RESUMO
Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria in a post-translational reaction. Mitochondrial precursor proteins which use the ER-SURF pathway employ the surface of the endoplasmic reticulum (ER) as an important sorting platform. How they reach the mitochondrial import machinery from the ER is not known. Here we show that mitochondrial contact sites play a crucial role in the ER-to-mitochondria transfer of precursor proteins. The ER mitochondria encounter structure (ERMES) and Tom70, together with Djp1 and Lam6, are part of two parallel and partially redundant ER-to-mitochondria delivery routes. When ER-to-mitochondria transfer is prevented by loss of these two contact sites, many precursors of mitochondrial inner membrane proteins are left stranded on the ER membrane, resulting in mitochondrial dysfunction. Our observations support an active role of the ER in mitochondrial protein biogenesis.
Assuntos
Mitocôndrias , Proteínas de Saccharomyces cerevisiae , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Transporte Proteico , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Retrograde transport of membranes and proteins from the cell surface to the Golgi and beyond is essential to maintain homeostasis, compartment identity, and physiological functions. To study retrograde traffic biochemically, by live-cell imaging or by electron microscopy, we engineered functionalized anti-GFP nanobodies (camelid VHH antibody domains) to be bacterially expressed and purified. Tyrosine sulfation consensus sequences were fused to the nanobody for biochemical detection of trans-Golgi arrival, fluorophores for fluorescence microscopy and live imaging, and APEX2 (ascorbate peroxidase 2) for electron microscopy and compartment ablation. These functionalized nanobodies are specifically captured by GFP-modified reporter proteins at the cell surface and transported piggyback to the reporters' homing compartments. As an application of this tool, we have used it to determine the contribution of adaptor protein-1/clathrin in retrograde transport kinetics of the mannose-6-phosphate receptors from endosomes back to the trans-Golgi network. Our experiments establish functionalized nanobodies as a powerful tool to demonstrate and quantify retrograde transport pathways.
Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Membrana Celular/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endossomos/metabolismo , Receptor IGF Tipo 2/metabolismo , Anticorpos de Domínio Único/metabolismo , Rede trans-Golgi/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Camelus , Endonucleases , Endossomos/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia Eletrônica , Microscopia de Fluorescência , Enzimas Multifuncionais , Rede trans-Golgi/ultraestruturaRESUMO
Mitochondrial biogenesis relies on the synthesis of hundreds of different precursor proteins in the cytosol and their subsequent import into the organelle. Recent studies suggest that the surface of the endoplasmic reticulum (ER) actively contributes to the targeting of some mitochondrial precursors. In the past, in vitro import experiments with isolated mitochondria proved to be extremely powerful to elucidate the individual reactions of the mitochondrial import machinery. However, this in vitro approach is not well suited to study the influence of non-mitochondrial membranes. In this study, we describe an in vitro system using semi-intact yeast cells to test a potential import relevance of the ER proteins Erg3, Lcb5 and Ssh1, all being required for efficient mitochondrial respiration. We optimized the conditions of this experimental test system and found that cells lacking Ssh1, a paralog of the Sec61 translocation pore, show a reduced import efficiency of mitochondrial precursor proteins. Our results suggest that Ssh1, directly or indirectly, increases the efficiency of the biogenesis of mitochondrial proteins. Our findings are compatible with a functional interdependence of the mitochondrial and the ER protein translocation systems.
Assuntos
Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Biogênese de Organelas , Canais de Translocação SEC/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Citosol/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
BACKGROUND: Aggregation of peptide hormone precursors in the trans-Golgi network is an essential process in the biogenesis of secretory granules in endocrine cells. It has recently been proposed that this aggregation corresponds to the formation of functional amyloids. Our previous finding that dominant mutations in provasopressin, which cause cell degeneration and diabetes insipidus, prevent native folding and produce fibrillar aggregates in the endoplasmic reticulum (ER) might thus reflect mislocalized amyloid formation by sequences that evolved to mediate granule sorting. RESULTS: Here we identified two sequences responsible for fibrillar aggregation of mutant precursors in the ER: the N-terminal vasopressin nonapeptide and the C-terminal glycopeptide. To test their role in granule sorting, the glycopeptide was deleted and/or vasopressin mutated to inactivate ER aggregation while still permitting precursor folding and ER exit. These mutations strongly reduced sorting into granules and regulated secretion in endocrine AtT20 cells. CONCLUSION: The same sequences - vasopressin and the glycopeptide - mediate physiological aggregation of the wild-type hormone precursor into secretory granules and the pathological fibrillar aggregation of disease mutants in the ER. These findings support the amyloid hypothesis for secretory granule biogenesis.
Assuntos
Amiloide/metabolismo , Diabetes Insípido/metabolismo , Agregados Proteicos , Vesículas Secretórias/metabolismo , Vasopressinas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Genes Reporter , Glicopeptídeos/metabolismo , Humanos , Camundongos , Proteínas Mutantes/metabolismo , Dobramento de Proteína , Transporte Proteico , Deleção de SequênciaRESUMO
Numerous mRNAs are degraded in processing bodies (P bodies) in Saccharomyces cerevisiae. In logarithmically growing cells, only 0-1 P bodies per cell are detectable. However, the number and appearance of P bodies change once the cell encounters stress. Here, we show that the polysome-associated mRNA-binding protein Scp160 interacts with P body components, such as the decapping protein Dcp2 and the scaffold protein Pat1, presumably, on polysomes. Loss of either Scp160 or its interaction partner Bfr1 caused the formation of Dcp2-positive structures. These Dcp2-positive foci contained mRNA, because their formation was inhibited by the presence of cycloheximide. In addition, Scp160 was required for proper P body formation because only a subset of bona fide P body components could assemble into the Dcp2-positive foci in Δscp160 cells. In either Δbfr1 or Δscp160 cells, P body formation was uncoupled from translational attenuation as the polysome profile remained unchanged. Collectively, our data suggest that Bfr1 and Scp160 prevent P body formation under normal growth conditions.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Imunoprecipitação , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Polirribossomos/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismoRESUMO
The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of growth. Mammalian TOR complex 2 (mTORC2) regulates AGC kinase family members and is implicated in various disorders, including cancer and diabetes. Here we report that mTORC2 is localized to the endoplasmic reticulum (ER) subcompartment termed mitochondria-associated ER membrane (MAM). mTORC2 localization to MAM was growth factor-stimulated, and mTORC2 at MAM interacted with the IP3 receptor (IP3R)-Grp75-voltage-dependent anion-selective channel 1 ER-mitochondrial tethering complex. mTORC2 deficiency disrupted MAM, causing mitochondrial defects including increases in mitochondrial membrane potential, ATP production, and calcium uptake. mTORC2 controlled MAM integrity and mitochondrial function via Akt mediated phosphorylation of the MAM associated proteins IP3R, Hexokinase 2, and phosphofurin acidic cluster sorting protein 2. Thus, mTORC2 is at the core of a MAM signaling hub that controls growth and metabolism.
Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Retículo Endoplasmático/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Complexos Multiproteicos/genética , Proteínas Proto-Oncogênicas c-akt/genética , Serina-Treonina Quinases TOR/genéticaRESUMO
Lipid mobilization through fatty acid ß-oxidation is a central process essential for energy production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where ß-oxidation products enter mitochondria and fuel the tricarboxylic acid cycle. Little is known about the physical and metabolic cooperation between these organelles. Here we found that expression of fatty acid transporters and of the rate-limiting enzyme involved in ß-oxidation is decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. Consequently, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although ß-oxidation occurs in both mitochondria and peroxisomes in mammals, Arf1's role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact sites.
Assuntos
Mitocôndrias , Peroxissomos , Animais , Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Ácidos Graxos/metabolismo , Oxirredução , Metabolismo dos Lipídeos/genética , Homeostase , Mamíferos/metabolismoRESUMO
ADP-ribosylation factors (Arfs) are small GTPases regulating membrane traffic in the secretory pathway. They are closely related and appear to have overlapping functions, regulators, and effectors. The functional specificity of individual Arfs and the extent of redundancy are still largely unknown. We addressed these questions by CRISPR/Cas9-mediated genomic deletion of the human class I (Arf1/3) and class II (Arf4/5) Arfs, either individually or in combination. Most knockout cell lines were viable with slight growth defects only when lacking Arf1 or Arf4. However, Arf1+4 and Arf4+5 could not be deleted simultaneously. Class I Arfs are nonessential, and Arf4 alone is sufficient for viability. Upon Arf1 deletion, the Golgi was enlarged, and recruitment of vesicle coats decreased, confirming a major role of Arf1 in vesicle formation at the Golgi. Knockout of Arf4 caused secretion of ER-resident proteins, indicating specific defects in coatomer-dependent ER protein retrieval by KDEL receptors. The knockout cell lines will be useful tools to study other Arf-dependent processes.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Técnicas de Inativação de Genes , Complexo de Golgi/metabolismo , Forma Celular , Retículo Endoplasmático/metabolismo , Deleção de Genes , Complexo de Golgi/ultraestrutura , Células HeLa , HumanosRESUMO
Unlike constitutively secreted proteins, peptide hormones are stored in densely packed secretory granules, before regulated release upon stimulation. Secretory granules are formed at the TGN by self-aggregation of prohormones as functional amyloids. The nonapeptide hormone vasopressin, which forms a small disulfide loop, was shown to be responsible for granule formation of its precursor in the TGN as well as for toxic fibrillar aggregation of unfolded mutants in the ER. Several other hormone precursors also contain similar small disulfide loops suggesting their function as a general device to mediate aggregation for granule sorting. To test this hypothesis, we studied the capacity of small disulfide loops of different hormone precursors to mediate aggregation in the ER and the TGN. They indeed induced ER aggregation in Neuro-2a and COS-1 cells. Fused to a constitutively secreted reporter protein, they also promoted sorting into secretory granules, enhanced stimulated secretion, and increased Lubrol insolubility in AtT20 cells. These results support the hypothesis that small disulfide loops act as novel signals for sorting into secretory granules by self-aggregation.
Assuntos
Hormônios Peptídicos/metabolismo , Vesículas Secretórias/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Grânulos Citoplasmáticos/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Complexo de Golgi/metabolismo , Hormônios/genética , Hormônios/metabolismo , Humanos , Hormônios Peptídicos/genética , Transporte Proteico , Vesículas Secretórias/fisiologia , Vasopressinas/metabolismo , Rede trans-Golgi/metabolismoRESUMO
Autosomal dominant neurohypophyseal diabetes insipidus results from mutations in the precursor protein of the antidiuretic hormone arginine vasopressin. Mutant prohormone is retained in the endoplasmic reticulum of vasopressinergic neurons and causes their progressive degeneration by an unknown mechanism. Here, we show that several dominant pro-vasopressin mutants form disulfide-linked homo-oligomers and develop large aggregations visible by immunofluorescence and immunogold electron microscopy, both in a fibroblast and a neuronal cell line. Double-labeling showed the pro-vasopressin aggregates to colocalize with the chaperone calreticulin, indicating that they originated from the endoplasmic reticulum. The aggregates revealed a remarkable fibrillar substructure. Bacterially expressed and purified mutant pro-vasopressin spontaneously formed fibrils under oxidizing conditions. Mutagenesis experiments showed that the presence of cysteines, but no specific single cysteine, is essential for disulfide oligomerization and aggregation in vivo. Our findings assign autosomal dominant diabetes insipidus to the group of neurodegenerative diseases associated with the formation of fibrillar protein aggregates.
Assuntos
Diabetes Insípido Neurogênico/metabolismo , Retículo Endoplasmático/metabolismo , Mutação , Precursores de Proteínas/química , Precursores de Proteínas/genética , Vasopressinas/química , Vasopressinas/genética , Animais , Células COS , Chlorocebus aethiops , Diabetes Insípido Neurogênico/genética , Dissulfetos/química , Dissulfetos/metabolismo , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Humanos , Conformação Proteica , Dobramento de Proteína , Precursores de Proteínas/metabolismo , Vasopressinas/metabolismoRESUMO
Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1 and Rab11-positive recycling endosome recruitment occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.
Assuntos
Endossomos/metabolismo , Lisossomos/metabolismo , Microscopia de Fluorescência/métodos , Células HeLa , Humanos , Microscopia de Fluorescência/instrumentaçãoRESUMO
For the biogenesis of mitochondria, hundreds of proteins need to be targeted from the cytosol into the various compartments of this organelle. The intramitochondrial targeting routes these proteins take to reach their respective location in the organelle are well understood. However, the early targeting processes, from cytosolic ribosomes to the membrane of the organelle, are still largely unknown. In this study, we present evidence that an integral membrane protein of the endoplasmic reticulum (ER), Ema19, plays a role in this process. Mutants lacking Ema19 show an increased stability of mitochondrial precursor proteins, indicating that Ema19 promotes the proteolytic degradation of nonproductive precursors. The deletion of Ema19 improves the growth of respiration-deficient cells, suggesting that Ema19-mediated degradation can compete with productive protein import into mitochondria. Ema19 is the yeast representative of a conserved protein family. The human Ema19 homologue is known as sigma 2 receptor or TMEM97. Though its molecular function is not known, previous studies suggested a role of the sigma 2 receptor as a quality control factor in the ER, compatible with our observations about Ema19. More globally, our data provide an additional demonstration of the important role of the ER in mitochondrial protein targeting.
Assuntos
Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Proteólise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
In endocrine cells, prohormones and granins are segregated in the TGN (trans-Golgi network) from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner on specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures that resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of CgA (chromogranin A) is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP (green fluorescent protein) or a short epitope tag was expressed in COS-1 fibroblast cells and analysed by fluorescence and electron microscopy and pulse-chase labelling. Full-length CgA as well as deletion constructs containing the N-terminal 77 residues generated granule-like structures in the cell periphery that co-localized with co-expressed SgII (secretogranin II). These are essentially the same segments of the protein that were previously shown to be required for granule sorting in wild-type PC12 (pheochromocytoma cells) cells and for rescuing a regulated secretory pathway in A35C cells, a variant PC12 line deficient in granule formation. The results support the notion that self-aggregation is at the core of granule formation and sorting into the regulated pathway.
Assuntos
Cromogranina A/metabolismo , Células Endócrinas/metabolismo , Via Secretória , Vesículas Secretórias/metabolismo , Animais , Biomarcadores , Linhagem Celular , Chlorocebus aethiops , Cromogranina A/genética , Células Endócrinas/ultraestrutura , Epitopos/imunologia , Deleção de Genes , Camundongos , Microscopia Imunoeletrônica , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
This review focuses on the cellular and molecular aspects underlying familial neurohypophyseal diabetes insipidus (DI), a rare disorder that is usually transmitted in an autosomal-dominant fashion. The disease, manifesting in infancy or early childhood and gradually progressing in severity, is caused by fully penetrant heterozygous mutations in the gene encoding prepro-vasopressin-neurophysin II, the precursor of the antidiuretic hormone arginine vasopressin (AVP). Post mortem studies in affected adults have shown cell degeneration in vasopressinergic hypothalamic nuclei. Studies in cells expressing pathogenic mutants and knock-in rodent models have shown that the mutant precursors are folding incompetent and fail to exit the endoplasmic reticulum (ER), as occurs normally with proteins that have entered the regulated secretory pathway. A portion of these mutants is eliminated via ER-associated degradation (ERAD) by proteasomes after retrotranslocation to the cytosol. Another portion forms large disulfide-linked fibrillar aggregates within the ER, in which wild-type precursor is trapped. Aggregation capacity is independently conferred by two domains of the prohormone, namely the AVP moiety and the C-terminal glycopeptide (copeptin). The same domains are also required for packaging into dense-core secretory granules and regulated secretion, suggesting a disturbed balance between the physiological self-aggregation at the trans-Golgi network and avoiding premature aggregate formation at the ER in the disease. The critical role of ERAD in maintaining physiological water balance has been underscored by experiments in mice expressing wild-type AVP but lacking critical components of the ERAD machinery. These animals also develop DI and show amyloid-like aggregates in the ER lumen. Thus, the capacity of the ERAD is exceeded in autosomal dominant DI, which can be viewed as a neurodegenerative disorder associated with the formation of amyloid ER aggregates. While DI symptoms develop prior to detectable cell death in transgenic DI mice, the eventual loss of vasopressinergic neurons is accompanied by autophagy, but the mechanism leading to cell degeneration in autosomal dominant neurohypophyseal DI still remains unknown.
Assuntos
Diabetes Insípido Neurogênico/metabolismo , Agregados Proteicos/fisiologia , Proteólise , Animais , Autofagia/fisiologia , Diabetes Insípido Neurogênico/patologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Glicopeptídeos/metabolismo , Humanos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Vasopressinas/metabolismoRESUMO
Endosomal transport is essential for cellular organization and compartmentalization and cell-cell communication. Sorting endosomes provide a crossroads for various trafficking pathways and determine recycling, secretion or degradation of proteins. The organization of these processes requires membrane-tethering factors to coordinate Rab GTPase function with membrane fusion. Here, we report a conserved tethering platform that acts in the Rab11 recycling pathways at sorting endosomes, which we name factors for endosome recycling and Rab interactions (FERARI). The Rab-binding module of FERARI consists of Rab11FIP5 and rabenosyn-5/RABS-5, while the SNARE-interacting module comprises VPS45 and VIPAS39. Unexpectedly, the membrane fission protein EHD1 is also a FERARI component. Thus, FERARI appears to combine fusion activity through the SM protein VPS45 with pinching activity through EHD1 on SNX-1-positive endosomal membranes. We propose that coordination of fusion and pinching through a kiss-and-run mechanism drives cargo at endosomes into recycling pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Endocitose/genética , Endossomos/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Comunicação Celular , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Intestinos/citologia , Imagem Molecular , Oócitos/citologia , Oócitos/metabolismo , Transporte Proteico , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
The antidiuretic hormone vasopressin is synthesized as a longer precursor protein. After folding in the endoplasmic reticulum (ER), provasopressin is transported through the secretory pathway, forms secretory granules in the trans-Golgi network (TGN), is processed, and finally secreted into the circulation. Mutations in provasopressin cause autosomal dominant diabetes insipidus. They prevent native protein folding and cause fibrillar, amyloid-like aggregation in the ER, which eventually results in cell death. Secretory granules of peptide hormones were proposed to constitute functional amyloids and thus might be the cause of amyloid formation of misfolded mutant protein in the ER. Indeed, the same two segments in the precursor-vasopressin and a C-terminal glycopeptide-were found to be responsible for pathological aggregation in the ER and physiological aggregation in granule formation in the TGN. Furthermore, even wild-type provasopressin tends to aggregate in the ER, but is controlled by ER-associated degradation. When essential components thereof, Sel1L or Hrd1, were inactivated, wild-type provasopressin accumulated as fibrillar aggregates in vasopressinergic neurons in mice, causing diabetes insipidus. Evolution of amyloidogenic sequences for granule formation thus made provasopressin dependent on ER quality control mechanisms. These principles may similarly apply to other peptide hormones.
Assuntos
Amiloide/metabolismo , Diabetes Insípido Neurogênico/metabolismo , Agregados Proteicos/fisiologia , Vasopressinas/metabolismo , Animais , Modelos Animais de Doenças , CamundongosRESUMO
Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1-1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1-1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.
Assuntos
Retículo Endoplasmático/metabolismo , Células Eucarióticas/metabolismo , Membranas Intracelulares/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular , Células Cultivadas , Retículo Endoplasmático/ultraestrutura , Células Eucarióticas/ultraestrutura , Glicosilfosfatidilinositóis/metabolismo , Membranas Intracelulares/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/metabolismo , Microscopia Eletrônica , Mutação/genética , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , Receptores de Superfície Celular/metabolismo , Proteínas SNARE , Saccharomyces cerevisiae , Vesículas Transportadoras/genética , Vesículas Transportadoras/ultraestruturaRESUMO
Neurohypophyseal diabetes insipidus (DI) is most often caused by trauma, including operations, and infiltrating processes in the hypothalamic-pituitary region. Irradiation, ischemia, infections, or autoimmunity can also underlie the disease. Since the middle of the nineteenth century, familial forms of neurohypophyseal DI have been described. Most commonly, the disease is transmitted in an autosomal dominant fashion; very rarely, autosomal recessive inheritance has been observed. Hereditary neurohypophyseal DI is caused by mutations in the gene encoding the antidiuretic hormone vasopressin (AVP) and its carrier protein neurophysin II (NPII). Symptoms result from the lack of hormone, or from the inability of mutant AVP to activate its renal receptor, and respond to treatment with desmopressin (DDAVP). Dominant mutations cause retention of the hormone precursor in the endoplasmic reticulum (ER) of vasopressinergic neurons in the hypothalamus, resulting in cellular dysfunction and eventually neuronal death. This so-called neurotoxicity hypothesis was initially established on the basis of autopsy studies in affected humans and has been supported by heterologous cell culture expression experiments and murine knock-in models. Current data show that retained mutants fail to be eliminated by the cell's quality control system and accumulate in fibrillar aggregations within the ER. Autosomal dominant neurohypophyseal DI may thus be viewed as a neurodegenerative disease confined to vasopressinergic neurons.
Assuntos
Diabetes Insípido Neurogênico/genética , Doenças Neurodegenerativas/genética , Animais , Retículo Endoplasmático/patologia , Humanos , Camundongos , MutaçãoRESUMO
Positively charged nanogold was used as a probe to trace the internalization of plasma membrane (PM) domains carrying negatively charged residues at an ultrastructural level. The probe revealed distinct endocytic pathways within tobacco protoplasts and allowed the morphology of the organelles involved in endocytosis to be characterized in great detail. Putative early endosomes with a tubulo-vesicular structure, similar to that observed in animal cells, are described and a new compartment, characterized by interconnected vesicles, was identified as a late endosome using the Arabidopsis anti-syntaxin family Syp-21 antibody. Endocytosis dissection using Brefeldin A (BFA), pulse chase, temperature- and energy-dependent experiments combined with quantitative analysis of nanogold particles in different compartments, suggested that recycling to the PM predominated with respect to degradation. Further experiments using ikarugamycin (IKA), an inhibitor of clathrin-dependent endocytosis, and negatively charged nanogold confirmed that distinct endocytic pathways coexist in tobacco protoplasts.
Assuntos
Clatrina/fisiologia , Endocitose , Nicotiana/fisiologia , Azidas/farmacologia , Membrana Celular/metabolismo , Células Cultivadas , Temperatura Baixa , Endocitose/efeitos dos fármacos , Exocitose , Ouro , Lactamas/farmacologia , Nanopartículas Metálicas , Protoplastos/fisiologia , Fatores de TempoRESUMO
The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and gamma-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.