RESUMEN

The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control crucial biological processes. HS chains are synthesized in a non-template driven process mainly in the Golgi apparatus, involving a large number of enzymes capable of subtly modifying its substitution pattern, hence, its interactions and biological effects. Changes in the localization of HS-modifying enzymes throughout the Golgi were found to correlate with changes in the structure of HS, rather than protein expression levels. Following BFA treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Shortly after heparin treatment, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS sulfation. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings demonstrate that knowledge of the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking is a critical prerequisite for the complete delineation of HS biosynthesis.

Asunto(s)

Vesículas Cubiertas por Proteínas de Revestimiento/enzimología , Retículo Endoplásmico/enzimología , Aparato de Golgi/enzimología , Heparitina Sulfato/biosíntesis , Transporte Biológico/efectos de los fármacos , Brefeldino A/farmacología , Vesículas Cubiertas por Proteínas de Revestimiento/genética , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Retículo Endoplásmico/química , Retículo Endoplásmico/efectos de los fármacos , Regulación de la Expresión Génica , Aparato de Golgi/química , Aparato de Golgi/efectos de los fármacos , Heparina/farmacología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Plásmidos/química , Plásmidos/metabolismo , Cultivo Primario de Células , Transfección , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo

RESUMEN

Extracellular pools of intracellular molecular chaperones are increasingly evident. The peri/epicellular(pec) pool of the endoplasmic reticulum redox chaperone protein disulfide isomerase-A1(PDI) is involved in thrombosis and vascular remodeling, while PDI externalization routes remain elusive. In endothelial cells, vesicular-type PDI secretion involves classical and unconventional pathways, while in platelets PDI exocytosis involves actin cytoskeleton. However, little is known about pecPDI in vascular smooth muscle cells(VSMC). Here, we showed that VSMC display a robust cell-surface(cs) PDI pool, which binds to cs independently of electrostatic forces. However, contrarily to other cells, soluble secreted PDI pool was undetectable in VSMC. Calcium ionophore A23187 and TNFα enhanced VSMC csPDI. Furthermore, VSMC PDI externalization occurred via Golgi-bypass unconventional route, which was independent of cytoskeleton or lysosomes. Secreted PDI was absent in ex vivo wild-type mice aortas but markedly enhanced in PDI-overexpressing mice. Such characterization of VSMC pecPDI reinforces cell-type and context specific routes of PDI externalization.

Asunto(s)

Aparato de Golgi/enzimología , Músculo Liso Vascular/enzimología , Proteína Disulfuro Isomerasas/metabolismo , Animales , Calcimicina/farmacología , Células Cultivadas , Aparato de Golgi/efectos de los fármacos , Ratones , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/enzimología , Conejos , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

UDP-Glc entrance into the endoplasmic reticulum (ER) of eukaryotic cells is a key step in the quality control of glycoprotein folding, a mechanism requiring transfer of a Glc residue from the nucleotide sugar (NS) to glycoprotein folding intermediates by the UDP-Glc:glycoprotein glucosyltransferase (UGGT). According to a bioinformatics search there are only eight genes in the Schizosaccharomyces pombe genome belonging to the three Pfam families to which all known nucleotide-sugar transporters (NSTs) of the secretory pathway belong. The protein products of two of them (hut1+ and yea4+) localize to the ER, those of genes gms1+, vrg4+, pet1+, pet2+ and pet3+ to the Golgi, whereas that of gms2+ has an unknown location. Here we demonstrate that (1) Δhut1 and Δgpt1 (UGGT null) mutants share several phenotypic features; (2) Δhut1 mutants show a 50% reduction in UDP-Glc transport into ER-derived membranes; (3) in vivo UDP-Glc ER entrance occurred in Δhut1Δyea4Δgms2 mutants and in cells in which Δhut1 disruption was combined with that of each of four of the genes encoding Golgi-located proteins. Therefore, disruption of all genes whose products localize to the ER or have an unknown location did not obliterate UDP-Glc ER entrance. We conclude that the hut1+ gene product is involved in UDP-Glc entrance into the ER, but that at least another as yet unknown NST displaying an unconventional sequence operates in the yeast secretory pathway. This conclusion agrees with our previous results showing that UDP-Glc entrance into the yeast ER does not follow the classical NST antiport mechanism.

Asunto(s)

Retículo Endoplásmico/enzimología , Glucosiltransferasas/genética , Glicoproteínas/genética , Proteínas Mutantes/genética , Retículo Endoplásmico/química , Glucosiltransferasas/química , Glicoproteínas/química , Aparato de Golgi/enzimología , Proteínas Mutantes/química , Pliegue de Proteína , Schizosaccharomyces/enzimología

RESUMEN

Extracellular protein disulfide isomerase (PDIA1) pool mediates thrombosis and vascular remodeling, however its externalization mechanisms remain unclear. We performed systematic pharmacological screening of secretory pathways affecting extracellular PDIA1 in endothelial cells (EC). We identified cell-surface (csPDIA1) and secreted non-particulated PDIA1 pools in EC. Such Golgi bypass also occurred for secreted PDIA1 in EC at baseline or after PMA, thrombin or ATP stimulation. Inhibitors of Type I, II and III unconventional routes, secretory lysosomes and recycling endosomes, including syntaxin-12 deletion, did not impair EC PDIA1 externalization. This suggests predominantly Golgi-independent unconventional secretory route(s), which were GRASP55-independent. Also, these data reinforce a vesicular-type traffic for PDIA1. We further showed that PDIA1 traffic is ATP-independent, while actin or tubulin cytoskeletal disruption markedly increased EC PDIA1 secretion. Clathrin inhibition enhanced extracellular soluble PDIA1, suggesting dynamic cycling. Externalized PDIA1 represents <2% of intracellular PDIA1. PDIA1 was robustly secreted by physiological levels of arterial laminar shear in EC and supported alpha 5 integrin thiol oxidation. Such results help clarify signaling and homeostatic mechanisms involved in multiple (patho)physiological extracellular PDIA1 functions.

Asunto(s)

Células Endoteliales de la Vena Umbilical Humana/enzimología , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Fenómenos Biomecánicos , Células Cultivadas , Aparato de Golgi/enzimología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Integrina alfa5/metabolismo , Mecanotransducción Celular , Oxidación-Reducción , Transporte de Proteínas

RESUMEN

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.

Asunto(s)

Aparato de Golgi/enzimología , Proteínas de Transporte de Membrana/genética , Trypanosoma cruzi/enzimología , Perfilación de la Expresión Génica , Prueba de Complementación Genética , Aparato de Golgi/genética , Kluyveromyces/genética , Kluyveromyces/metabolismo , Estadios del Ciclo de Vida , Proteínas de Transporte de Membrana/metabolismo , Especificidad por Sustrato

RESUMEN

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.

Asunto(s)

Retículo Endoplásmico/enzimología , Aparato de Golgi/enzimología , Sialiltransferasas/metabolismo , Animales , Asparagina/genética , Asparagina/metabolismo , Células CHO , Cricetinae , Cricetulus , Retículo Endoplásmico/genética , Evolución Molecular , Glicosilación , Aparato de Golgi/genética , Humanos , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Sialiltransferasas/genética , beta-Galactosida alfa-2,3-Sialiltransferasa

RESUMEN

All life cycle stages of the protozoan parasite Trypanosoma cruzi are enveloped by mucin-like glycoproteins which, despite major changes in their polypeptide cores, are extensively and similarly O-glycosylated. O-Glycan biosynthesis is initiated by the addition of αGlcNAc to Thr in a reaction catalyzed by Golgi UDP-GlcNAc:polypeptide O-α-N-acetyl-d-glucosaminyltransferases (ppαGlcNAcTs), which are encoded by TcOGNT1 and TcOGNT2. We now directly show that TcOGNT2 is associated with the Golgi apparatus of the epimastigote stage and is markedly downregulated in both differentiated metacyclic trypomastigotes (MCTs) and cell culture-derived trypomastigotes (TCTs). The significance of downregulation was examined by forced continued expression of TcOGNT2, which resulted in a substantial increase of TcOGNT2 protein levels but only modestly increased ppαGlcNAcT activity in extracts and altered cell surface glycosylation in TCTs. Constitutive TcOGNT2 overexpression had no discernible effect on proliferating epimastigotes but negatively affected production of both types of trypomastigotes. MCTs differentiated from epimastigotes at a low frequency, though they were apparently normal based on morphological and biochemical criteria. However, these MCTs exhibited an impaired ability to produce amastigotes and TCTs in cell culture monolayers, most likely due to a reduced infection frequency. Remarkably, inhibition of MCT production did not depend on TcOGNT2 catalytic activity, whereas TCT production was inhibited only by active TcOGNT2. These findings indicate that TcOGNT2 downregulation is important for proper differentiation of MCTs and functioning of TCTs and that TcOGNT2 regulates these functions by using both catalytic and noncatalytic mechanisms.

Asunto(s)

Glicoproteínas/genética , Mucinas/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/biosíntesis , Aparato de Golgi/enzimología , Estadios del Ciclo de Vida/genética , Mucinas/genética , Péptidos/genética , Péptidos/metabolismo , Polisacáridos/biosíntesis , Proteínas Protozoarias/genética , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/crecimiento & desarrollo

RESUMEN

BACKGROUND INFORMATION: Heterotrimeric GTP-binding proteins play a key role in cell trafficking regulation. Above all, specific Gßγ subunits have been shown to be a major component of a signal transduction pathway, which also involves phospholipases C (PLC), protein kinases C (PKC) and D (PKD), whose main function is to regulate transport between Golgi and plasma membrane. It was the involvement of PLC which led us to study the role of the other member of this G protein family, the α subunits, in the regulation of membrane fission at the Golgi apparatus. RESULTS: Among constitutive active (QL) variants of different G protein α subunit sub-families, only GαqQL subunits were able to induce Golgi fragmentation, a phenotype that mainly reflects a membrane fission increase at this organelle. This phenotype was not observed with a GαqQL palmitoylation mutant, showing the need for a membrane-bounded subunit. Besides, GαqQL-dependent Golgi fission was blocked by specific PLC and PKC inhibitors, and in the presence of a PKD1-kinase dead variant. In addition, GαqQL was the only α subunit capable of inducing PKD1 phosphorylation. Finally, Vesicular Stomatitis Virus thermosensitive mutant glycoprotein (VSVG tsO45) transport assays have demonstrated that GαqQL acts directly on Golgi membranes to regulate trafficking between this organelle and plasma membrane. CONCLUSIONS: All these results indicate Gαq subunits for the first time as a regulator of PKD-mediated intracellular trafficking between Golgi apparatus and plasma membrane, opening new perspectives in the understanding of internal trafficking regulation by external signals through G protein-coupled receptors.

Asunto(s)

Membrana Celular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Aparato de Golgi/metabolismo , Proteína Quinasa C/metabolismo , Membrana Celular/enzimología , Citoesqueleto/metabolismo , Aparato de Golgi/enzimología , Células HeLa , Humanos , Microtúbulos/metabolismo , Modelos Biológicos , Fosfoinositido Fosfolipasa C/metabolismo , Fosforilación , Proteína Quinasa C/química , Estructura Terciaria de Proteína , Transporte de Proteínas , Red trans-Golgi/metabolismo

RESUMEN

Protein palmitoylation is a post-translational modification that contributes to determining protein localization and function. Palmitoylation has been described in trypanosomatid protozoa, but no zDHHC palmitoyl transferase has been identified in Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. In this study we identify and show the subcellular localization of TcHIP (Tc00.1047053508199.50), a putative T. cruzi zDHHC palmitoyl transferase. Analysis of the deduced protein sequence indicates that it contains ankyrin repeats (Ank and Ank2) and the zDHHC conserved domain, typical of zDHHC palmitoyl transferases. A TcHIP polyclonal antiserum obtained from mice immunized with the purified recombinant protein was used to study the presence and subcellular localization of the native enzyme. In western blots this antiserum recognized a protein of about 95 kDa, consistent with the predicted molecular mass of TcHIP (95.4 kDa), in whole extracts of T. cruzi epimastigotes, metacyclic trypomastigotes and intracellular amastigotes. Immunolocalization by confocal microscopy showed TcHIP labeling at the Golgi complex, co-localizing with the T. cruzi Golgi marker TcRab7-GFP. Transfectant T. cruzi epimastigotes containing a construct encoding TcHIP fused to proteins A and C (TcHIP/AC) were obtained. In western blotting experiments, the TcHIP polyclonal antiserum recognized both native and TcHIP/AC proteins in extracts of the transfectants. Confocal microscopy showed co-localization of native TcHIP with TcHIP/AC. These findings demonstrate the presence of a putative zDHHC palmitoyl transferase (TcHIP) containing ankyrin and zDHHC domains in different developmental forms of T. cruzi, and its association with the Golgi complex.

Asunto(s)

Aciltransferasas/aislamiento & purificación , Aparato de Golgi/enzimología , Trypanosoma cruzi/enzimología , Aciltransferasas/química , Aciltransferasas/genética , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Lipoilación , Microscopía Confocal , Microscopía Fluorescente , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Trypanosoma cruzi/ultraestructura

RESUMEN

AIMS: S-nitrosylation of Cys118 is a redox-based mechanism for Ras activation mediated by nitric oxide (NO) at the plasma membrane. RESULTS: Ras signaling pathway stimulation by 50 and/or 100 µM of S-nitrosoglutathione (GSNO) causes proliferation of HeLa cells. Proliferation was not observed in HeLa cells overexpressing non-nitrosatable H-Ras(C118S). HeLa cells overexpressing H-Ras(wt) containing the spatiotemporal probe green fluorescent protein (GFP) fused to the Ras-binding domain of Raf-1 (GFP-RBD) incubated with 100 µM GSNO stimulated a rapid and transient redistribution of GFP-RBD to the plasma membrane, followed by a delayed and sustained recruitment to the Golgi. No activation of H-Ras at the plasma membrane occurred in cells overexpressing H-Ras(C118S), contrasting with a robust and sustained activation of the GTPase at the Golgi. Inhibition of Src kinase prevented cell proliferation and activation of H-Ras by GSNO at the Golgi. Human umbilical vein endothelial cells (HUVECs) stimulated with bradykinin to generate NO were used to differentiate cell proliferation and Ras activation at the plasma membrane versus Golgi. In this model, Src kinase was not involved in cell proliferation, whereas Ras activation proceeded only at the plasma membrane, indicating that HUVEC proliferation induced by NO resulted only from stimulation of Ras. INNOVATION: The present work is the first to demonstrate that NO-mediated activation of Ras in different subcellular compartments regulates different downstream signaling pathways. CONCLUSION: S-nitrosylation of H-Ras at Cys(118) and the activation of Src kinase are spatiotemporally linked events of the S-nitrosothiol-mediated signaling pathway that occurs at the plasma membrane and at the Golgi. The nonparticipation of Src kinase and the localized production of NO by endothelial NO synthase at the plasma membrane limited NO-mediated Ras activation to the plasma membrane.

Asunto(s)

Proliferación Celular , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , S-Nitrosoglutatión/farmacología , Animales , Bradiquinina/farmacología , Células COS , Señalización del Calcio , Membrana Celular/enzimología , Chlorocebus aethiops , Cisteína/análogos & derivados , Cisteína/metabolismo , Activación Enzimática , Aparato de Golgi/enzimología , Células HeLa , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Óxido Nítrico/fisiología , Oxidación-Reducción , Fosfolipasa C gamma/antagonistas & inhibidores , Fosfolipasa C gamma/metabolismo , Procesamiento Proteico-Postraduccional , S-Nitrosotioles/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismo

RESUMEN

The functional link between glycolipid glycosyltransferases (GT) relies on the ability of these proteins to form organized molecular complexes. The organization, stoichiometry and composition of these complexes may impact their sorting properties, sub-Golgi localization, and may determine relative efficiency of GT in different glycolipid biosynthetic pathways. In this work, by using Förster resonance energy transfer microscopy in live CHO-K1 cells, we investigated homo- and hetero-complex formation by different GT as well as their spatial organization and molecular stoichiometry on Golgi membranes. We find that GalNAcT and GalT2 Ntd are able to form hetero-complexes in a 1:2 molar ratio at the trans-Golgi network and that GalT2 but not GalNAcT forms homo-complexes. Also, GalNAcT/GalT2 complexes exhibit a stable behavior reflected by its clustered lateral organization. These results reveals that particular topological organization of GTs may have functional implications in determining the composition of glycolipids in cellular membranes.

Asunto(s)

Galactosiltransferasas/metabolismo , Aparato de Golgi/enzimología , Complejos Multienzimáticos/metabolismo , N-Acetilgalactosaminiltransferasas/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Transferencia Resonante de Energía de Fluorescencia , Red trans-Golgi/enzimología

RESUMEN

The current study examined the role of PLD2 in the maintenance of mast cell structure. Phospholipase D (PLD) catalyzes hydrolysis of phosphatidylcholine to produce choline and phosphatidic acid (PA). PLD has two isoforms, PLD1 and PLD2, which vary in expression and localization depending on the cell type. The mast cell line RBL-2H3 was transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2. The results of this study show that PLD2CI cells have a distinct star-shaped morphology, whereas PLD2CA and RBL-2H3 cells are spindle shaped. In PLD2CI cells, the Golgi complex was also disorganized with dilated cisternae, and more Golgi-associated vesicles were present as compared with the PLD2CA and RBL-2H3 cells. Treatment with exogenous PA led to the restoration of the wild-type Golgi complex phenotype in PLD2CI cells. Conversely, treatment of RBL-2H3 and PLD2CA cells with 1% 1-Butanol led to a disruption of the Golgi complex. The distribution of acidic compartments, including secretory granules and lysosomes, was also modified in PLD2CI cells, where they concentrated in the perinuclear region. These results suggest that the PA produced by PLD2 plays an important role in regulating cell morphology in mast cells.

Asunto(s)

Mastocitos/citología , Fosfolipasa D/metabolismo , Animales , Línea Celular , Aparato de Golgi/enzimología , Aparato de Golgi/ultraestructura , Isoenzimas/genética , Isoenzimas/metabolismo , Lisosomas/enzimología , Lisosomas/ultraestructura , Mastocitos/enzimología , Mastocitos/ultraestructura , Ácidos Fosfatidicos/farmacología , Fosfolipasa D/genética , ARN Mensajero/metabolismo , Ratas , Vesículas Secretoras/enzimología , Vesículas Secretoras/ultraestructura

RESUMEN

Glycolipids constitute a complex family of amphipathic molecules structurally characterized by a hydrophilic mono- or oligo-saccharide moiety linked to a hydrophobic ceramide moiety. Due to their asymmetric distribution in cell membranes, exposing the saccharide moiety to the extracytoplasmic side of the cell, glycolipids participate in a variety of cell-cell and cell-ligand interactions. Here we summarize aspects of the cell biology of the stepwise synthesis of the saccharide moiety in the Golgi complex of cells from vertebrates. In particular we refer to the participant glycosyltransferases, with emphasis on their trafficking along the secretory pathway, their retention and organization in the Golgi complex membranes and their dependence on the Golgi complex ultra structural organization for proper function.

Asunto(s)

Glucolípidos/química , Aparato de Golgi/metabolismo , Oligosacáridos/biosíntesis , Animales , Retículo Endoplásmico/metabolismo , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Aparato de Golgi/enzimología , Humanos

RESUMEN

Copper-stimulated P-type ATPases are essential in the fine-tuning of intracellular copper. In the present work we characterized a copper-dependent ATPase hydrolysis in a native Golgi-enriched preparation from liver and investigated its modulation by cyclic AMP-dependent protein kinase (PKA). The very high-affinity Atp7b copper pump presented here shows a K(0.5) for free copper of 2.5×10(-17) M in bathocuproine disulfonate/copper buffer and ATP hydrolysis was inhibited 50% upon stimulation of PKA pathway, using forskolin, cAMP or cholera toxin. Incubation with PKA inhibitor (PKAi(5-24) peptide) raises Cu(I)-ATPase activity by 50%. Addition of purified PKA α-catalytic subunit increases K(0.5) for free copper (6.2×10(-17) M) without modification in the affinity for ATP in the low-affinity range of the substrate curve (∼1 mM). The Hill coefficient for free copper activation also remains unchanged if exogenous PKA is added (2.7 and 2.3 in the absence and presence of PKA, respectively). The results demonstrate that this high-affinity copper pump in its natural environment is a target of the liver PKA pathway, being regulatory phosphorylation able to influence both turnover rate and ion affinity.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Cobre/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Aparato de Golgi/enzimología , Membranas Intracelulares/enzimología , Hígado/enzimología , Adenosina Trifosfato/metabolismo , Animales , Biocatálisis , ATPasas Transportadoras de Cobre , Fosforilación , Sus scrofa , Factores de Tiempo

RESUMEN

The conserved oligomeric Golgi (COG) complex is a eight subunit (COG1 to 8) tethering complex involved in the retrograde trafficking of multiple Golgi processing proteins. Here we studied the glycolipid synthesis status in ldlC cells, a Cog2 null mutant CHO cell line. Biochemical studies revealed a block in the coupling between LacCer and GM3 synthesis, resulting in decreased levels of GM3 in these cells. Uncoupling was not attributable to decreased activity of the glycosyltransferase that uses LacCer as acceptor substrate (SialT1). Rather, immunocytochemical experiments evidenced a mislocalization of SialT1 as consequence of the lack of Cog2 in these cells. Co-immunoprecipitation experiments disclose a Cog2 mediated interaction of SialT1 with the COG complex member Cog1. Results indicate that cycling of some Golgi glycolipid glycosyltransferases depends on the participation of the COG complex and that deficiencies in COG complex subunits, by altering their traffic and localization, affect glycolipid composition.

Asunto(s)

Gangliósido G(M3)/biosíntesis , Aparato de Golgi/enzimología , Mutación , Sialiltransferasas/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Microscopía Fluorescente , Unión Proteica

RESUMEN

Glycolipid glycosyltransferases (GGT) are transported from the endoplasmic reticulum (ER) to the Golgi, their site of residence, via COPII vesicles. An interaction of a (R/K)X(R/K) motif at their cytoplasmic tail (CT) with Sar1 is critical for the selective concentration in the transport vesicles. In this work using computational docking, we identify three putative binding pockets in Sar1 (sites A, B, and C) involved in the interaction with the (R/K)X(R/K) motif. Sar1 mutants with alanine replacement of amino acids in site A were tested in vitro and in cells. In vitro, mutant versions showed a reduced ability to bind immobilized peptides with the CT sequence of GalT2. In cells, Sar1 mutants (Sar1(D198A)) specifically affect the exiting of GGT from the ER, resulting in an ER/Golgi concentration ratio favoring the ER. Neither the typical Golgi localization of GM130 nor the exiting and transport of the G protein of the vesicular stomatitis virus were affected. The protein kinase inhibitor H89 produced accumulation of Sec23, Sar1, and GalT2 at the ER exit sites; Sar1(D189A) also accumulated at these sites, but in this case GalT2 remained disperse along ER membranes. The results indicate that amino acids in site A of Sar1 are involved in the interaction with the CT of GGT for concentration at ER exiting sites.

Asunto(s)

Retículo Endoplásmico/enzimología , Galactosiltransferasas/metabolismo , Aparato de Golgi/enzimología , Modelos Moleculares , Proteínas de Unión al GTP Monoméricas/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Células CHO , Vesículas Cubiertas por Proteínas de Revestimiento/enzimología , Vesículas Cubiertas por Proteínas de Revestimiento/genética , Cricetinae , Cricetulus , Retículo Endoplásmico/genética , Galactosiltransferasas/genética , Aparato de Golgi/genética , Isoquinolinas/farmacología , Ratones , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/genética , Mutación , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Sulfonamidas/farmacología , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo

RESUMEN

PDI, a redox chaperone, is involved in host cell uptake of bacteria/viruses, phagosome formation, and vascular NADPH oxidase regulation. PDI involvement in phagocyte infection by parasites has been poorly explored. Here, we investigated the role of PDI in in vitro infection of J774 macrophages by amastigote and promastigote forms of the protozoan Leishmania chagasi and assessed whether PDI associates with the macrophage NADPH oxidase complex. Promastigote but not amastigote phagocytosis was inhibited significantly by macrophage incubation with thiol/PDI inhibitors DTNB, bacitracin, phenylarsine oxide, and neutralizing PDI antibody in a parasite redox-dependent way. Binding assays indicate that PDI preferentially mediates parasite internalization. Bref-A, an ER-Golgi-disrupting agent, prevented PDI concentration in an enriched macrophage membrane fraction and promoted a significant decrease in infection. Promastigote phagocytosis was increased further by macrophage overexpression of wild-type PDI and decreased upon transfection with an antisense PDI plasmid or PDI siRNA. At later stages of infection, PDI physically interacted with L. chagasi, as revealed by immunoprecipitation data. Promastigote uptake was inhibited consistently by macrophage preincubation with catalase. Additionally, loss- or gain-of-function experiments indicated that PMA-driven NADPH oxidase activation correlated directly with PDI expression levels. Close association between PDI and the p22phox NADPH oxidase subunit was shown by confocal colocalization and coimmunoprecipitation. These results provide evidence that PDI not only associates with phagocyte NADPH oxidase but also that PDI is crucial for efficient macrophage infection by L. chagasi.

Asunto(s)

Aparato de Golgi/enzimología , Leishmania , Leishmaniasis/enzimología , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Fagocitosis , Proteína Disulfuro Isomerasas/metabolismo , Animales , Antibacterianos , Brefeldino A/farmacología , Cricetinae , Inhibidores Enzimáticos/farmacología , Masculino , Ratones , Proteína Disulfuro Isomerasas/antagonistas & inhibidores

RESUMEN

Cadmium is a nonessential, highly toxic heavy metal that shows ionic properties similar to calcium. These ionic similarities imply that the cadmium ion, Cd2+, is a calcium ion, Ca2+, receptor-agonist, affecting the same biochemical pathways involved in Ca2+ homeostasis. In the yeast Saccharomyces cerevisiae, the PMC1 and PMR1 genes encode vacuolar and Golgi Ca2+-ATPases, respectively. The PMR1 protein product Pmr1p is involved in both Ca2+ and Mn2+ homeostasis. This study investigated the importance of Pmc1p and Pmr1p for Cd2+ cellular detoxification. Using the standard techniques of yeast molecular research and a multielemental procedure named particle-induced X-ray emission, Pmr1p was identified as a protein that directly participates in the detoxification of Cd2+, possibly through the secretory pathway. The results allow us to posit a model of Cd2+ detoxification where Pmr1p has a central role in cell survival in a Cd2+-rich environment.

Asunto(s)

Cadmio/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Aparato de Golgi/enzimología , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Cadmio/toxicidad , ATPasas Transportadoras de Calcio/genética , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Modelos Biológicos , Chaperonas Moleculares/genética , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacuolas/enzimología , Vacuolas/genética , Vacuolas/metabolismo

RESUMEN

Ganglioside glycosyltransferases organize as multienzyme complexes that localize in different sub-Golgi compartments. Here we studied whether in CHO-K1 cells lacking CMP-NeuAc: GM3 sialyltransferase (SialT2), the sub-Golgi localization of UDP-Gal:glucosylceramide beta-1,4-galactosyltransferase (GalT1) and CMP-NeuAc:lactosylceramide sialyltransferase (SialT1) complex is affected when SialT2, another member of this complex, is coexpressed. GalT1 and SialT1 sub-Golgi localization was determined by studying the effect of brefeldin A (BFA) and monensin on the synthesis of glycolipids and on the sub-Golgi localization of GalT1(1-52)-CFP (cyan fluorescent protein) and SialT1(1-54)-YFP (yellow fluorescent protein) chimeras by single cell fluorescence microscopy and by isopycnic subfractionation. We found that BFA, and also monensin, impair the synthesis of glycolipids beyond GM3 ganglioside in wild type (WT) cells but beyond GlcCer in SialT2(+) cells. Although BFA redistributed GalT1-CFP and SialT1-YFP to the endoplasmic reticulum in WT cells, a fraction of these chimeras remained associated with a distal Golgi compartment, enriched in trans Golgi network, and recycling endosome markers in SialT2(+) cells. In BFA-treated cells, the percentage of GalT1-CFP and SialT1-YFP associated with Golgi-like membrane fractions separated by isopycnic subfractionation was higher in SialT2(+) cells than in WT cells. These effects were reverted by knocking down the expression of SialT2 with specific siRNA. Results indicate that sub-Golgi localization of glycosyltransferase complexes may change according to the relative levels of the expression of participating enzymes and reveal a capacity of the organelle to adapt the topology of the glycolipid synthesis machinery to functional states of the cell.

Asunto(s)

Galactosiltransferasas/metabolismo , Glucolípidos/biosíntesis , Aparato de Golgi/enzimología , N-Acetilgalactosaminiltransferasas/metabolismo , Sialiltransferasas/metabolismo , Animales , Antibacterianos/farmacología , Antiprotozoarios/farmacología , Biomarcadores/metabolismo , Brefeldino A/farmacología , Células CHO , Centrifugación Isopicnica , Células Clonales/enzimología , Cricetinae , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Monensina/farmacología , N-Acetilgalactosaminiltransferasas/química , N-Acetilgalactosaminiltransferasas/genética , ARN Interferente Pequeño/farmacología , Sialiltransferasas/química , Sialiltransferasas/genética , Fracciones Subcelulares/metabolismo , Transfección

RESUMEN

GEM (glycosphingolipid-enriched microdomains) are specialized detergent-resistant domains of the plasma membrane in which some gangliosides concentrate. Although genesis of GEM is considered to occur in the Golgi complex, where the synthesis of gangliosides also occurs, the issue concerning the incorporation of ganglioside species into GEM is still poorly understood. In this work, using Chinese hamster ovary K1 cell clones with different glycolipid compositions, we compared the behaviour with cold Triton X-100 solubilization of plasma membrane ganglioside species with the same species newly synthesized in Golgi membranes. We also investigated whether three ganglioside glycosyltransferases (a sialyl-, a N-acetylgalactosaminyl- and a galactosyl-transferase) are included or excluded from GEM in Golgi membranes. Our data show that an important fraction of plasma membrane G(M3), and most G(D3) and G(T3), reside in GEM. Immunocytochemical examination of G(D3)-expressing cells showed G(D3) to be distributed as cold-detergent-resistant patches in the plasma membrane. These patches did not co-localize with a glycosylphosphatidylinositol-anchored protein used as GEM marker, indicating a heterogeneous composition of plasma membrane GEM. In Golgi membranes we were unable to find evidence for GEM localization of either ganglioside glycosyltransferases or newly synthesized gangliosides. Since the same ganglioside species appear in plasma membrane GEM, it was concluded that in vivo nascent G(D3), G(T3) and G(M3) segregate from their synthesizing transferases and then enter GEM. This latter event could have taken place shortly after synthesis in the Golgi cisternae, along the secretory pathway and/or at the cell surface.

Asunto(s)

Detergentes/química , Gangliósidos/biosíntesis , Gangliósidos/metabolismo , Glicosiltransferasas/metabolismo , Aparato de Golgi/química , Membranas Intracelulares/química , Animales , Células CHO/química , Células CHO/enzimología , Células CHO/metabolismo , Extractos Celulares/química , Línea Celular , Membrana Celular/química , Cricetinae , Aparato de Golgi/enzimología , Humanos , Membranas Intracelulares/enzimología , Microdominios de Membrana/química , Octoxinol/metabolismo , Sialiltransferasas/biosíntesis