RESUMEN
The vesicular stomatitis virus glycoprotein (VSV G) is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway. The cytoplasmic domain of VSV G contains information for several intracellular sorting steps including efficient export from the ER, basolateral delivery, and endocytosis. In order to identify proteins that potentially interact with the polypeptide sorting motifs in the VSV G tail, the carboxy-terminal 27 amino acids of VSV G were used as bait in a yeast two-hybrid system. The protein identified most frequently in the screen is a novel protein of 38 kDa, p38. In the present work, the initial molecular and biochemical characterization of p38 is described. Preliminary evidence suggests that p38 may interact transiently with endoplasmic reticulum (ER) membranes, and thus may affect VSV G and other cargo movement at the step of ER to Golgi traffic.
Asunto(s)
Proteínas Portadoras/metabolismo , Glicoproteínas de Membrana , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Anticuerpos , Secuencia de Bases , Proteínas Portadoras/genética , Citoplasma/metabolismo , ADN Complementario/análisis , ADN Complementario/aislamiento & purificación , Células HeLa , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Fracciones Subcelulares , Distribución Tisular , Técnicas del Sistema de Dos Híbridos , Proteínas Quinasas p38 Activadas por MitógenosAsunto(s)
Aparato de Golgi/metabolismo , Virus de la Bronquitis Infecciosa/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas de la Matriz Viral/metabolismo , Ensamble de Virus , Animales , Línea Celular , Proteínas M de Coronavirus , Cricetinae , Aparato de Golgi/virología , Virus de la Bronquitis Infecciosa/genética , Microscopía Inmunoelectrónica , Recombinación Genética , Virus Vaccinia/genética , Proteínas del Envoltorio Viral/genética , Proteínas de la Matriz Viral/genéticaRESUMEN
Caspases are an extended family of cysteine proteases that play critical roles in apoptosis. Animals deficient in caspases-2 or -3, which share very similar tetrapeptide cleavage specificities, exhibit very different phenotypes, suggesting that the unique features of individual caspases may account for distinct regulation and specialized functions. Recent studies demonstrate that unique apoptotic stimuli are transduced by distinct proteolytic pathways, with multiple components of the proteolytic machinery clustering at distinct subcellular sites. We demonstrate here that, in addition to its nuclear distribution, caspase-2 is localized to the Golgi complex, where it cleaves golgin-160 at a unique site not susceptible to cleavage by other caspases with very similar tetrapeptide specificities. Early cleavage at this site precedes cleavage at distal sites by other caspases. Prevention of cleavage at the unique caspase-2 site delays disintegration of the Golgi complex after delivery of a pro-apoptotic signal. We propose that the Golgi complex, like mitochondria, senses and integrates unique local conditions, and transduces pro-apoptotic signals through local caspases, which regulate local effectors.
Asunto(s)
Apoptosis , Autoantígenos/metabolismo , Caspasas/metabolismo , Aparato de Golgi/enzimología , Proteínas de la Membrana , Caspasa 2 , Núcleo Celular/enzimología , Técnica del Anticuerpo Fluorescente , Proteínas de la Matriz de Golgi , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Cinética , Proteínas Luminiscentes , Microscopía Fluorescente , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Transducción de Señal , Especificidad por SustratoRESUMEN
In addition to serving as membrane anchors for cell surface proteins, glycosylphosphatidylinositols (GPIs) can be found abundantly as free glycolipids in mammalian cells. In this study we analyze the subcellular distribution and intracellular transport of metabolically radiolabeled GPIs in three different cell lines. We use a variety of membrane isolation techniques (subcellular fractionation, plasma membrane vesiculation to isolate pure plasma membrane fractions, and enveloped viruses to sample cellular membranes) to provide direct evidence that free GPIs are not confined to their site of synthesis, the endoplasmic reticulum, but can redistribute to populate other subcellular organelles. Over short labeling periods (2.5 h), radiolabeled GPIs were found at similar concentration in all subcellular fractions with the exception of a mitochondria-enriched fraction where GPI concentration was low. Pulse-chase experiments over extended chase periods showed that although the total amount of cellular radiolabeled GPIs decreased, the plasma membrane complement of labeled GPIs increased. GPIs at the plasma membrane were found to populate primarily the exoplasmic leaflet as detected using periodate oxidation of the cell surface. Transport of GPIs to the cell surface was inhibited by Brefeldin A and blocked at 15 degrees C, suggesting that GPIs are transported to the plasma membrane via a vesicular mechanism. The rate of transport of radiolabeled GPIs to the cell surface was found to be comparable with the rate of secretion of newly synthesized soluble proteins destined for the extracellular space.
Asunto(s)
Membrana Celular/química , Glicosilfosfatidilinositoles/análisis , Animales , Transporte Biológico , Brefeldino A/farmacología , Perros , Retículo Endoplásmico/química , Glicosilfosfatidilinositoles/metabolismo , Manosa/metabolismo , Ratones , Mitocondrias/química , Temperatura , Timoma/química , Células Tumorales Cultivadas , Virus/químicaRESUMEN
The vesicular stomatitis virus (VSV) G protein is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway. A signal in the cytoplasmic tail of VSV G (DxE or Asp-x-Glu, where x is any amino acid) was recently proposed to mediate efficient export of the protein from the endoplasmic reticulum (ER). In this study, we show that the DxE motif only partially accounts for efficient ER exit of VSV G. We have identified a six-amino-acid signal, which includes the previously identified Asp and Glu residues, that is required for efficient exit of VSV G from the ER. This six-residue signal also includes the targeting sequence YxxO (where x is any amino acid and O is a bulky, hydrophobic residue) implicated in several different sorting pathways. The only defect in VSV G proteins with mutations in the six-residue signal is slow exit from the ER; folding and oligomerization in the ER are normal, and the mutants eventually reach the plasma membrane. Addition of this six-residue motif to an inefficiently transported reporter protein is sufficient to confer an enhanced ER export rate. The signal we have identified is highly conserved among divergent VSV G proteins, and we suggest this reflects the importance of this motif in the evolution of VSV G as a proficient exocytic protein.
Asunto(s)
Exocitosis/fisiología , Glicoproteínas de Membrana , Señales de Clasificación de Proteína/metabolismo , Tirosina/metabolismo , Virus de la Estomatitis Vesicular Indiana/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Membrana Celular/metabolismo , Cricetinae , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Líquido Intracelular/metabolismo , Cinética , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/química , Señales de Clasificación de Proteína/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
The coronavirus E protein is a poorly characterized small envelope protein present in low levels in virions. We are interested in the role of E in the intracellular targeting of infectious bronchitis virus (IBV) membrane proteins. We generated a cDNA clone of IBV E and antibodies to the E protein to study its cell biological properties in the absence of virus infection. We show that IBV E is an integral membrane protein when expressed in cells from cDNA. Epitope-specific antibodies revealed that the C terminus of IBV E is cytoplasmic and the N terminus is translocated. The short luminal N terminus of IBV E contains a consensus site for N-linked glycosylation, but the site is not used. When expressed using recombinant vaccinia virus, the IBV E protein is released from cells at low levels in sedimentable particles that have a density similar to that of coronavirus virions. The IBV M protein is incorporated into these particles when present. Indirect immunofluorescence microscopy showed that E is localized to the Golgi complex in cells transiently expressing IBV E. When coexpressed with IBV M, both from cDNA and in IBV infection, the two proteins are colocalized in Golgi membranes, near the coronavirus budding site. Thus, even though IBV E is present at low levels in virions, it is apparently expressed at high levels in infected cells near the site of virus assembly.
Asunto(s)
Aparato de Golgi/metabolismo , Virus de la Bronquitis Infecciosa/fisiología , Proteínas del Envoltorio Viral/metabolismo , Animales , Transporte Biológico , Línea Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Proteínas M de Coronavirus , Cricetinae , Aparato de Golgi/virología , Células HeLa , Humanos , Virus de la Bronquitis Infecciosa/genética , Procesamiento Proteico-Postraduccional , Transfección , Virus Vaccinia , Células Vero , Proteínas del Envoltorio Viral/genética , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Virión/fisiología , Ensamble de VirusRESUMEN
Golgi resident proteins maintain their localization despite a continual protein and lipid flux through the organelle. To study Golgi retention mechanisms, we have focused upon the chimeric protein Gm1. This protein contains the Golgi transmembrane domain targeting signal from the infectious bronchitis virus M protein and the lumenal and cytoplasmic domain of the vesicular stomatitis virus glycoprotein (VSV G). The Gm1 protein is targeted to the Golgi where it forms an unusually stable detergent-resistant oligomer. The formation of oligomeric structures may aid retention of Golgi resident proteins. Thus, determining the stabilization mechanism may shed light on Golgi protein retention. Previous work determined that the transmembrane domain is required for the targeting and oligomerization of Gm1, but it is the cytoplasmic tail that stabilizes the complexes [Weisz, O. A., Swift, A. M., and Machamer, C. E. (1993) J. Cell Biol. 122, 1185-1196]. However, further study of the oligomer has been difficult due to its insolubility. Here we report that fragmenting the Gm1 protein into several pieces facilitates solubilization by sodium dodecyl sulfate (SDS). By analyzing the fragments produced after cleavage, we determined that the stability of the oligomer is not caused by covalent linkage of Gm1 to itself or other proteins. The fragment corresponding to the transmembrane domain and tail of Gm1 had an enhanced mobility in SDS gels relative to the same fragment of the parent VSV G protein. The enhanced migration of the tail fragment does not reflect sequence differences or post-translational modification, but correlates with Golgi localization and oligomerization. We suggest that the enhanced mobility of the Gm1 tail fragment reflects an altered conformation which serves to stabilize the detergent-resistant oligomers.
Asunto(s)
Citoplasma/química , Detergentes/farmacología , Aparato de Golgi/metabolismo , Glicoproteínas de Membrana , Fragmentos de Péptidos/metabolismo , Conformación Proteica/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Citoplasma/efectos de los fármacos , Citoplasma/genética , Aparato de Golgi/química , Aparato de Golgi/genética , Células HeLa , Hexosaminidasas/metabolismo , Humanos , Virus de la Bronquitis Infecciosa/genética , Datos de Secuencia Molecular , Oligosacáridos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Polímeros/metabolismo , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/efectos de los fármacos , Dodecil Sulfato de Sodio/farmacología , Solubilidad , Virus de la Estomatitis Vesicular Indiana/genética , Proteínas del Envoltorio Viral/genética , Proteínas de la Matriz Viral/genéticaRESUMEN
Distinct lipid compositions of intracellular organelles could provide a physical basis for targeting of membrane proteins, particularly where transmembrane domains have been shown to play a role. We tested the possibility that cholesterol is required for targeting of membrane proteins to the Golgi complex. We used insect cells for our studies because they are cholesterol auxotrophs and can be depleted of cholesterol by growth in delipidated serum. We found that two well-characterized mammalian Golgi proteins were targeted to the Golgi region of Aedes albopictus cells, both in the presence and absence of cellular cholesterol. Our results imply that a cholesterol gradient through the secretory pathway is not required for membrane protein targeting to the Golgi complex, at least in insect cells.
Asunto(s)
Aedes/química , Colesterol/fisiología , Aparato de Golgi/química , Proteínas de la Membrana/análisis , Animales , Transporte Biológico , Bovinos , Línea Celular , Manosidasas/análisis , Manosidasas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , N-Acetil-Lactosamina Sintasa/análisis , N-Acetil-Lactosamina Sintasa/metabolismo , Proteínas Recombinantes de Fusión , alfa-ManosidasaRESUMEN
To investigate the distribution of lipids through the Golgi complex, we analyzed the envelopes of several viruses that assemble in different subcompartments of the Golgi, as well as subcellular fractions. Our results indicate that each Golgi subcompartment has a distinct phospholipid composition due mainly to differences in the relative amounts of semilysobisphosphatidic acid (SLBPA), sphingomyelin, phosphatidylserine, and phosphatidylinositol. Interestingly, SLBPA is enriched in the adjacent Golgi networks compared with the Golgi stack, and this enrichment varies with cell type. The heterogeneous distribution of SLBPA through the Golgi complex suggests it may play an important role in the structure and/or function of this organelle.
Asunto(s)
Aparato de Golgi/química , Ácidos Fosfatidicos/análisis , Animales , Extractos Celulares , Fraccionamiento Celular , Línea Celular , Cricetinae , Perros , Aparato de Golgi/virología , Riñón , Fosfolípidos/análisis , Virus ARN/metabolismo , Receptor IGF Tipo 2/análisis , Ensamble de VirusRESUMEN
Expression of the human gene A4 is enriched in the colonic epithelium and is transcriptionally activated on differentiation of colonic epithelial cells in vitro (M. M. Oliva, T. C. Wu, and V. W. Yang. Arch. Biochem. Biophys. 302: 183-192, 1993). A4 cDNA contains an open reading frame that predicts a polypeptide of 17 kDa. To determine the function of the A4 protein, we characterized its biochemical and physiological properties. Hydropathy analysis of deduced A4 amino acid sequence revealed four putative membrane-spanning alpha-helices. The hydrophobic nature of A4 was confirmed by its being extractable with organic solvents. Immunocytochemical studies of cells expressing A4 localized it to the endoplasmic reticulum. Moreover, A4 multimerized in vivo as determined by coimmunoprecipitation experiments. The four-transmembrane topology and biophysical characteristics of A4 suggest that it belongs to a family of integral membrane proteins called proteolipids, some of which multimerize to form ion channels. Subsequent electrophysiological studies of nuclei isolated from microinjected Xenopus laevis oocytes transiently expressing A4 showed the appearance of a 28-pS channel. Thus our studies indicate that A4 is a colonic epithelium-enriched protein localized to the endoplasmic reticulum and that, similar to other proteolipids, A4 multimerizes and exhibits characteristics of an ion channel.
Asunto(s)
Mucosa Intestinal/fisiología , Canales Iónicos/fisiología , Proteínas de la Membrana/fisiología , Proteolípidos/fisiología , Secuencia de Aminoácidos , Animales , Western Blotting , Compartimento Celular , Retículo Endoplásmico/metabolismo , Epitelio/fisiología , Humanos , Membranas Intracelulares/metabolismo , Proteínas con Dominio MARVEL , Sustancias Macromoleculares , Potenciales de la Membrana , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Pruebas de Precipitina , Proteínas Recombinantes , Solubilidad , Células Tumorales Cultivadas , Xenopus laevisRESUMEN
The M glycoprotein from the avian coronavirus, infectious bronchitis virus (IBV), contains information for localization to the cis-Golgi network in its first transmembrane domain. We hypothesize that localization to the Golgi complex may depend in part on specific interactions between protein transmembrane domains and membrane lipids. Because the site of sphingolipid synthesis overlaps the localization of IBV M, we asked whether perturbation of sphingolipids affected localization of IBV M. Short-term treatment with two inhibitors of sphingolipid synthesis had no effect on localization of IBV M or other Golgi markers. Thus, ongoing synthesis of these lipids was not required for proper localization. Surprisingly, a third inhibitor, d,l-threo-1-phenyl-2-decanoylamino-3-morpholino- 1-propanol (PDMP), shifted the steady-state distribution of IBV M from the Golgi complex to the ER. This effect was rapid and reversible and was also observed for ERGIC-53 but not for Golgi stack proteins. At the concentration of PDMP used, conversion of ceramide into both glucosylceramide and sphingomyelin was inhibited. Pretreatment with upstream inhibitors partially reversed the effects of PDMP, suggesting that ceramide accumulation mediates the PDMP-induced alterations. Indeed, an increase in cellular ceramide was measured in PDMP-treated cells. We propose that IBV M is at least in part localized by retrieval mechanisms. Further, ceramide accumulation reveals this cycle by upsetting the balance of anterograde and retrograde traffic and/ or disrupting retention by altering bilayer dynamics.
Asunto(s)
Ceramidas/metabolismo , Aparato de Golgi/metabolismo , Virus de la Bronquitis Infecciosa , Esfingolípidos/biosíntesis , Proteínas de la Matriz Viral/biosíntesis , Animales , Biomarcadores , Línea Celular , Cricetinae , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/farmacología , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/ultraestructura , Riñón , Cinética , Modelos Biológicos , Morfolinas/farmacología , Proteínas de la Matriz Viral/análisisRESUMEN
We isolated forms of enveloped vaccinia virus from infected HeLa cells to obtain membranes for the analysis of lipids of the cis-Golgi network and trans-Golgi network. The intracellular mature virus obtains its envelope by wrapping itself in the membranes of the cis-Golgi network. A fraction of these virions then acquires a second envelope by enwrapping trans-Golgi network membranes to form the intracellular enveloped virus. Lipids were analyzed by high performance thin layer chromatography and digital densitometry to establish a steady-state lipid profile of viral membranes, which should reflect the compositions of the cis-Golgi network and trans-Golgi network. Phosphatidyl-inositol was slightly enriched in the cis-Golgi network of HeLa cells, whereas the trans-Golgi network showed a minor increase in phosphatidylserine and sphingomyelin. Similarly, cholesterol was only slightly more abundant in the trans-Golgi compared to the cis-Golgi. An unusual lipid, semilysobisphosphatidic acid, was present in significant amounts in vaccinia envelopes. Semilysobisphosphatidic acid was present in similar levels in infected and uninfected cells, and was therefore not induced by vaccinia infection. Subcellular fractionation of HeLa cells indicated that the recovery of semilysobisphosphatidic acid paralleled the recovery of a Golgi marker. Furthermore, a lipid species that comigrated with semilysobisphosphatidic acid was also present in lipids extracted from highly purified, intact Golgi complexes from rat liver. Together, these results suggest that semilysobisphosphatidic acid is a normal component of Golgi membranes.
Asunto(s)
Aparato de Golgi/química , Ácidos Fosfatidicos/química , Fosfolípidos/química , Virus Vaccinia/química , Animales , Colesterol/química , Aparato de Golgi/ultraestructura , Células HeLa , Humanos , Microscopía Electrónica , Ratas , Virus Vaccinia/ultraestructuraAsunto(s)
Clonación Molecular/métodos , Vectores Genéticos , Virus Vaccinia/genética , Animales , Línea Celular , Chlorocebus aethiops , Contención de Riesgos Biológicos , ARN Polimerasas Dirigidas por ADN , Técnicas de Transferencia de Gen , Células HeLa , Humanos , Plásmidos , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Virus Vaccinia/crecimiento & desarrollo , Virus Vaccinia/aislamiento & purificación , Virus Vaccinia/fisiología , Proteínas Virales , Cultivo de Virus/métodos , Replicación ViralRESUMEN
T lymphocytes are activated upon binding of their Ag receptors to a complex of Ag-derived peptides and MHC class I or class II molecules expressed on the surface of APC. It is now well established that APC degrade exogenous Ag in acidic endosomal compartments, and that Ag fragments bind to class II molecules moving through these compartments on their way to the surface of the APC. Although peptides derived from some endogenous Ag can also bind to class II molecules and subsequently be recognized by class II-restricted T cells, the intracellular trafficking pathways that enable endogenous proteins to be processed for association with class II molecules remain controversial. We have analyzed the mechanism by which the envelope (env) protein of the HIV-1 is processed in infected cells for recognition by class II-restricted T cells. A large number of env-specific class II-restricted human CTL clones were shown to lyse B-lymphoblastoid cell lines expressing the env. A novel dilutional assay proved that A novel dilutional assay proved that recognition of endogenous env protein was not a consequence of release and re-uptake of the env protein and subsequent processing by the standard class II-restricted pathway. Processing of endogenous env protein required that the protein be co-translationally translocated into the endoplasmic reticulum (ER) and then exit the ER, since the class II-restricted CTL did not recognize env protein localized to the cytosol or retained in the ER of target cells. Under these conditions, however, class I-restricted recognition was readily demonstrated. Finally, class II-restricted recognition was strikingly dependent upon the steady state level of surface env protein, since extracellular reagents that removed intact env protein from the surface of target cells inhibited recognition. This inhibition operated at the Ag-processing level rather than at the level of subsequent Ag recognition. These results provide the first direct evidence that endogenously synthesized membrane proteins enter the class II-restricted Ag-processing pathway after expression on the cell surface in an intact form.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Infecciones por VIH/inmunología , Linfocitos T/inmunología , Células Presentadoras de Antígenos/inmunología , Antígenos de Superficie/inmunología , Secuencia de Bases , Antígenos CD4/química , Antígenos CD4/metabolismo , Membrana Celular/inmunología , Membrana Celular/metabolismo , Células Cultivadas , Retículo Endoplásmico/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Antígenos HLA-D/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Técnicas In Vitro , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , SolubilidadRESUMEN
The first membrane-spanning domain (m1) of the M glycoprotein of avian coronavirus (formerly called E1) is sufficient to retain this protein in the cis-Golgi. When the membrane-spanning domain of a protein which is efficiently delivered to the plasma membrane (VSV G protein) is replaced with m1, the resulting chimera (Gm1) is retained in the Golgi (Swift, A. M., and C. E. Machamer. 1991. J. Cell Biol. 115:19-30). When assayed in sucrose gradients, we observed that Gm1 formed a large oligomer, and that much of this oligomer was SDS resistant and stayed near the top of the stacking gel of an SDS-polyacrylamide gel. The unusual stability of the oligomer allowed it to be detected easily. Gm1 mutants with single amino acid substitutions in the m1 domain that were retained in the Golgi complex formed SDS-resistant oligomers, whereas mutants that were rapidly released to the plasma membrane did not. Oligomerization was not detected immediately after synthesis of Gm1, but occurred gradually with a lag of approximately 10 min, suggesting that it is not merely aggregation of misfolded proteins. Furthermore, oligomerization did not occur under several conditions that block ER to Golgi transport. The lumenal domain was not required for oligomerization since another chimera (alpha m1G), where the lumenal domain of Gm1 was replaced by the alpha subunit of human chorionic gonadotropin, also formed an SDS-resistant oligomer, and was able to form hetero-oligomers with Gm1 as revealed by coprecipitation experiments. SDS resistance was conferred by the cytoplasmic tail of VSV G, because proteolytic digestion of the tail in microsomes containing Gm1 oligomers resulted in loss of SDS resistance, although the protease-treated material continued to migrate as a large oligomer on sucrose gradients. Interestingly, treatment of cells with cytochalasin D blocked formation of SDS-resistant (but not SDS-sensitive) oligomers. Our data suggest that SDS-resistant oligomers form as newly synthesized molecules of Gm1 arrive at the Golgi complex and may interact (directly or indirectly) with an actin-based cytoskeletal matrix. The oligomerization of Gm1 and other resident proteins could serve as a mechanism for their retention in the Golgi complex.
Asunto(s)
Membrana Celular/metabolismo , Glicoproteínas/metabolismo , Aparato de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Glicoproteínas de Membrana , Proteínas del Envoltorio Viral/metabolismo , Actinas/química , Secuencia de Aminoácidos , Animales , Biotina , Centrifugación por Gradiente de Densidad , Quimera , Citocalasina D/farmacología , Glicoproteínas/química , Glicoproteínas/genética , Aparato de Golgi/química , Aparato de Golgi/ultraestructura , Células HeLa , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Conformación Proteica , Dodecil Sulfato de Sodio , Tripsina , Virus de la Estomatitis Vesicular Indiana , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
Recent cloning of genes encoding membrane proteins of the Golgi complex has allowed investigation of protein targeting to this organelle. Targeting signals have been identified in three glycosyltransferases, a viral envelope protein and several proteins of the trans-Golgi network. Interestingly, the targeting signals for membrane proteins of the Golgi stacks seem to be contained in transmembrane domains. Information in the cytoplasmic tails is required for the targeting of trans-Golgi network proteins. Mechanisms involving both retention and retrieval have been invoked.
Asunto(s)
Aparato de Golgi/química , Proteínas de la Membrana/metabolismo , Transporte Biológico/fisiología , Aparato de Golgi/fisiología , Humanos , Transducción de Señal/fisiología , Relación Estructura-ActividadRESUMEN
The first membrane-spanning domain (m1) of the model cis Golgi protein M (formerly called E1) from the avian coronavirus infectious bronchitis virus is required for targeting to the Golgi complex. When inserted in place of the membrane-spanning domain of a plasma membrane protein (vesicular stomatitis virus G protein), the chimeric protein ("Gm1") is retained in the Golgi complex of transfected cells. To determine the precise features of the m1 domain responsible for Golgi targeting, we produced single amino acid substitutions in m1 and analyzed their effects on localization of Gm1. Expression at the plasma membrane was used as the criterion for loss of Golgi retention. Rates of oligosaccharide processing were used as a measure of rate and efficiency of transport through the Golgi complex. We identified four uncharged polar residues that are critical for Golgi retention of Gm1 (Asn465, Thr469, Thr476, and Gln480). These residues line one face of a predicted alpha-helix. Interestingly, when the m1 domain of the homologous M protein from mouse hepatitis virus is inserted into the G protein reporter, the chimeric protein is not efficiently retained in the Golgi complex, but transported to the cell surface. Although it possesses three of the four residues we identified as important in the avian m1 sequence, other residues in the membrane-spanning domain from the mouse protein must prevent efficient recognition of the polar face within the lipid bilayer of the cis Golgi.
Asunto(s)
Aparato de Golgi/metabolismo , Estructura Secundaria de Proteína , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Línea Celular , Cricetinae , Femenino , Virus de la Bronquitis Infecciosa/genética , Virus de la Bronquitis Infecciosa/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligosacáridos/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transfección , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/químicaRESUMEN
Vaccinia virus, the prototype of the Poxviridae, is a large DNA virus which replicates in the cytoplasm of the host cell. The assembly pathway of vaccinia virus displays several unique features, such as the production of two structurally distinct, infectious forms. One of these, termed intracellular naked virus (INV), remains cells associated while the other, termed extracellular enveloped virus (EEV), is released from the cell. In addition, it has long been believed that INVs acquire their lipid envelopes by a unique example of de novo membrane biogenesis. To examine the structure and assembly of vaccinia virus we have used immunoelectron microscopy using antibodies to proteins of different subcellular compartments as well as a phospholipid analysis of purified INV and EEV. Our data are not consistent with the de novo model of viral membrane synthesis but rather argue that the vaccinia virus DNA becomes enwrapped by a membrane cisterna derived from the intermediate compartment between the ER and the Golgi stacks, thus acquiring two membranes in one step. Phospholipid analysis of purified INV supports its derivation from an early biosynthetic compartment. This unique assembly process is repeated once more when the INV becomes enwrapped by an additional membrane cisterna, in agreement with earlier reports. The available data suggest that after fusion between the outer envelope and the plasma membrane, mature EEV is released from the cell.
Asunto(s)
Membranas Intracelulares/microbiología , Virus Vaccinia/crecimiento & desarrollo , Retículo Endoplásmico/microbiología , Aparato de Golgi/microbiología , Células HeLa/microbiología , Células HeLa/ultraestructura , Humanos , Membranas Intracelulares/ultraestructura , Modelos Biológicos , Virus Vaccinia/patogenicidad , Virus Vaccinia/ultraestructura , Esparcimiento de VirusRESUMEN
Madin-Darby canine kidney (MDCK) cells deliver endogenous apical and basolateral proteins directly to the appropriate domains. We are investigating the molecular signals on a model plasma membrane hydrolase, dipeptidylpeptidase IV (DPPIV). Most newly synthesized rat liver DPPIV is delivered directly to the apical surface of transfected MDCK cells; however, about 20% is delivered first to the basolateral surface and reaches the apical surface via transcytosis (Casanova, J. E., Mishumi, Y., Ikehara, Y., Hubbard, A. L., and Mostov, K. E. (1991) J. Biol. Chem. 266, 24428-24432). A soluble form of DPPIV (solDPPIV) containing only the lumenal domain of the protein was efficiently transported and secreted by stably transfected MDCK cells. If this domain contains apical sorting information, we would expect 80% of the soluble protein to be secreted apically. Surprisingly, 95% of the secreted solDPPIV was found in the apical medium. The high efficiency of apical secretion suggested that the transmembrane domain and cytoplasmic tail of DPPIV might contain competing basolateral targeting information. To test this hypothesis, we investigated the trafficking of a chimera in which the cytoplasmic tail and transmembrane domains of DPPIV were joined to lysozyme, an exogenous protein which should not contain sorting information. This protein was delivered predominantly to the basolateral surface. Our results suggest that the lumenal domain of DPPIV carries dominant apical sorting information while the transmembrane domain and cytoplasmic tail of the molecule contains competing basolateral sorting information.
Asunto(s)
Polaridad Celular , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Hígado/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Compartimento Celular , Membrana Celular/metabolismo , Células Cultivadas , Dipeptidil Peptidasa 4 , Perros , Técnicas In Vitro , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos/química , Ratas , Relación Estructura-ActividadRESUMEN
We investigated the effects of an inhibitor of sphingolipid biosynthesis, 1-phenyl-2-(decanoyl-amino)-3-morpholino-1-propanol (PDMP), on cells in culture. Two Golgi-associated enzymes were affected by incubation of cells with PDMP. The synthesis of glucosylceramide was inhibited at low concentrations of PDMP (2.5-10 microM), and in the presence of higher concentrations (greater than or equal to 25 microM), synthesis of sphingomyelin was also reduced. Transport of vesicular stomatitis virus G protein through the Golgi complex was progressively retarded by increasing concentrations of PDMP. In the presence of 75 microM PDMP, the half-times of VSV-G protein arrival at the cis, medial, and trans Golgi and the cell surface were increased 1.5-, 2.1-, 2.4-, and 2.8-fold, respectively, compared to control values. Transport of fluorescent sphingolipids, synthesized de novo at the Golgi complex from fluorescent ceramide precursors, to the cell surface was retarded by approximately 20% in the presence of 50 microM PDMP and by approximately 50% in the presence of 100 microM PDMP. Control experiments demonstrated that PDMP had minimal effects on cell morphology and physiology (including microtubule and endoplasmic reticulum structure, mitochondrial function, and endocytosis). Although incubation of cells with relatively high concentrations of PDMP was required to see the effects on protein and sphingolipid transport, use of a fluorescent analogue of PDMP demonstrated that most cell-associated PDMP was sequestered in lysosomes, while the concentration at the Golgi complex, the site of the target synthetic enzymes, was relatively low. Taken together, these results suggest that transport of proteins and sphingolipids through the secretory pathway may be coupled to sphingolipid synthesis.