RESUMO
SREBPs are master regulators of lipid homeostasis and undergo sterol-regulated export from ER to Golgi apparatus for processing and activation via COPII-coated vesicles. While COPII recognizes SREBP through its escort protein SCAP, factor(s) specifically promoting SREBP/SCAP loading to the COPII machinery remains unknown. Here, we show that the ER/lipid droplet-associated protein Cideb selectively promotes the loading of SREBP/SCAP into COPII vesicles. Sterol deprivation releases SCAP from Insig and enhances ER export of SREBP/SCAP by inducing SCAP-Cideb interaction, thereby modulating sterol sensitivity. Moreover, Cideb binds to the guanine nucleotide exchange factor Sec12 to enrich SCAP/SREBP at ER exit sites, where assembling of COPII complex initiates. Loss of Cideb inhibits the cargo loading of SREBP/SCAP, reduces SREBP activation, and alleviates diet-induced hepatic steatosis. Our data point to a linchpin role of Cideb in regulated ER export of SREBP and lipid homeostasis.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/fisiologia , Retículo Endoplasmático/fisiologia , Complexo de Golgi/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Esteróis/farmacologia , Animais , Proteínas Reguladoras de Apoptose/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Homeostase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Transporte Proteico , Proteína de Ligação a Elemento Regulador de Esterol 1/genéticaRESUMO
Native cargo proteins exit the endoplasmic reticulum (ER) in COPII-coated vesicles, whereas resident and misfolded proteins are substantially excluded from vesicles by a retention mechanism that remains unresolved. We probed the ER retention process using the proteostasis regulator 4-phenylbutyrate (4-PBA), which we show targets COPII protein to reduce the stringency of retention. 4-PBA competes with p24 proteins to bind COPII. When p24 protein uptake is blocked, COPII vesicles package resident proteins and an ER-trapped mutant LDL receptor. We further show that 4-PBA triggers the secretion of a KDEL-tagged luminal resident, implying that a compromised retention mechanism causes saturation of the KDEL retrieval system. The results indicate that stringent ER retention requires the COPII coat machinery to actively sort biosynthetic cargo from diffusible misfolded and resident ER proteins.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Fenilbutiratos/metabolismo , Transporte Proteico/efeitos dos fármacos , Antineoplásicos , Humanos , Ligação ProteicaRESUMO
Stimulation of cells with epidermal growth factor (EGF) induces internalization and partial degradation of the EGF receptor (EGFR) by the endo-lysosomal pathway. For continuous cell functioning, EGFR plasma membrane levels are maintained by transporting newly synthesized EGFRs to the cell surface. The regulation of this process is largely unknown. In this study, we find that EGF stimulation specifically increases the transport efficiency of newly synthesized EGFRs from the endoplasmic reticulum to the plasma membrane. This coincides with an up-regulation of the inner coat protein complex II (COPII) components SEC23B, SEC24B, and SEC24D, which we show to be specifically required for EGFR transport. Up-regulation of these COPII components requires the transcriptional regulator RNF11, which localizes to early endosomes and appears additionally in the cell nucleus upon continuous EGF stimulation. Collectively, our work identifies a new regulatory mechanism that integrates the degradation and transport of EGFR in order to maintain its physiological levels at the plasma membrane.
Assuntos
Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Receptores ErbB/metabolismo , Proteólise , Transcrição Gênica , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA , Endossomos/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Because the functional borders of the intermediate compartment (IC) are not well defined, the spatial map of the transport machineries operating between the endoplasmic reticulum (ER) and the Golgi apparatus remains incomplete. Our previous studies showed that the IC consists of interconnected vacuolar and tubular parts with specific roles in pre-Golgi trafficking. Here, using live cell imaging, we demonstrate that the tubules containing the GTPase Rab1A create a long-lived membrane compartment around the centrosome. Separation of this pericentrosomal domain of the IC from the Golgi ribbon, due to centrosome motility, revealed that it contains a distinct pool of COPI coats and acts as a temperature-sensitive way station in post-ER trafficking. However, unlike the Golgi, the pericentrosomal IC resists the disassembly of COPI coats by brefeldin A, maintaining its juxtaposition with the endocytic recycling compartment, and operation as the focal point of a dynamic tubular network that extends to the cell periphery. These results provide novel insight into the compartmental organization of the secretory pathway and Golgi biogenesis. Moreover, they reveal a direct functional connection between the IC and the endosomal system, which evidently contributes to unconventional transport of the cystic fibrosis transmembrane conductance regulator to the cell surface.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Centrossomo/fisiologia , Complexo I de Proteína do Envoltório/fisiologia , Transporte Proteico/fisiologia , Proteínas rab1 de Ligação ao GTP/metabolismo , Rede trans-Golgi/fisiologia , Animais , Brefeldina A/farmacologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Linhagem Celular/virologia , Centrossomo/ultraestrutura , Cricetinae , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Endocitose , Complexo de Golgi/efeitos dos fármacos , Células HeLa , Humanos , Membranas Intracelulares/fisiologia , Membranas Intracelulares/ultraestrutura , Rim/citologia , Mesocricetus , Microscopia de Vídeo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Vírus da Floresta de Semliki/fisiologia , Proteínas Virais de Fusão/metabolismo , Proteínas rab1 de Ligação ao GTP/genética , Rede trans-Golgi/ultraestruturaRESUMO
In eukaryotic cells several physiologic and pathologic conditions generate the accumulation of unfolded proteins in the endoplasmic reticulum (ER), leading to ER stress. To restore normal function, some ER transmembrane proteins sense the ER stress and activate coordinated signalling pathways collectively called the Unfolded Protein Response (UPR). Little is known on how the UPR relates to post-ER compartments and to the export from the ER of newly synthesized proteins. Here, we report that the ER stress response induced by either thapsigargin or nitric oxide modifies the dynamics of the intracellular distribution of ERGIC-53 and GM130, two markers of the ER Golgi Intermediate Compartment and of the cis-Golgi, respectively. In addition, induction of ER stress alters the morphology of the ERGIC and the Golgi complex and interferes with the reformation of both compartments. Moreover, ER stress rapidly reduces the transport to the Golgi complex of the temperature sensitive mutant of the Vesicular Stomatitis Virus G Glycoprotein (VSV-G) fused with the Green Fluorescent Protein (ts045G), without apparently decreasing the amount of the protein competent for export. Interestingly, a parallel rapid reduction of the number of Sec31 labelled fluorescent puncta on the ER membranes does occur, thus suggesting that the ER stress alters the ER export and the dynamic of post-ER compartments by rapidly targeting the formation of COPII-coated transport intermediates.
Assuntos
Autoantígenos/metabolismo , Biomarcadores/metabolismo , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Lectinas de Ligação a Manose/metabolismo , Proteínas de Membrana/metabolismo , Tapsigargina/farmacologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Linhagem Celular , Estruturas Celulares/efeitos dos fármacos , Estruturas Celulares/metabolismo , Estruturas Celulares/ultraestrutura , Retículo Endoplasmático , Complexo de Golgi , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Humanos , Glicoproteínas de Membrana/metabolismo , Engenharia de Proteínas , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Resposta a Proteínas não Dobradas , Proteínas do Envelope Viral/metabolismoRESUMO
The GABA transporter-1 (GAT1) is a prototypical protein of the synaptic specialization. Export of GAT1 from the endoplasmic reticulum (ER) is contingent on its interaction with the COPII (coatomer protein-II) coat subunit Sec24D. Here we show that silencing all four Sec24 isoforms strongly inhibits transport of GAT1 to the cell surface. In contrast, transport of GAT1-RL/AS, a mutant that is deficient in Sec24D recruitment, was not inhibited, suggesting a nonconventional, COPII-independent pathway. However, ARFGAP1 bound directly to the C terminus of both GAT1-RL/AS and wild-type GAT1. Surface expression of GAT1-RL/AS involved ARFGAP1. GAT1-RL/AS appeared to bypass the ER-Golgi-intermediate compartment, but its pathway to the plasma membrane still involved passage through the Golgi. Thus, the GAT1-RL/AS mutant allowed to test whether COPII-dependent ER-export is required for correct sorting of GAT1 to the axon terminal in neuronal cells. In contrast to wild-type GAT1, GAT1-RL/AS failed to be specifically enriched at the tip of neurite extensions of CAD.a cells (a neuroblastoma cell line that can be differentiated into a neuron-like phenotype) and in the axon terminals of hippocampal neurons. These findings indicate that correct sorting to the axon is contingent on ER export via the COPII machinery and passage through the ER-Golgi-intermediate compartment.
Assuntos
Axônios/fisiologia , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Neurônios/citologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Animais Recém-Nascidos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Células Cultivadas , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Proteínas Ativadoras de GTPase/genética , Hipocampo/citologia , Humanos , Imunoprecipitação/métodos , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/metabolismo , Microscopia Confocal/métodos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Wistar , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Temperatura , Transfecção/métodos , Trítio/metabolismo , Proteínas de Transporte Vesicular/genética , Ácido gama-Aminobutírico/metabolismoRESUMO
Angiogenic switch in renal cell carcinoma (RCC) is attributed to the inactivation of the von Hippel-Lindau tumor suppressor, stabilization of hypoxia inducible factor-1 transcription factor and increased vascular endothelial growth factor. To evaluate the role of an angiogenesis inhibitor, thrombopsondin-1 (TSP1), we compared TSP1 production in human RCC and normal tissue and secretion by the normal renal epithelium (human normal kidney, HNK) and RCC cells. Normal and RCC tissues stained positive for TSP1, and the levels of TSP1 mRNA and total protein were similar in RCC and HNK cells. However, HNK cells secreted high TSP1, which rendered them nonangiogenic, whereas RCC cells secreted little TSP1 and were angiogenic. Western blot and immunostaining revealed TSP1 in the cytoplasm of RCC cells on serum withdrawal, whereas, in HNK cells, it was rapidly exported. Seeking mechanisms of defective TSP1 secretion, we discovered impaired calcium uptake by RCC in response to vascular endothelial growth factor. In HNK cells, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, a calcium chelator, simulated TSP1 retention, mimicking the RCC phenotype. Further analysis revealed a profound decrease in transient receptor potential canonical ion channel 4 (TRPC4) Ca(2+) channel expression in RCC cells. TRPC4 silencing in HNK cells caused TSP1 retention and impaired secretion. Double labeling of the secretory system components revealed TSP1 colocalization with coatomer protein II (COPII) anterograde vesicles in HNK cells. In contrast, in RCC cells, TSP1 colocalized with COPI vesicles, pointing to the retrograde transport to the endoplasmic reticulum caused by misfolding. Our study indicates that TRPC4 loss in RCC leads to impaired Ca(2+) intake, misfolding, retrograde transport and diminished secretion of antiangiogenic TSP1, thus enabling angiogenic switch during RCC progression.
Assuntos
Carcinoma de Células Renais/irrigação sanguínea , Neoplasias Renais/irrigação sanguínea , Neovascularização Patológica/patologia , Trombospondina 1/metabolismo , Animais , Western Blotting , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Cálcio/metabolismo , Cálcio/farmacocinética , Carcinoma de Células Renais/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Quelantes/farmacologia , Neovascularização da Córnea/genética , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Neoplasias Renais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Trombospondina 1/genética , TransfecçãoRESUMO
The endoplasmic reticulum (ER) is a central processor that controls the expression of functional proteins, required for the communication of the cell with the external environment. Plasma membranes receptors, ion channels, secreted hormones, catabolic and metabolic enzymes are folded and assembled in the ER. Key metabolic functions are also regulated from the ER. Molecular quality control monitors ER processing activities and co-ordinates these activities with cell and organism demands. Recent understandings of the molecular basis for ER processing activities illuminate the key role of the ER in the development of a variety of diseases. ER derived diseases include specific genetic disorders such as cystic fibrosis or highly prevalent diseases including diabetes and a range of neurodegenerative diseases. ER processing also plays a key role in the development of cancer. This review summarizes the molecular basis for ER processing functions and current avenues in ER-targeted drug development.