex) contains 23,744 interactions among 7,668 proteins with 86% previously undocumented. BioPlex accurately depicts known complexes, attaining 80%-100% coverage for most CORUM complexes. The network readily subdivides into communities that correspond to complexes or clusters of functionally related proteins. More generally, network architecture reflects cellular localization, biological process, and molecular function, enabling functional characterization of thousands of proteins. Network structure also reveals associations among thousands of protein domains, suggesting a basis for examining structurally related proteins. Finally, BioPlex, in combination with other approaches, can be used to reveal interactions of biological or clinical significance. For example, mutations in the membrane protein VAPB implicated in familial amyotrophic lateral sclerosis perturb a defined community of interactors.
Asunto(s)
Mapas de Interacción de Proteínas , Proteómica/métodos , Esclerosis Amiotrófica Lateral/genética , Humanos , Espectrometría de Masas , Mapeo de Interacción de Proteínas , Proteínas/química , Proteínas/aislamiento & purificación , Proteínas/metabolismoRESUMEN
Most available information on endoplasmic reticulum (ER)-plasma membrane (PM) contacts in cells of higher eukaryotes concerns proteins implicated in the regulation of Ca(2+) entry. However, growing evidence suggests that such contacts play more general roles in cell physiology, pointing to the existence of additionally ubiquitously expressed ER-PM tethers. Here, we show that the three extended synaptotagmins (E-Syts) are ER proteins that participate in such tethering function via C2 domain-dependent interactions with the PM that require PI(4,5)P2 in the case of E-Syt2 and E-Syt3 and also elevation of cytosolic Ca(2+) in the case of E-Syt1. As they form heteromeric complexes, the E-Syts confer cytosolic Ca(2+) regulation to ER-PM contact formation. E-Syts-dependent contacts, however, are not required for store-operated Ca(2+) entry. Thus, the ER-PM tethering function of the E-Syts (tricalbins in yeast) mediates the formation of ER-PM contacts sites, which are functionally distinct from those mediated by STIM1 and Orai1.
Asunto(s)
Calcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Sinaptotagminas/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/ultraestructura , Retículo Endoplásmico/química , Retículo Endoplásmico/ultraestructura , Células HeLa , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Sinaptotagminas/química , Sinaptotagminas/genética , Levaduras/citología , Levaduras/metabolismoRESUMEN
Synaptojanin-1 (SJ1) is a major neuronal-enriched PI(4, 5)P2 4- and 5-phosphatase implicated in the shedding of endocytic factors during endocytosis. A mutation (R258Q) that impairs selectively its 4-phosphatase activity causes Parkinsonism in humans and neurological defects in mice (SJ1RQKI mice). Studies of these mice showed, besides an abnormal assembly state of endocytic factors at synapses, the presence of dystrophic nerve terminals selectively in a subset of nigro-striatal dopamine (DA)-ergic axons, suggesting a special lability of DA neurons to the impairment of SJ1 function. Here we have further investigated the impact of SJ1 on DA neurons using iPSC-derived SJ1 KO and SJ1RQKI DA neurons and their isogenic controls. In addition to the expected enhanced clustering of endocytic factors in nerve terminals, we observed in both SJ1 mutant neuronal lines increased cilia length. Further analysis of cilia of SJ1RQDA neurons revealed abnormal accumulation of the Ca2+ channel Cav1.3 and of ubiquitin chains, suggesting a defect in the clearing of ubiquitinated proteins at the ciliary base, where a focal concentration of SJ1 was observed. We suggest that SJ1 may contribute to the control of ciliary protein dynamics in DA neurons, with implications on cilia-mediated signaling.
Asunto(s)
Células Madre Pluripotentes Inducidas , Proteínas del Tejido Nervioso , Enfermedad de Parkinson , Trastornos Parkinsonianos , Humanos , Ratones , Animales , Enfermedad de Parkinson/metabolismo , Neuronas Dopaminérgicas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , MutaciónRESUMEN
Members of the synaptophysin and synaptogyrin family are vesicle proteins with four transmembrane domains. In spite of their abundance in synaptic vesicle (SV) membranes, their role remains elusive and only mild defects at the cellular and organismal level are observed in mice lacking one or more family members. Here, we show that coexpression with synapsin in fibroblasts of each of the four brain-enriched members of this family-synaptophysin, synaptoporin, synaptogyrin 1, and synaptogyrin 3-is sufficient to generate clusters of small vesicles in the same size range of SVs. Moreover, mice lacking all these four proteins have larger SVs. We conclude that synaptophysin and synaptogyrin family proteins play an overlapping function in the biogenesis of SVs and in determining their small size.
Asunto(s)
Vesículas Sinápticas , Sinaptogirinas , Sinaptofisina , Animales , Sinaptofisina/metabolismo , Sinaptofisina/genética , Vesículas Sinápticas/metabolismo , Ratones , Sinaptogirinas/metabolismo , Sinaptogirinas/genética , Sinapsinas/metabolismo , Sinapsinas/genética , Ratones Noqueados , Fibroblastos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratas , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genéticaRESUMEN
Mutations in Leucine-rich repeat kinase 2 (LRRK2) are responsible for late-onset autosomal dominant Parkinson's disease. LRRK2 has been implicated in a wide range of physiological processes including membrane repair in the endolysosomal system. Here, using cell-free systems, we report that purified LRRK2 directly binds acidic lipid bilayers with a preference for highly curved bilayers. While this binding is nucleotide independent, LRRK2 can also deform low-curvature liposomes into narrow tubules in a guanylnucleotide-dependent but Adenosine 5'-triphosphate-independent way. Moreover, assembly of LRRK2 into scaffolds at the surface of lipid tubules can constrict them. We suggest that an interplay between the membrane remodeling and signaling properties of LRRK2 may be key to its physiological function. LRRK2, via its kinase activity, may achieve its signaling role at sites where membrane remodeling occurs.
Asunto(s)
Enfermedad de Parkinson , Proteínas Serina-Treonina Quinasas , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Fosforilación , MutaciónRESUMEN
The spine apparatus is a specialized compartment of the neuronal smooth endoplasmic reticulum (ER) located in a subset of dendritic spines. It consists of stacks of ER cisterns that are interconnected by an unknown dense matrix and are continuous with each other and with the ER of the dendritic shaft. While this organelle was first observed over 60 y ago, its molecular organization remains a mystery. Here, we performed in vivo proximity proteomics to gain some insight into its molecular components. To do so, we used the only known spine apparatus-specific protein, synaptopodin, to target a biotinylating enzyme to this organelle. We validated the specific localization in dendritic spines of a small subset of proteins identified by this approach, and we further showed their colocalization with synaptopodin when expressed in nonneuronal cells. One such protein is Pdlim7, an actin binding protein not previously identified in spines. Pdlim7, which we found to interact with synaptopodin through multiple domains, also colocalizes with synaptopodin on the cisternal organelle, a peculiar stack of ER cisterns resembling the spine apparatus and found at axon initial segments of a subset of neurons. Moreover, Pdlim7 has an expression pattern similar to that of synaptopodin in the brain, highlighting a functional partnership between the two proteins. The components of the spine apparatus identified in this work will help elucidate mechanisms in the biogenesis and maintenance of this enigmatic structure with implications for the function of dendritic spines in physiology and disease.
Asunto(s)
Espinas Dendríticas , Proteómica , Espinas Dendríticas/metabolismo , Retículo Endoplásmico/metabolismo , Hipocampo/metabolismo , Proteínas de Microfilamentos/metabolismoRESUMEN
VPS13 is a eukaryotic lipid transport protein localized at membrane contact sites. Previous studies suggested that it may transfer lipids between adjacent bilayers by a bridge-like mechanism. Direct evidence for this hypothesis from a full-length structure and from electron microscopy (EM) studies in situ is still missing, however. Here, we have capitalized on AlphaFold predictions to complement the structural information already available about VPS13 and to generate a full-length model of human VPS13C, the Parkinson's disease-linked VPS13 paralog localized at contacts between the endoplasmic reticulum (ER) and endo/lysosomes. Such a model predicts an â¼30-nm rod with a hydrophobic groove that extends throughout its length. We further investigated whether such a structure can be observed in situ at ER-endo/lysosome contacts. To this aim, we combined genetic approaches with cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) to examine HeLa cells overexpressing this protein (either full length or with an internal truncation) along with VAP, its anchoring binding partner at the ER. Using these methods, we identified rod-like densities that span the space separating the two adjacent membranes and that match the predicted structures of either full-length VPS13C or its shorter truncated mutant, thus providing in situ evidence for a bridge model of VPS13 in lipid transport.
Asunto(s)
Retículo Endoplásmico , Metabolismo de los Lípidos , Proteínas , Transportadoras de Casetes de Unión a ATP , Proteínas de la Membrana Bacteriana Externa , Transporte Biológico , Membrana Celular/química , Microscopía por Crioelectrón , Retículo Endoplásmico/química , Células HeLa , Humanos , Lisosomas/química , Proteínas/químicaRESUMEN
Chorea-acanthocytosis (ChAc) and McLeod syndrome are diseases with shared clinical manifestations caused by mutations in VPS13A and XK, respectively. Key features of these conditions are the degeneration of caudate neurons and the presence of abnormally shaped erythrocytes. XK belongs to a family of plasma membrane (PM) lipid scramblases whose action results in exposure of PtdSer at the cell surface. VPS13A is an endoplasmic reticulum (ER)-anchored lipid transfer protein with a putative role in the transport of lipids at contacts of the ER with other membranes. Recently VPS13A and XK were reported to interact by still unknown mechanisms. So far, however, there is no evidence for a colocalization of the two proteins at contacts of the ER with the PM, where XK resides, as VPS13A was shown to be localized at contacts between the ER and either mitochondria or lipid droplets. Here we show that VPS13A can also localize at ER-PM contacts via the binding of its PH domain to a cytosolic loop of XK, that such interaction is regulated by an intramolecular interaction within XK, and that both VPS13A and XK are highly expressed in the caudate neurons. Binding of the PH domain of VPS13A to XK is competitive with its binding to intracellular membranes that mediate other tethering functions of VPS13A. Our findings support a model according to which VPS13A-dependent lipid transfer between the ER and the PM is coupled to lipid scrambling within the PM. They raise the possibility that defective cell surface exposure of PtdSer may be responsible for neurodegeneration.
Asunto(s)
Proteínas Portadoras , Membrana Celular , Lípidos , Proteínas de Transporte Vesicular , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Humanos , Neuroacantocitosis/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
PURPOSE: The function of FAM177A1 and its relationship to human disease is largely unknown. Recent studies have demonstrated FAM177A1 to be a critical immune-associated gene. One previous case study has linked FAM177A1 to a neurodevelopmental disorder in 4 siblings. METHODS: We identified 5 individuals from 3 unrelated families with biallelic variants in FAM177A1. The physiological function of FAM177A1 was studied in a zebrafish model organism and human cell lines with loss-of-function variants similar to the affected cohort. RESULTS: These individuals share a characteristic phenotype defined by macrocephaly, global developmental delay, intellectual disability, seizures, behavioral abnormalities, hypotonia, and gait disturbance. We show that FAM177A1 localizes to the Golgi complex in mammalian and zebrafish cells. Intersection of the RNA sequencing and metabolomic data sets from FAM177A1-deficient human fibroblasts and whole zebrafish larvae demonstrated dysregulation of pathways associated with apoptosis, inflammation, and negative regulation of cell proliferation. CONCLUSION: Our data shed light on the emerging function of FAM177A1 and defines FAM177A1-related neurodevelopmental disorder as a new clinical entity.
RESUMEN
Extended synaptotagmins (E-Syts) mediate lipid exchange between the endoplasmic reticulum (ER) and the plasma membrane (PM). Anchored on the ER, E-Syts bind the PM via an array of C2 domains in a Ca2+- and lipid-dependent manner, drawing the two membranes close to facilitate lipid exchange. How these C2 domains bind the PM and regulate the ER-PM distance is not well understood. Here, we applied optical tweezers to dissect PM binding by E-Syt1 and E-Syt2. We detected Ca2+- and lipid-dependent membrane-binding kinetics of both E-Syts and determined the binding energies and rates of individual C2 domains or pairs. We incorporated these parameters in a theoretical model to recapitulate salient features of E-Syt-mediated membrane contacts observed in vivo, including their equilibrium distances and probabilities. Our methods can be applied to study other proteins containing multiple membrane-binding domains linked by disordered polypeptides.
Asunto(s)
Calcio , Pinzas Ópticas , Calcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Lípidos/análisisRESUMEN
Dynamin, the founding member of a family of dynamin-like proteins (DLPs) implicated in membrane remodelling, has a critical role in endocytic membrane fission events. The use of complementary approaches, including live-cell imaging, cell-free studies, X-ray crystallography and genetic studies in mice, has greatly advanced our understanding of the mechanisms by which dynamin acts, its essential roles in cell physiology and the specific function of different dynamin isoforms. In addition, several connections between dynamin and human disease have also emerged, highlighting specific contributions of this GTPase to the physiology of different tissues.
Asunto(s)
Membrana Celular/fisiología , Dinaminas/fisiología , Animales , Membrana Celular/metabolismo , Dinaminas/química , Dinaminas/genética , Dinaminas/metabolismo , Endocitosis/genética , Endocitosis/fisiología , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/fisiología , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiología , Mamíferos/genética , Mamíferos/metabolismo , Fluidez de la Membrana/genética , Ratones , Modelos Biológicos , Modelos Moleculares , Conformación ProteicaRESUMEN
Membrane-shaping proteins of the BAR domain superfamily are determinants of organelle biogenesis, membrane trafficking, cell division, and cell migration. An upsurge of research now reveals new principles of BAR domain-mediated membrane remodeling, enhancing our understanding of membrane curvature-mediated information processing.
Asunto(s)
Proteínas de la Membrana/metabolismo , Animales , Membrana Celular/química , Membrana Celular/ultraestructura , Citoesqueleto/metabolismo , GTP Fosfohidrolasas/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/ultraestructura , Estructura Terciaria de ProteínaRESUMEN
The recent identification of several novel endocytic compartments has challenged our current understanding of the topological and functional organization of the endocytic pathway. Using quantitative single vesicle imaging and acute manipulation of phosphoinositides we show that APPL endosomes, which participate in growth factor receptor trafficking and signaling, represent an early endocytic intermediate common to a subset of clathrin derived endocytic vesicles and macropinosomes. Most APPL endosomes are precursors of classical PI3P positive endosomes, and PI3P plays a critical role in promoting this conversion. Depletion of PI3P causes a striking reversion of Rab5 positive endosomes to the APPL stage, and results in enhanced growth factor signaling. These findings reveal a surprising plasticity of the early endocytic pathway. Importantly, PI3P functions as a switch to dynamically regulate maturation and signaling of APPL endosomes.
Asunto(s)
Endosomas/metabolismo , Fosfatidilinositoles/metabolismo , Animales , Células COS , Chlorocebus aethiops , Vesículas Cubiertas por Clatrina/metabolismo , Endocitosis , Transducción de SeñalRESUMEN
Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using 'salvaged fluorescence'. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.
Asunto(s)
Imagen Óptica , Fracciones Subcelulares/metabolismo , Animales , Humanos , Orgánulos/metabolismoRESUMEN
BAR superfamily domains shape membranes through poorly understood mechanisms. We solved structures of F-BAR modules bound to flat and curved bilayers using electron (cryo)microscopy. We show that membrane tubules form when F-BARs polymerize into helical coats that are held together by lateral and tip-to-tip interactions. On gel-state membranes or after mutation of residues along the lateral interaction surface, F-BARs adsorb onto bilayers via surfaces other than their concave face. We conclude that membrane binding is separable from membrane bending, and that imposition of the module's concave surface forces fluid-phase bilayers to bend locally. Furthermore, exposure of the domain's lateral interaction surface through a change in orientation serves as the crucial trigger for assembly of the helical coat and propagation of bilayer bending. The geometric constraints and sequential assembly of the helical lattice explain how F-BAR and classical BAR domains segregate into distinct microdomains, and provide insight into the spatial regulation of membrane invagination.
Asunto(s)
Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Animales , Células COS , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Membrana Celular/ultraestructura , Chlorocebus aethiops , Microscopía por Crioelectrón , Dinaminas/metabolismo , Proteínas de Unión a Ácidos Grasos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Liposomas/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/ultraestructura , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Antígenos de Histocompatibilidad Menor , Modelos Biológicos , Modelos Moleculares , Estructura Terciaria de Proteína , TransfecciónRESUMEN
Numerous genes whose mutations cause, or increase the risk of, Parkinson's disease (PD) have been identified. An inactivating mutation (R258Q) in the Sac inositol phosphatase domain of synaptojanin 1 (SJ1/PARK20), a phosphoinositide phosphatase implicated in synaptic vesicle recycling, results in PD. The gene encoding Sac2/INPP5F, another Sac domain-containing protein, is located within a PD risk locus identified by genome-wide association studies. Knock-In mice carrying the SJ1 patient mutation (SJ1RQKI) exhibit PD features, while Sac2 knockout mice (Sac2KO) do not have obvious neurologic defects. We report a "synthetic" effect of the SJ1 mutation and the KO of Sac2 in mice. Most mice with both mutations died perinatally. The occasional survivors had stunted growth, died within 3 wk, and showed abnormalities of striatal dopaminergic nerve terminals at an earlier stage than SJ1RQKI mice. The abnormal accumulation of endocytic factors observed at synapses of cultured SJ1RQKI neurons was more severe in double-mutant neurons. Our results suggest that SJ1 and Sac2 have partially overlapping functions and are consistent with a potential role of Sac2 as a PD risk gene.
Asunto(s)
Inositol Polifosfato 5-Fosfatasas/genética , Enfermedad de Parkinson/enzimología , Animales , Dopamina/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Inositol Polifosfato 5-Fosfatasas/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Terminaciones Nerviosas/metabolismo , Enfermedad de Parkinson/genética , Fenotipo , Sinapsis/metabolismoRESUMEN
The extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and transport lipids between them via SMP domains. E-Syt1 tethers and transports lipids in a Ca2+-dependent manner, but the role of Ca2+ in this regulation is unclear. Of the five C2 domains of E-Syt1, only C2A and C2C contain Ca2+-binding sites. Using liposome-based assays, we show that Ca2+ binding to C2C promotes E-Syt1-mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P2-rich membranes, as previously suggested by studies in semi-permeabilized cells. Importantly, Ca2+ binding to C2A enables lipid transport by releasing a charge-based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E-Syt1 constructs defective in Ca2+ binding in either C2A or C2C failed to rescue two defects in PM lipid homeostasis observed in E-Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca2+-triggered phosphatidylserine scrambling. Thus, a main effect of Ca2+ on E-Syt1 is to reverse an autoinhibited state and to couple membrane tethering with lipid transport.