RESUMO
The transmembrane emp24 domain-containing (TMED) proteins, also called p24 proteins, are members of a family of sorting receptors present in all representatives of the Eukarya and abundantly present in all subcompartments of the early secretory pathway, namely the endoplasmic reticulum (ER), the Golgi, and the intermediate compartment. Although essential during the bidirectional transport between the ER and the Golgi, there is still a lack of information regarding the TMED's structure across different subfamilies. Besides, although the presence of a TMED homo-oligomerization was suggested previously based on crystallographic contacts observed for the isolated Golgi Dynamics (GOLD) domain, no further analyses of its presence in solution were done. Here, we describe the first high-resolution structure of a TMED1 GOLD representative and its biophysical characterization in solution. The crystal structure showed a dimer formation that is also present in solution in a salt-dependent manner, suggesting that the GOLD domain can form homodimers in solution even in the absence of the TMED1 coiled-coil region. A molecular dynamics description of the dimer stabilization, with a phylogenetic analysis of the residues important for the oligomerization and a model for the orientation towards the lipid membrane, are also presented.
Assuntos
Complexo de Golgi/química , Simulação de Acoplamento Molecular , Filogenia , Proteínas de Transporte Vesicular/química , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Humanos , Domínios Proteicos , Termodinâmica , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Golgi phosphoprotein 3 (GOLPH3) was the first reported oncoprotein of the Golgi apparatus. It was identified as an evolutionarily conserved protein upon its discovery about 20 years ago, but its function remains puzzling in normal and cancer cells. The GOLPH3 gene is part of a group of genes that also includes the GOLPH3L gene. Because cancer has deep roots in multicellular evolution, studying the evolution of the GOLPH3 gene family in non-model species represents an opportunity to identify new model systems that could help better understand the biology behind this group of genes. The main goal of this study is to explore the evolution of the GOLPH3 gene family in birds as a starting point to understand the evolutionary history of this oncoprotein. We identified a repertoire of three GOLPH3 genes in birds. We found duplicated copies of the GOLPH3 gene in all main groups of birds other than paleognaths, and a single copy of the GOLPH3L gene. We suggest there were at least three independent origins for GOLPH3 duplicates. Amino acid divergence estimates show that most of the variation is located in the N-terminal region of the protein. Our transcript abundance estimations show that one paralog is highly and ubiquitously expressed, and the others were variable. Our results are an example of the significance of understanding the evolution of the GOLPH3 gene family, especially for unraveling its structural and functional attributes.
Assuntos
Aves/genética , Evolução Molecular , Complexo de Golgi/genética , Proteínas de Membrana/genética , Proteínas Oncogênicas/genética , Sequência de Aminoácidos/genética , Animais , Carcinogênese/genética , Duplicação Gênica , Humanos , Neoplasias/genética , Fosfoproteínas/genética , Alinhamento de SequênciaRESUMO
Protein trafficking is altered when normal cells acquire a tumor phenotype. A key subcellular compartment in regulating protein trafficking is the Golgi apparatus, but its role in carcinogenesis is still not well defined. Golgi phosphoprotein 3 (GOLPH3), a peripheral membrane protein mostly localized at the trans-Golgi network, is overexpressed in several tumor types including glioblastoma multiforme (GBM), the most lethal primary brain tumor. Moreover, GOLPH3 is currently considered an oncoprotein, however its precise function in GBM is not fully understood. Here, we analyzed in T98G cells of GBM, which express high levels of epidermal growth factor receptor (EGFR), the effect of stable RNAi-mediated knockdown of GOLPH3. We found that silencing GOLPH3 caused a significant reduction in the proliferation of T98G cells and an unexpected increase in total EGFR levels, even at the cell surface, which was however less prone to ligand-induced autophosphorylation. Furthermore, silencing GOLPH3 decreased EGFR sialylation and fucosylation, which correlated with delayed ligand-induced EGFR downregulation and its accumulation at endo-lysosomal compartments. Finally, we found that EGF failed at promoting EGFR ubiquitylation when the levels of GOLPH3 were reduced. Altogether, our results show that GOLPH3 in T98G cells regulates the endocytic trafficking and activation of EGFR likely by affecting its extent of glycosylation and ubiquitylation.
Assuntos
Carcinogênese/genética , Glioblastoma/genética , Proteínas de Membrana/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/patologia , Glicosilação , Complexo de Golgi/genética , Humanos , Proteínas de Membrana/antagonistas & inibidores , Transporte Proteico/genética , Ubiquitinação/genética , Rede trans-Golgi/genéticaRESUMO
The cellular cytoskeleton is involved with multiple biological processes and is tightly regulated by multiple proteins and effectors. Among these, the RhoGTPases family is one of the most important players. RhoGTPAses are, in turn, regulated by many other elements. In the past decade, one of those regulators, the RhoGAP Rho GTPase Activating Protein 21 (ARHGAP21), has been overlooked, despite being implied as having an important role on many of those processes. In this paper, we aimed to review the available literature regarding ARHGAP21 to highlight its importance and the mechanisms of action that have been found so far for this still unknown protein involved with cell adhesion, migration, Golgi regulation, cell trafficking, and even insulin secretion.
Assuntos
Citoesqueleto/genética , Proteínas Ativadoras de GTPase/genética , Complexo de Golgi/genética , Proteínas rho de Ligação ao GTP/genética , Adesão Celular/genética , Movimento Celular/genética , Citoesqueleto/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Secreção de Insulina/genética , Transporte Proteico/genéticaRESUMO
Many secretory cells increase the synthesis and secretion of cargo proteins in response to specific stimuli. How cells couple increased cargo load with a coordinate rise in secretory capacity to ensure efficient transport is not well understood. We used thyroid cells stimulated with thyrotropin (TSH) to demonstrate a coordinate increase in the production of thyroid-specific cargo proteins and ER-Golgi transport factors, and a parallel expansion of the Golgi complex. TSH also increased expression of the CREB3L1 transcription factor, which alone caused amplified transport factor levels and Golgi enlargement. Furthermore, CREB3L1 potentiated the TSH-induced increase in Golgi volume. A dominant-negative CREB3L1 construct hampered the ability of TSH to induce Golgi expansion, implying that this transcription factor contributes to Golgi expansion. Our findings support a model in which CREB3L1 acts as a downstream effector of TSH to regulate the expression of cargo proteins, and simultaneously increases the synthesis of transport factors and the expansion of the Golgi to synchronize the rise in cargo load with the amplified capacity of the secretory pathway.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Complexo de Golgi/genética , Proteínas do Tecido Nervoso/genética , Glândula Tireoide/metabolismo , Tireotropina/genética , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/genética , Complexo de Golgi/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Via Secretória/genética , Tireotropina/metabolismoRESUMO
The cell wall is a complex extracellular matrix composed primarily of polysaccharides. Noncellulosic polysaccharides, glycoproteins and proteoglycans are synthesized in the Golgi apparatus by glycosyltransferases (GTs), which use nucleotide sugars as donors to glycosylate nascent glycan and glycoprotein acceptors that are subsequently exported to the extracellular space. Many nucleotide sugars are synthesized in the cytosol, leading to a topological issue because the active sites of most GTs are located in the Golgi lumen. Nucleotide sugar transporters (NSTs) overcome this problem by translocating nucleoside diphosphate sugars from the cytosol into the lumen of the organelle. The structures of the cell wall components synthesized in the Golgi are diverse and complex; therefore, transporter activities are necessary so that the nucleotide sugars can provide substrates for the GTs. In this review, we describe the topology of reactions involved in polysaccharide biosynthesis in the Golgi and focus on the roles of NSTs as well as their impacts on cell wall structure when they are altered.
Assuntos
Parede Celular/genética , Células Vegetais/metabolismo , Polissacarídeos/biossíntese , Açúcares/metabolismo , Transporte Biológico/genética , Parede Celular/química , Parede Celular/metabolismo , Glicosilação , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Proteínas de Transporte de Monossacarídeos , Nucleotídeos/química , Nucleotídeos/metabolismo , Polissacarídeos/genéticaRESUMO
BACKGROUND: Nucleotide sugar transporters (NSTs) play an essential role in translocating nucleotide sugars into the lumen of the endoplasmic reticulum and Golgi apparatus to be used as substrates in glycosylation reactions. This intracellular transport is an essential step in the biosynthesis of glycoconjugates. RESULTS: We have identified a family of 11 putative NSTs in Trypanosoma cruzi, the etiological agent of Chagas' disease. A UDP-N-acetylglucosamine transporter, TcNST1, was identified by a yeast complementation approach. Based on a phylogenetic analysis four candidate genes were selected and used for complementation assays in a Kluyveromyces lactis mutant strain. The transporter is likely expressed in all stages of the parasite life cycle and during differentiation of epimastigotes to infective metacyclics. Immunofluorescence analyses of a GFP-TcNST1 fusion protein indicate that the transporter is localized to the Golgi apparatus. As many NSTs are multisubstrate transporters, we also tested the capacity of TcNST1 to transport GDP-Man. CONCLUSIONS: We have identified a UDP-N-acetylglucosamine transporter in T. cruzi, which is specifically localized to the Golgi apparatus and seems to be expressed, at the mRNA level, throughout the parasite life cycle. Functional studies of TcNST1 will be important to unravel the role of NSTs and specific glycoconjugates in T. cruzi survival and infectivity.
Assuntos
Complexo de Golgi/enzimologia , Proteínas de Membrana Transportadoras/genética , Trypanosoma cruzi/enzimologia , Perfilação da Expressão Gênica , Teste de Complementação Genética , Complexo de Golgi/genética , Kluyveromyces/genética , Kluyveromyces/metabolismo , Estágios do Ciclo de Vida , Proteínas de Membrana Transportadoras/metabolismo , Especificidade por SubstratoRESUMO
The acquisition of a migratory phenotype is central in processes as diverse as embryo differentiation and tumor metastasis. An early event in this phenomenon is the generation of a nucleus-centrosome-Golgi back-to-front axis. AKAP350 (also known as AKAP9) is a Golgi and centrosome scaffold protein that is involved in microtubule nucleation. AKAP350 interacts with CIP4 (also known as TRIP10), a cdc42 effector that regulates actin dynamics. The present study aimed to characterize the participation of centrosomal AKAP350 in the acquisition of migratory polarity, and the involvement of CIP4 in the pathway. The decrease in total or in centrosomal AKAP350 led to decreased formation of the nucleus-centrosome-Golgi axis and defective cell migration. CIP4 localized at the centrosome, which was enhanced in migratory cells, but inhibited in cells with decreased centrosomal AKAP350. A decrease in the CIP4 expression or inhibition of the CIP4-AKAP350 interaction also led to defective cell polarization. Centrosome positioning, but not nuclear movement, was affected by loss of CIP4 or AKAP350 function. Our results support a model in which AKAP350 recruits CIP4 to the centrosome, providing a centrosomal scaffold to integrate microtubule and actin dynamics, thus enabling centrosome polarization and ensuring cell migration directionality.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Centrossomo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Complexo de Golgi/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Animais , Proteínas do Citoesqueleto/genética , Cães , Complexo de Golgi/genética , Células Hep G2 , Humanos , Células Madin Darby de Rim Canino , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Antígenos de Histocompatibilidade MenorRESUMO
ST3Gal-II, a type II transmembrane protein, is the main mammalian sialyltransferase responsible for GD1a and GT1b ganglioside biosynthesis in brain. It contains two putative N-glycosylation sites (Asn(92) and Asn(211)). Whereas Asn(92) is only conserved in mammalian species, Asn(211) is highly conserved in mammals, birds and fish. The present study explores the occupancy and relevance for intracellular trafficking and enzyme activity of these potential N-glycosylations in human ST3Gal-II. We found that ST3Gal-II distributes along the Golgi complex, mainly in proximal compartments. By pharmacological, biochemical and site-directed mutagenesis, we observed that ST3Gal-II is mostly N-glycosylated at Asn(211) and that this co-translational modification is critical for its exit from the endoplasmic reticulum and proper Golgi localization. The individual N-glycosylation sites had different effects on ST3Gal-II enzymatic activity. Whereas the N-glycan at position Asn(211) seems to negatively influence the activity of the enzyme using both glycolipid and glycoprotein as acceptor substrates, the single N-glycan mutant at Asn(92) had only a moderate effect. Lastly, we demonstrated that the N-terminal ST3Gal-II domain containing the cytosolic, transmembrane and stem region (amino acids 1-51) is able to drive a protein reporter out of the endoplasmic reticulum and to retain it in the Golgi complex. This suggests that the C-terminal domain of ST3Gal-II depends on N-glycosylation to attain an optimum conformation for proper exit from the endoplasmic reticulum, but it does not represent an absolute requirement for Golgi complex retention of the enzyme.
Assuntos
Retículo Endoplasmático/enzimologia , Complexo de Golgi/enzimologia , Sialiltransferases/metabolismo , Animais , Asparagina/genética , Asparagina/metabolismo , Células CHO , Cricetinae , Cricetulus , Retículo Endoplasmático/genética , Evolução Molecular , Glicosilação , Complexo de Golgi/genética , Humanos , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Sialiltransferases/genética , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
The Golgi apparatus is thought to play a role in calcium homeostasis in plant cells. However, the calcium dynamics in this organelle is unknown in plants. To monitor the [Ca2+]Golgiin vivo, we obtained and analyzed Arabidopsis thaliana plants that express aequorin in the Golgi. Our results show that free [Ca2+] levels in the Golgi are higher than in the cytosol (0.70 µM vs. 0.05 µM, respectively). Stimuli such as cold shock, mechanical stimulation and hyperosmotic stress, led to a transient increase in cytosolic calcium; however, no instant change in the [Ca2+]Golgi concentration was detected. Nevertheless, a delayed increase in the [Ca2+]Golgi up to 2-3 µM was observed. Cyclopiazonic acid and thapsigargin inhibited the stimuli-induced [Ca2+]Golgi increase, suggesting that [Ca2+]Golgi levels are dependent upon the activity of Ca2+-ATPases. Treatment of these plants with the synthetic auxin analog, 2,4-dichlorophenoxy acetic acid (2,4-D), produced a slow decrease of free calcium in the organelle. Our results indicate that the plant Golgi apparatus is not involved in the generation of cytosolic calcium transients and exhibits its own dynamics modulated in part by the activity of Ca2+ pumps and hormones.