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1.
Proc Natl Acad Sci U S A ; 117(18): 9884-9895, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32321832

RESUMO

The factors and mechanisms involved in vacuolar transport in plants, and in particular those directing vesicles to their target endomembrane compartment, remain largely unknown. To identify components of the vacuolar trafficking machinery, we searched for Arabidopsis modified transport to the vacuole (mtv) mutants that abnormally secrete the synthetic vacuolar cargo VAC2. We report here on the identification of 17 mtv mutations, corresponding to mutant alleles of MTV2/VSR4, MTV3/PTEN2A MTV7/EREL1, MTV8/ARFC1, MTV9/PUF2, MTV10/VPS3, MTV11/VPS15, MTV12/GRV2, MTV14/GFS10, MTV15/BET11, MTV16/VPS51, MTV17/VPS54, and MTV18/VSR1 Eight of the MTV proteins localize at the interface between the trans-Golgi network (TGN) and the multivesicular bodies (MVBs), supporting that the trafficking step between these compartments is essential for segregating vacuolar proteins from those destined for secretion. Importantly, the GARP tethering complex subunits MTV16/VPS51 and MTV17/VPS54 were found at endoplasmic reticulum (ER)- and microtubule-associated compartments (EMACs). Moreover, MTV16/VPS51 interacts with the motor domain of kinesins, suggesting that, in addition to tethering vesicles, the GARP complex may regulate the motors that transport them. Our findings unveil a previously uncharacterized compartment of the plant vacuolar trafficking pathway and support a role for microtubules and kinesins in GARP-dependent transport of soluble vacuolar cargo in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Transporte Proteico/genética , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/genética , Alelos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Vesículas Citoplasmáticas/genética , Vesículas Citoplasmáticas/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Cinesina/genética , Cinesina/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Corpos Multivesiculares/genética , Corpos Multivesiculares/metabolismo , Mutação , Vacúolos/genética , Proteínas de Transporte Vesicular/metabolismo
2.
Life Sci ; 253: 117700, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32335164

RESUMO

AIMS: Although previous studies elaborated that selective autophagy was involved in quality control of some organelles, including nucleus, mitochondria, the endoplasmic reticulum and peroxisomes, it remained unclear whether the selective autophagy of the Golgi apparatus (Golgiphagy) existed or not. MAIN METHODS: In this study, H9c2 cells, HUVECs, HA-VSMCs and HEK293T cells were treated with autophagy inducers, Golgi stress inducers and cardiomyocytes hypertrophy stimulators. The Golgiphagy was evaluated by analysing the co-localization of Golgi markers and LC3B. Furthermore, the transmission electron microscope was used to observe the occurrence of Golgiphagy. The co-immunoprecipitation assay was used to evaluate the interaction of GOLPH3 and LC3B. KEY FINDINGS: Results showed that starvation promoted the co-localization of both GM130-positive and TGN46-positive Golgi fragments with LC3B-positive autophagosomes in H9c2 cells, HUVECs, HA-VSMCs and HEK293T cells. Transmission electron microscopy images showed that Golgi apparatus was sequestered into the autophagosomes in the starvation group. Moreover, Golgi stress inducers also facilitated the co-localization of Golgi markers and LC3B in H9c2 cells, HUVECs, HA-VSMCs and HEK293T cells. Furthermore, cardiomyocyte hypertrophy stimulators also triggered the appearance of Golgiphagy in H9c2 cells. Importantly, the co-immunoprecipitation assay indicated endogenous GOLPH3 interacted with LC3B in H9c2 cells, HUVECs, HA-VSMCs. However, knocking down GOLPH3 inhibited the Golgiphagy. SIGNIFICANCE: This study unveiled a new selective autophagy of the Golgi apparatus (Golgiphagy). In addition, GOLPH3 might act as a novel cargo receptor to regulate Golgiphagy. Maintaining homeostasis of the Golgi apparatus via GOLPH3-mediated autophagy was indispensable for cell survival.


Assuntos
Autofagia/fisiologia , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Microscopia Eletrônica de Transmissão , Miócitos Cardíacos/metabolismo , Ratos
3.
Nature ; 580(7804): 530-535, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32322062

RESUMO

Cancer cells increase lipogenesis for their proliferation and the activation of sterol regulatory element-binding proteins (SREBPs) has a central role in this process. SREBPs are inhibited by a complex composed of INSIG proteins, SREBP cleavage-activating protein (SCAP) and sterols in the endoplasmic reticulum. Regulation of the interaction between INSIG proteins and SCAP by sterol levels is critical for the dissociation of the SCAP-SREBP complex from the endoplasmic reticulum and the activation of SREBPs1,2. However, whether this protein interaction is regulated by a mechanism other than the abundance of sterol-and in particular, whether oncogenic signalling has a role-is unclear. Here we show that activated AKT in human hepatocellular carcinoma (HCC) cells phosphorylates cytosolic phosphoenolpyruvate carboxykinase 1 (PCK1), the rate-limiting enzyme in gluconeogenesis, at Ser90. Phosphorylated PCK1 translocates to the endoplasmic reticulum, where it uses GTP as a phosphate donor to phosphorylate INSIG1 at Ser207 and INSIG2 at Ser151. This phosphorylation reduces the binding of sterols to INSIG1 and INSIG2 and disrupts the interaction between INSIG proteins and SCAP, leading to the translocation of the SCAP-SREBP complex to the Golgi apparatus, the activation of SREBP proteins (SREBP1 or SREBP2) and the transcription of downstream lipogenesis-related genes, proliferation of tumour cells, and tumorigenesis in mice. In addition, phosphorylation of PCK1 at Ser90, INSIG1 at Ser207 and INSIG2 at Ser151 is not only positively correlated with the nuclear accumulation of SREBP1 in samples from patients with HCC, but also associated with poor HCC prognosis. Our findings highlight the importance of the protein kinase activity of PCK1 in the activation of SREBPs, lipogenesis and the development of HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Gluconeogênese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipogênese , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Animais , Carcinogênese , Carcinoma Hepatocelular/patologia , Proliferação de Células , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Neoplasias Hepáticas/patologia , Masculino , Proteínas de Membrana/química , Camundongos , Camundongos Nus , Oxisteróis/metabolismo , Fosforilação , Prognóstico , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
4.
Nat Commun ; 11(1): 1754, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32273498

RESUMO

Alternative autophagy is an autophagy-related protein 5 (Atg5)-independent type of macroautophagy. Unc51-like kinase 1 (Ulk1) is an essential initiator not only for Atg5-dependent canonical autophagy but also for alternative autophagy. However, the mechanism as to how Ulk1 differentially regulates both types of autophagy has remained unclear. In this study, we identify a phosphorylation site of Ulk1 at Ser746, which is phosphorylated during genotoxic stress-induced alternative autophagy. Phospho-Ulk1746 localizes exclusively on the Golgi and is required for alternative autophagy, but not canonical autophagy. We also identify receptor-interacting protein kinase 3 (RIPK3) as the kinase responsible for genotoxic stress-induced Ulk1746 phosphorylation, because RIPK3 interacts with and phosphorylates Ulk1 at Ser746, and loss of RIPK3 abolishes Ulk1746 phosphorylation. These findings indicate that RIPK3-dependent Ulk1746 phosphorylation on the Golgi plays a pivotal role in genotoxic stress-induced alternative autophagy.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/fisiologia , Dano ao DNA , Complexo de Golgi/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Animais , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Sítios de Ligação/genética , Células Cultivadas , Embrião de Mamíferos/citologia , Etoposídeo/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos Knockout , Microscopia Confocal , Fosforilação , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Homologia de Sequência de Aminoácidos , Serina/genética
5.
Parasitol Res ; 119(5): 1629-1640, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32219551

RESUMO

Rab proteins constitute the largest group of small GTPases and act as molecular switches in a wide variety of cellular processes, including proliferation, cytoskeleton assembly, and membrane trafficking in all eukaryotic cells. Rab21 has been reported in several eukaryotic cells, and our results suggest that in Entamoeba histolytica, Rab21 is involved in the vesicular traffic associated with the Golgi apparatus, where its function appears to be important to maintain the structure of this organelle. In addition, proteins such as Rab1A and Sec24, identified in this work associated with EhRab21, participate in the traffic of COPII vesicles from the endoplasmic reticulum to the Golgi apparatus and are necessary to maintain the latter's structure in human cells. In addition, EhRab21 probably affects the lysosome biogenesis, as indicated by an increase in the number of lysosomes as a result of the increase in EhRab21 activity. The participation of EhRab21 in the pathogenesis of amebiasis was verified on the amoebic liver abscess formation model using hamsters (Mesocricetus auratus), in which the overexpression of EhRab21Q64L (positive dominant mutant protein) decreased the number of liver abscesses formed.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Entamoeba histolytica/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Amebíase/patologia , Animais , Cricetinae , Retículo Endoplasmático/metabolismo , Humanos , Abscesso Hepático Amebiano/patologia , Lisossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo
6.
Nat Commun ; 11(1): 1304, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161259

RESUMO

The integrated stress response (ISR) converges on eIF2α phosphorylation to regulate protein synthesis. ISR is activated by several stress conditions, including endoplasmic reticulum (ER) stress, executed by protein kinase R-like endoplasmic reticulum kinase (PERK). We report that ER stress combined with ISR inhibition causes an impaired maturation of several tyrosine kinase receptors (RTKs), consistent with a partial block of their trafficking from the ER to the Golgi. Other proteins mature or are secreted normally, indicating selective retention in the ER (sERr). sERr is relieved upon protein synthesis attenuation and is accompanied by the generation of large mixed disulfide bonded complexes, including ERp44. sERr was pharmacologically recapitulated by combining the HIV-protease inhibitor nelfinavir with ISRIB, an experimental drug that inhibits ISR. Nelfinavir/ISRIB combination is highly effective to inhibit the growth of RTK-addicted cell lines and hepatocellular (HCC) cells in vitro and in vivo. Thus, pharmacological sERr can be utilized as a modality for cancer treatment.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , eIF-2 Quinase/metabolismo , Acetamidas/farmacologia , Acetamidas/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Sistemas CRISPR-Cas/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Cicloexilaminas/farmacologia , Cicloexilaminas/uso terapêutico , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Técnicas de Inativação de Genes , Complexo de Golgi/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Nelfinavir/farmacologia , Nelfinavir/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/genética
7.
Nat Commun ; 11(1): 1418, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184397

RESUMO

The Golgi apparatus plays a central role in the intracellular transport of macromolecules. However, molecular mechanisms of Golgi-mediated lipid transport remain poorly understood. Here, we show that genetic inactivation of the Golgi-resident protein GRASP55 in mice reduces whole-body fat mass via impaired intestinal fat absorption and evokes resistance to high-fat diet induced body weight gain. Mechanistic analyses reveal that GRASP55 participates in the Golgi-mediated lipid droplet (LD) targeting of some LD-associated lipases, such as ATGL and MGL, which is required for sustained lipid supply for chylomicron assembly and secretion. Consequently, GRASP55 deficiency leads to reduced chylomicron secretion and abnormally large LD formation in intestinal epithelial cells upon exogenous lipid challenge. Notably, deletion of dGrasp in Drosophila causes similar defects of lipid accumulation in the midgut. These results highlight the importance of the Golgi complex in cellular lipid regulation, which is evolutionary conserved, and uncover potential therapeutic targets for obesity-associated diseases.


Assuntos
Gorduras/metabolismo , Proteínas da Matriz do Complexo de Golgi/genética , Obesidade/genética , Obesidade/prevenção & controle , Animais , Transporte Biológico , Dieta Hiperlipídica , Drosophila , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Obesidade/fisiopatologia , Ganho de Peso
8.
Nat Commun ; 11(1): 1128, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111832

RESUMO

The sterol-regulatory element binding proteins (SREBP) are central transcriptional regulators of lipid metabolism. Using haploid genetic screens we identify the SREBP Regulating Gene (SPRING/C12ORF49) as a determinant of the SREBP pathway. SPRING is a glycosylated Golgi-resident membrane protein and its ablation in Hap1 cells, Hepa1-6 hepatoma cells, and primary murine hepatocytes reduces SREBP signaling. In mice, Spring deletion is embryonic lethal yet silencing of hepatic Spring expression also attenuates the SREBP response. Mechanistically, attenuated SREBP signaling in SPRINGKO cells results from reduced SREBP cleavage-activating protein (SCAP) and its mislocalization to the Golgi irrespective of the cellular sterol status. Consistent with limited functional SCAP in SPRINGKO cells, reintroducing SCAP restores SREBP-dependent signaling and function. Moreover, in line with the role of SREBP in tumor growth, a wide range of tumor cell lines display dependency on SPRING expression. In conclusion, we identify SPRING as a previously unrecognized modulator of SREBP signaling.


Assuntos
Colesterol/metabolismo , Glicoproteínas de Membrana/metabolismo , Transdução de Sinais , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Animais , Linhagem Celular , Desenvolvimento Embrionário/genética , Retículo Endoplasmático/metabolismo , Expressão Gênica , Complexo de Golgi/metabolismo , Haploidia , Hepatócitos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Elemento Regulador de Esterol/genética
9.
Nat Chem Biol ; 16(3): 327-336, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32080624

RESUMO

The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.


Assuntos
Benzamidas/metabolismo , Proteínas Qa-SNARE/metabolismo , Tiofenos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Benzamidas/farmacologia , Transporte Biológico , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Transporte Proteico , Ricina/metabolismo , Toxina Shiga/metabolismo , Toxinas Shiga/metabolismo , Tiofenos/farmacologia , Proteínas de Transporte Vesicular/fisiologia
10.
Nat Rev Mol Cell Biol ; 21(3): 151-166, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32034394

RESUMO

During division, eukaryotic cells undergo a dramatic, complex and coordinated remodelling of their cytoskeleton and membranes. For cell division to occur, chromosomes must be segregated and new cellular structures, such as the spindle apparatus, must be assembled. Pre-existing organelles, such as the nuclear envelope, endoplasmic reticulum and Golgi apparatus, must be disassembled or remodelled, distributed and reformed. Smaller organelles such as mitochondria as well as cytoplasmic content must also be properly distributed between daughter cells. This mixture of organelles and cytoplasm is bound by a plasma membrane that is itself subject to remodelling as division progresses. The lipids resident in these different membrane compartments play important roles in facilitating the division process. In recent years, we have begun to understand how membrane remodelling is coordinated during division; however, there is still much to learn. In this Review, we discuss recent insights into how these important cellular events are performed and regulated.


Assuntos
Divisão Celular/fisiologia , Membranas/metabolismo , Organelas/fisiologia , Animais , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Células Eucarióticas/citologia , Complexo de Golgi/metabolismo , Humanos , Membranas/fisiologia , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Organelas/metabolismo , Fuso Acromático/metabolismo
11.
Nat Commun ; 11(1): 409, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31964869

RESUMO

The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown. Here we demonstrate that 26S proteasomes are associated with the cytosolic surface of Golgi membranes to facilitate Golgi Apparatus-Related Degradation (GARD) and degradation of GM130 in response to Golgi stress. The degradation of GM130 is dependent on p97/VCP and 26S proteasomes, and required for Golgi dispersal. Finally, we show that perturbation of Golgi homeostasis induces cell death of multiple myeloma in vitro and in vivo, offering a therapeutic strategy for this malignancy. Taken together, this work reveals a mechanism of Golgi-localized proteasomal degradation, providing a functional link between proteostasis control and Golgi architecture, which may be critical in various secretion-related pathologies.


Assuntos
Complexo de Golgi/metabolismo , Ionóforos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteostase/fisiologia , Animais , Apoptose/efeitos dos fármacos , Autoantígenos/metabolismo , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Complexo de Golgi/efeitos dos fármacos , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Ionóforos/farmacologia , Proteínas de Membrana/metabolismo , Camundongos , Monensin/farmacologia , Monensin/uso terapêutico , Mieloma Múltiplo/patologia , Proteólise/efeitos dos fármacos , Proteostase/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Proteína com Valosina/metabolismo
12.
PLoS One ; 15(1): e0227435, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31961879

RESUMO

Wnt/ß-catenin signalling is crucial for maintaining the balance between cell proliferation and differentiation, both during tissue morphogenesis and in tissue maintenance throughout postnatal life. Whereas the signalling activities of the core Wnt/ß-catenin pathway components are understood in great detail, far less is known about the precise role and regulation of the many different modulators of Wnt/ß-catenin signalling that have been identified to date. Here we describe TMEM98, a putative transmembrane protein of unknown function, as an interaction partner and regulator of the GSK3-binding protein FRAT2. We show that TMEM98 reduces FRAT2 protein levels and, accordingly, inhibits the FRAT2-mediated induction of ß-catenin/TCF signalling. We also characterize the intracellular trafficking of TMEM98 in more detail and show that it is recycled between the plasma membrane and the Golgi. Together, our findings not only reveal a new layer of regulation for Wnt/ß-catenin signalling, but also a new biological activity for TMEM98.


Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Via de Sinalização Wnt , Animais , Proteínas de Transporte/genética , Membrana Celular/genética , Complexo de Golgi/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Camundongos , Transporte Proteico , beta Catenina/genética , beta Catenina/metabolismo
13.
Mol Cell Biol ; 40(7)2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-31932478

RESUMO

Epidermal growth factor receptor (EGFR) is a prototype receptor tyrosine kinase and an oncoprotein in many solid tumors. Cell surface display of EGFR is essential for cellular responses to its ligands. While postactivation endocytic trafficking of EGFR has been well elucidated, little is known about mechanisms of basal/preactivation surface display of EGFR. Here, we identify a novel role of the endocytic regulator EHD1 and a potential EHD1 partner, RUSC2, in cell surface display of EGFR. EHD1 and RUSC2 colocalize with EGFR in vesicular/tubular structures and at the Golgi compartment. Inducible EHD1 knockdown reduced the cell surface EGFR expression with accumulation at the Golgi compartment, a phenotype rescued by exogenous EHD1. RUSC2 knockdown phenocopied the EHD1 depletion effects. EHD1 or RUSC2 depletion impaired the EGF-induced cell proliferation, demonstrating that the novel, EHD1- and RUSC2-dependent transport of unstimulated EGFR from the Golgi compartment to the cell surface that we describe is functionally important, with implications for physiologic and oncogenic roles of EGFR and targeted cancer therapies.


Assuntos
Proteínas de Transporte/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Comunicação Celular/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Proliferação de Células/fisiologia , Receptores ErbB/metabolismo , Humanos , Camundongos , Transporte Proteico/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Transporte Vesicular/genética
14.
Nucleic Acids Res ; 48(3): 1372-1391, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31840180

RESUMO

Release of phosphorothioate antisense oligonucleotides (PS-ASOs) from late endosomes (LEs) is a rate-limiting step and a poorly defined process for productive intracellular ASO drug delivery. Here, we examined the role of Golgi-endosome transport, specifically M6PR shuttling mediated by GCC2, in PS-ASO trafficking and activity. We found that reduction in cellular levels of GCC2 or M6PR impaired PS-ASO release from endosomes and decreased PS-ASO activity in human cells. GCC2 relocated to LEs upon PS-ASO treatment, and M6PR also co-localized with PS-ASOs in LEs or on LE membranes. These proteins act through the same pathway to influence PS-ASO activity, with GCC2 action preceding that of M6PR. Our data indicate that M6PR binds PS-ASOs and facilitates their vesicular escape. The co-localization of M6PR and of GCC2 with ASOs is influenced by the PS modifications, which have been shown to enhance the affinity of ASOs for proteins, suggesting that localization of these proteins to LEs is mediated by ASO-protein interactions. Reduction of M6PR levels also decreased PS-ASO activity in mouse cells and in livers of mice treated subcutaneously with PS-ASO, indicating a conserved mechanism. Together, these results demonstrate that the transport machinery between LE and Golgi facilitates PS-ASO release.


Assuntos
Endossomos/genética , Proteínas da Matriz do Complexo de Golgi/genética , Oligonucleotídeos Antissenso/genética , Receptor IGF Tipo 2/genética , Animais , Endocitose/genética , Endossomos/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Células HeLa , Humanos , Camundongos , Oligonucleotídeos Fosforotioatos/genética , Transporte Proteico/genética , Receptor IGF Tipo 2/metabolismo
15.
Cell Mol Life Sci ; 77(3): 511-529, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31218450

RESUMO

The sperm acrosome is a lysosome-related organelle that develops using membrane trafficking from the Golgi apparatus as well as the endolysosomal compartment. How vesicular trafficking is regulated in spermatids to form the acrosome remains to be elucidated. VPS13B, a RAB6-interactor, was recently shown involved in endomembrane trafficking. Here, we report the generation of the first Vps13b-knockout mouse model and show that male mutant mice are infertile due to oligoasthenoteratozoospermia. This phenotype was explained by a failure of Vps13b deficient spermatids to form an acrosome. In wild-type spermatids, immunostaining of Vps13b and Rab6 revealed that they transiently locate to the acrosomal inner membrane. Spermatids lacking Vps13b did not present with the Golgi structure that characterizes wild-type spermatids and showed abnormal targeting of PNA- and Rab6-positive Golgi-derived vesicles to Eea1- and Lamp2-positive structures. Altogether, our results uncover a function of Vps13b in the regulation of the vesicular transport between Golgi apparatus, acrosome, and endolysosome.


Assuntos
Acrossomo/metabolismo , Transporte Biológico/fisiologia , Complexo de Golgi/metabolismo , Espermatogênese/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Transporte Proteico/fisiologia , Espermátides/metabolismo , Espermatozoides/metabolismo
16.
Biochim Biophys Acta Mol Cell Res ; 1867(2): 118620, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31812495

RESUMO

Protein S-palmitoylation, the covalent lipid modification of the side chain of Cys residues with the 16­carbon fatty acid palmitate, is the most common acylation, and it enhances the membrane stability of ion channels. This post-translational modification (PTM) determines a functional mechanism of ion channel life cycle from maturation and membrane trafficking to localization. Especially, neurodevelopment is regulated by balancing the level of synaptic protein palmitoylation/depalmitoylation. Recently, we revealed the pathological role of the transient receptor potential canonical type 5 (TRPC5) channel in striatal neuronal loss during Huntington's disease (HD), which is abnormally activated by oxidative stress. Here, we report a mechanism of TRPC5 palmitoylation at a conserved cysteine residue, that is critical for intrinsic channel activity. Furthermore, we identified the therapeutic effect of TRPC5 depalmitoylation by enhancing the TRPC5 membrane instability on HD striatal cells in order to lower TRPC5 toxicity. Collectively, these findings suggest that controlling S-palmitoylation of the TRPC5 channel as a potential risk factor can modulate TRPC5 channel expression and activity, providing new insights into a therapeutic strategy for neurodegenerative diseases.


Assuntos
Neurônios/metabolismo , Estresse Oxidativo , Canais de Cátion TRPC/metabolismo , Motivos de Aminoácidos , Animais , Antineoplásicos Alquilantes/toxicidade , Apoptose/efeitos dos fármacos , Carmustina/toxicidade , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Proteína Huntingtina/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Lipoilação/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Estresse Oxidativo/efeitos dos fármacos , Palmitatos/farmacologia , Estabilidade Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Canais de Cátion TRPC/química , Canais de Cátion TRPC/genética
17.
Mol Cell ; 77(3): 618-632.e5, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-31806350

RESUMO

TMEM39A, encoding an ER-localized transmembrane protein, is a susceptibility locus for multiple autoimmune diseases. The molecular function of TMEM39A remains completely unknown. Here we demonstrated that TMEM39A, also called SUSR2, modulates autophagy activity by regulating the spatial distribution and levels of PtdIns(4)P. Depletion of SUSR2 elevates late endosomal/lysosomal PtdIns(4)P levels, facilitating recruitment of the HOPS complex to promote assembly of the SNARE complex for autophagosome maturation. SUSR2 knockdown also increases the degradative capability of lysosomes. Mechanistically, SUSR2 interacts with the ER-localized PtdIns(4)P phosphatase SAC1 and also the COPII SEC23/SEC24 subunits to promote the ER-to-Golgi transport of SAC1. Retention of SAC1 on the ER in SUSR2 knockdown cells increases the level of PtdIns(3)P produced by the VPS34 complex, promoting autophagosome formation. Our study reveals that TMEM39A/SUSR2 acts as an adaptor protein for efficient export of SAC1 from the ER and provides insights into the pathogenesis of diseases associated with TMEM39A mutations.


Assuntos
Autofagia/fisiologia , Proteínas de Membrana/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Proteínas de Membrana/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/fisiologia , Transporte Proteico/fisiologia
18.
Plant Mol Biol ; 102(1-2): 19-38, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31786704

RESUMO

KEY MESSAGE: Plant possesses particular Golgi-resident cyclophilin 21 proteins (CYP21s) and the catalytic isomerase activities have a negative effect on ABA signalling gene expression during early seedling development. Cyclophilins (CYPs) are essential for diverse cellular process, as these catalyse a rate-limiting step in protein folding. Although Golgi proteomics in Arabidopsis thaliana suggests the existence of several CYPs in the Golgi apparatus, only one putative Golgi-resident CYP protein has been reported in rice (Oryza sativa L.; OsCYP21-4). Here, we identified the Golgi-resident CYP21 family genes and analysed their molecular characteristics in Arabidopsis and rice. The CYP family genes (CYP21-1, CYP21-2, CYP21-3, and CYP21-4) are plant-specific, and their appearance and copy numbers differ among plant species. CYP21-1 and CYP21-4 are common to all angiosperms, whereas CYP21-2 and CYP21-3 evolved in the Malvidae subclass. Furthermore, all CYP21 proteins localize to cis-Golgi, trans-Golgi or both cis- and trans-Golgi membranes in plant cells. Additionally, based on the structure, enzymatic function, and topological orientation in Golgi membranes, CYP21 proteins are divided into two groups. Genetic analysis revealed that Group I proteins (CYP21-1 and CYP21-2) exhibit peptidyl prolyl cis-trans isomerase (PPIase) activity and regulate seed germination and seedling growth and development by affecting the expression levels of abscisic acid signalling genes. Thus, we identified the Golgi-resident CYPs and demonstrated that their PPIase activities are required for early seedling growth and development in higher plants.


Assuntos
Ciclofilinas/genética , Ciclofilinas/metabolismo , Complexo de Golgi/metabolismo , Desenvolvimento Vegetal , Plântula/metabolismo , Transdução de Sinais , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclofilinas/classificação , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Oryza/genética , Oryza/metabolismo , Peptidilprolil Isomerase/metabolismo , Filogenia , Desenvolvimento Vegetal/genética , Desenvolvimento Vegetal/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteômica
19.
Exp Cell Res ; 386(1): 111709, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31704058

RESUMO

Regulation of phosphatidylinositol phosphates plays a crucial role in signal transduction, membrane trafficking or autophagy. Members of the myotubularin family of lipid phosphatases contribute to phosphoinositide metabolism by counteracting the activity of phosphoinositide kinases. The mechanisms determining their subcellular localization and targeting to specific membrane compartments are still poorly understood. We show here that the inactive phosphatase MTMR9 localizes to the intermediate compartment and to the Golgi apparatus and is able to recruit its active phosphatase partners MTMR6 and MTMR8 to these locations. Furthermore, MTMR8 and MTMR9 co-localize with the small GTPase RAB1A and regulate its localization. Loss of MTMR9 expression compromises the integrity of the Golgi apparatus and results in altered distribution of RAB1A and actin nucleation-promoting factor WHAMM. Loss or overexpression of MTMR9 leads to decreased rate of protein secretion. We demonstrate that secretion of physiologically relevant cargo exemplified by the WNT3A protein is affected after perturbation of MTMR9 levels.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Exocitose , Células HEK293 , Células HeLa , Humanos , Transporte Proteico , Proteínas Tirosina Fosfatases não Receptoras/genética , Via de Sinalização Wnt , Proteína Wnt3A/metabolismo , Proteínas rab1 de Ligação ao GTP/metabolismo
20.
Biochim Biophys Acta Mol Cell Res ; 1867(4): 118627, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31863790

RESUMO

COPII vesicles mediate anterograde ER-Golgi traffic of newly synthesized proteins in nutrient rich conditions. An accumulating body of results indicates that the secretory COPII vesicles can be shifted to the roles in autophagosome formation and selective ER-phagy (autophagy of ER), depending on their specific subunits, in response to environmental stresses. In this mini-review, we summarize and discuss the multifaceted roles of COPII vesicles in autophagy and the underlying molecular mechanisms.


Assuntos
Autofagia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos
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