Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Mol Biol Cell ; 10(7): 2191-7, 1999 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10397758

RESUMO

Late endosomes and the Golgi complex maintain their cellular localizations by virtue of interactions with the microtubule-based cytoskeleton. We study the transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network in vitro. We show here that this process is facilitated by microtubules and the microtubule-based motor cytoplasmic dynein; transport is inhibited by excess recombinant dynamitin or purified microtubule-associated proteins. Mapmodulin, a protein that interacts with the microtubule-associated proteins MAP2, MAP4, and tau, stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact Chinese hamster ovary cells. The present study shows that mapmodulin also stimulates the initial rate with which mannose 6-phosphate receptors are transported from late endosomes to the trans-Golgi network in vitro. These findings represent the first indication that mapmodulin can stimulate a vesicle transport process, and they support a model in which the microtubule-based cytoskeleton enhances the efficiency of vesicle transport between membrane-bound compartments in mammalian cells.


Assuntos
Dineínas/metabolismo , Endossomos/metabolismo , Microtúbulos/metabolismo , Proteínas/metabolismo , Receptor IGF Tipo 2/metabolismo , Proteínas de Transporte Vesicular , Animais , Transporte Biológico , Células CHO/efeitos dos fármacos , Células CHO/metabolismo , Proteínas de Transporte/metabolismo , Bovinos , Cricetinae , Citoplasma/metabolismo , Complexo Dinactina , Complexo de Golgi/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Proteínas Nucleares , Paclitaxel/farmacologia , Proteínas de Ligação a RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Proteínas tau/genética , Proteínas tau/metabolismo
2.
J Cell Biol ; 142(2): 377-89, 1998 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-9679138

RESUMO

ERGIC-53, a homo-oligomeric recycling protein associated with the ER-Golgi intermediate compartment (ERGIC), has properties of a mannose-selective lectin in vitro, suggesting that it may function as a transport receptor for glycoproteins in the early secretory pathway. To investigate if ERGIC-53 is involved in glycoprotein secretion, a mutant form of this protein was generated that is incapable of leaving the ER. If expressed in HeLa cells in a tetracycline-inducible manner, this mutant accumulated in the ER and retained the endogenous ERGIC-53 in this compartment, thus preventing its recycling. Mistargeting of ERGIC-53 to the ER did not alter the gross morphology of the early secretory pathway, including the distribution of beta'-COP. However, it impaired the secretion of one major glycoprotein, identified as the precursor of the lysosomal enzyme cathepsin C, while overexpression of wild-type ERGIC-53 had no effect on glycoprotein secretion. Transport of two other lysosomal enzymes and three post-Golgi membrane glycoproteins was unaffected by inactivating the recycling of ERGIC-53. The results suggest that the recycling of ERGIC-53 is required for efficient intracellular transport of a small subset of glycoproteins, but it does not appear to be essential for the majority of glycoproteins.


Assuntos
Retículo Endoplasmático Rugoso/metabolismo , Lectinas/metabolismo , Lisossomos/enzimologia , Lectinas de Ligação a Manose , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Transporte Biológico Ativo , Catepsina C , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Expressão Gênica , Glicoproteínas/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Lectinas/genética , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , Transformação Genética
3.
J Biol Chem ; 272(44): 27737-44, 1997 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-9346916

RESUMO

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (alpha-SNAP) is a soluble protein that enables the NSF ATPase to associate with membranes and facilitate membrane trafficking events. Although NSF and alpha-SNAP have been shown to be required for many membrane transport processes, their role in the transport of mannose 6-phosphate receptors from endosomes to the trans Golgi network was not established. We present here a novel in vitro assay that monitors the transport of cation-dependent mannose 6-phosphate receptors between endosomes and the trans Golgi network. The assay relies on the trans Golgi network localization of tyrosine sulfotransferase and monitors transport of mannose 6-phosphate receptors engineered to contain a consensus sequence for modification by this enzyme. Using this new assay we show that alpha-SNAP strongly stimulates transport in reactions containing limiting amounts of cytosol. Together with alpha-SNAP, NSF can increase the extent of transport. These data show that alpha-SNAP, a soluble component of the SNAP receptor machinery, facilitates transport from endosomes to the trans Golgi network.


Assuntos
Proteínas de Transporte/fisiologia , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/fisiologia , Receptor IGF Tipo 2/metabolismo , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP , Sequência de Aminoácidos , Animais , Transporte Biológico/fisiologia , Células CHO , Cricetinae , GTP Fosfo-Hidrolases/metabolismo , Dados de Sequência Molecular , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Ácidos Sulfúricos/metabolismo , Tirosina/metabolismo
4.
Curr Biol ; 7(4): R235-7, 1997 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-9162499

RESUMO

The budding of transport vesicles from the Golgi complex is initiated by activation of the small GTPase ARF; the discovery of enzymes that can convert soluble ARF-GDP to the active, membrane-associated form ARF-GTP will shed light on the mechanism and regulation of the formation of transport vesicles.


Assuntos
Vesículas Revestidas/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Complexo de Golgi/fisiologia , Fatores de Troca do Nucleotídeo Guanina , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Fatores de Ribosilação do ADP , Animais , Vesículas Revestidas/ultraestrutura , Proteínas Fúngicas/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Guanosina Difosfato/metabolismo , Modelos Biológicos , Modelos Estruturais , Saccharomyces cerevisiae/ultraestrutura
5.
Mol Biol Cell ; 7(3): 483-93, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8868475

RESUMO

Based on sequence homologies with leguminous lectins, the intermediate compartment marker ERGIC-53 was proposed to be a member of a putative new class of animal lectins associated with the secretory pathway. Independent, a promyelocytic protein, MR60, was purified by mannose-column chromatography, and a cDNA was isolated that matched MR60 peptide sequences. This cDNA was identical to that of ERGIC-53 and homologies with the animal lectin family of the galectins were noticed. Not all peptide sequences of MR60, however, were found in ERGIC-53, raising the possibility that another protein associated with ERGIC-53 may possess the lectin activity. Here, we provide the first direct evidence for a lectin function of ERGIC-53. Overexpressed ERGIC-53 binds to a mannose column in a calcium-dependent manner and also co-stains with mannosylated neoglycoprotein in a morphological binding assay. By using a sequential elution protocol we show that ERGIC-53 has selectivity for mannose and low affinity for glucose and GlcNAc, but no affinity for galactose. To experimentally address the putative homology of ERGIC-53 to leguminous lectins, a highly conserved protein family with an invariant asparagine essential for carbohydrate binding, we substituted the corresponding asparagine in ERGIC-53. This mutation, as well as a mutation affecting a second site in the putative carbohydrate recognition domain, abolished mannose-column binding and co-staining with mannosylated neoglycoprotein. These findings establish ERGIC-53 as a lectin and provide functional evidence for its relationship to leguminous lectins. Based on its monosaccharide specificity, domain organization, and recycling properties, we propose ERGIC-53 to function as a sorting receptor for glyco-proteins in the early secretory pathway.


Assuntos
Cálcio/metabolismo , Lectinas de Ligação a Manose , Manose/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Transformada , Chlorocebus aethiops , Epitopos , Expressão Gênica , Humanos , Lectinas/química , Manose/farmacologia , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , Relação Estrutura-Atividade
6.
J Cell Biol ; 131(1): 57-67, 1995 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7559786

RESUMO

ERGIC-53 is a lectin-type membrane protein that continuously recycles between the ER, ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi. To identify the targeting signals that mediate this recycling, N-glycosylated and myc-tagged variants of ERGIC-53 were constructed. By monitoring endoglycosidase H resistance, we measured the loss from the ER-ERGIC-cis-Golgi cycle of ERGIC-53. A domain exchange approach with the plasma membrane reporter protein CD4 showed that the transmembrane and the lumenal domains are not sufficient, while the cytoplasmic domain of ERGIC-53 is required and sufficient for pre-medial-Golgi localization. However, the ERGIC-53 cytoplasmic domain on CD4 lead to increased ER-staining by immunofluorescence microscopy indicating that this domain alone cannot provide for unbiased recycling through the ER-ERGIC-cis-Golgi compartments. Complete progress through the ER-ERGIC-cis-Golgi recycling pathway requires the cytoplasmic domain acting together with the lumenal domain of ERGIC-53. Dissection of the cytoplasmic domain revealed a COOH-terminal di-lysine ER-retrieval signal, KKFF, and an RSQQE targeting determinant adjacent to the transmembrane domain. Surprisingly, the two COOH-terminal phenylalanines influence the targeting. They reduce the ER-retrieval capacity of the di-lysine signal and modulate the RSQQE determinant.


Assuntos
Retículo Endoplasmático Rugoso/metabolismo , Complexo de Golgi/metabolismo , Lectinas de Ligação a Manose , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular/metabolismo , Epitopos/metabolismo , Glicosilação , Humanos , Proteínas de Membrana/ultraestrutura , Dados de Sequência Molecular , Fenilalanina/fisiologia , Proteínas Recombinantes/metabolismo
8.
EMBO J ; 14(10): 2250-6, 1995 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-7774583

RESUMO

Membrane proteins often contain a sorting signal in their cytoplasmic tail that promotes their clustering into coated vesicles at a specific cellular site. ERGIC-53 contains a cytoplasmic ER-retrieval signal, KKFF. However, overexpressed ERGIC-53 is transported to the cell surface and rapidly endocytosed. Here we report that ERGIC-53 carries a previously undescribed endocytosis signal. Surprisingly, the signal was KKFF and like the ER-retrieval signal required a C-terminal position. In fact, the minimal consensus sequence determined by substitutional mutagenesis (K-K/R-F/Y-F/Y) was related to the ER-retrieval consensus (K-K-X-X). Furthermore, we provide evidence that internalization of VIP36, a protein that cycles between plasma membrane and Golgi, is mediated by a signal at its C-terminus that matches the internalization consensus sequence. The relatedness of the two signals suggests that coatomer-mediated retrieval of proteins may be mechanistically more related to clathrin-dependent sorting than previously anticipated.


Assuntos
Compartimento Celular/fisiologia , Endocitose/fisiologia , Retículo Endoplasmático/metabolismo , Lectinas de Ligação a Manose , Proteínas de Membrana/metabolismo , Oligopeptídeos/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Compartimento Celular/genética , Membrana Celular/metabolismo , Células Cultivadas , Sequência Consenso , Genes Reporter , Complexo de Golgi , Proteínas de Membrana/genética , Dados de Sequência Molecular , Oligopeptídeos/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
9.
J Biol Chem ; 269(9): 6279-81, 1994 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-8119975

RESUMO

ERGIC-53 (former designation, p53) is a 53-kDa nonglycosylated, dimeric, and hexameric type I membrane protein that has been established as a marker protein for a tubulovesicular intermediate compartment in which protein transport from the endoplasmic reticulum to the Golgi apparatus is blocked at 15 degrees C. Although ERGIC-53 is not a resident protein of the rough endoplasmic reticulum its cDNA sequence carries a double lysine endoplasmic reticulum retention motif at the cytoplasmically exposed COOH terminus. Here we report that overexpression of ERGIC-53 in COS cells saturates its intracellular retention system leading to the appearance of ERGIC-53 at the cell surface. Cell surface ERGIC-53 is efficiently endocytosed by a mechanism that is disturbed when the two critical lysines of the endoplasmic reticulum retention motif are replaced by serines. The results suggest a mechanistic similarity of pre-Golgi retention by the double lysine motif and lysine-based endocytosis.


Assuntos
Endocitose , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Lisina , Lectinas de Ligação a Manose , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , DNA Complementar/metabolismo , Imunofluorescência , Cinética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Dados de Sequência Molecular , Serina , Transfecção
10.
Eur J Cell Biol ; 61(1): 1-9, 1993 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8223692

RESUMO

Overlapping cDNAs encoding the entire human ERGIC-53, a 53 kDa membrane protein of the ER-Golgi intermediate compartment, have been isolated and their nucleotide sequence determined. The isolated cDNA is about 2.7 kb in length. The deduced polypeptide chain for ERGIC-53 consists of 510 amino acids (M(r) 54217) including an N-terminal signal sequence of 30 amino acids, a single putative transmembrane segment of 18 amino acids, and a short cytoplasmic domain of 12 amino acids. Surprisingly, the cytoplasmic segment contains two lysines positioned three and four residues from the C-terminus. Such a double lysine motif is known to function as a retention signal for a group of membrane proteins associated with the ER. Expression of a full-length cDNA of ERGIC-53 in Vero cells revealed intracellular localization similar but not always identical to the endogenously expressed ERGIC-53. The presence of an ER retention motif in a protein of the ER-Golgi intermediate compartment has important implications for the retention mechanism mediated by this signal.


Assuntos
Compartimento Celular/fisiologia , Retículo Endoplasmático/química , Complexo de Golgi/química , Lisina/análise , Lectinas de Ligação a Manose , Proteínas de Membrana/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/química , Código Genético , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Peso Molecular , Oligonucleotídeos/síntese química
11.
Mol Biochem Parasitol ; 58(1): 135-43, 1993 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8459825

RESUMO

Aldolase of the human malaria parasite Plasmodium falciparum (PfAldo) may be a potential target for the development of novel antimalarial drugs. Using in vitro mutagenesis we analyzed the function of the carboxy-terminus of the recombinant enzyme. Deletion of the carboxy-terminus of PfAldo confirmed its critical role in catalysis; exchange of conserved residues minimally affected enzyme activity. We exchanged a pair of parasite specific lysine residues with corresponding amino acids of the host. These mutant enzymes exhibited an increased catalytic activity and reduced binding to erythrocyte band 3 protein. Homologous peptides of human band 3 protein and P. falciparum alpha-tubulin were competitive inhibitors of PfAldo. Selective inhibition of PfAldo by the alpha-tubulin peptide depends on the presence of tandem lysine residues and the fine structure of the inhibitor peptide. Our data support the concept of a matrix organisation of glycolytic enzymes in Plasmodium falciparum.


Assuntos
Frutose-Bifosfato Aldolase/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Plasmodium falciparum/enzimologia , Tubulina (Proteína)/farmacologia , Sequência de Aminoácidos , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Sítios de Ligação , Catálise , Sequência Conservada , Proteínas da Matriz Extracelular/metabolismo , Frutose-Bifosfato Aldolase/genética , Lisina/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/metabolismo , Plasmodium falciparum/genética , Deleção de Sequência , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo
13.
Acta Leiden ; 60(1): 135-40, 1991.
Artigo em Inglês | MEDLINE | ID: mdl-1820702

RESUMO

P. falciparum lacks a functional citric acid cycle. Unlike most tissues of the mammalian host, it is totally dependent on glycolysis for energy generation. A compound which selectively inhibits the parasite's ATP-generating machinery is therefore a potential antimalarial agent. Such a drug may interact in two ways: a) by inhibiting the activity of an enzyme or b) by disturbing the micro-organization of consecutive enzymes in a metabolic pathway. In mammalian tissues the glycolytic pathway involves the cytoskeleton as a matrix to keep phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase in an optimal sterical position for rapid substrate conversion. For instance, these three enzymes bind to the band 3 protein in erythrocytes or to actin in muscle cells. P. falciparum aldolase binds with very high affinity to the band 3 protein of human erythrocyte ghosts. However, the true in vivo site of association is believed to be actin II of P. falciparum. This actin has a sequence element which is almost identical to that of the band 3 aldolase binding site. We therefore suppose that plasmodia exploit a similar matrix organization. If true, the association of these enzymes with the cytoskeleton is a target for novel antimalarials. In contrast to all vertebrate aldolases, P. falciparum and P. berghei aldolases have two neighbouring lysine residues near the carboxy-terminus. We show here that mutagenesis of these basic residues has an effect on the catalytic constants Vmax and KM and moreover, the ability to bind to band 3 is reduced.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Frutose-Bifosfato Aldolase/antagonistas & inibidores , Malária/prevenção & controle , Plasmodium falciparum/enzimologia , Sequência de Aminoácidos , Antimaláricos , Sequência de Bases , Desenho de Fármacos , Resistência a Medicamentos/genética , Frutose-Bifosfato Aldolase/genética , Dados de Sequência Molecular , Plasmodium falciparum/efeitos dos fármacos
14.
Invest Urol ; 13(5): 346-50, 1976 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-815197

RESUMO

Previous reports have suggested that urease-producing bacteria play a prominent role in the formation of infection-induced urinary stones. We have carried out crystalization experiments in vitro which show that bacterial urease alkalinizes urine, thereby causing: (i) supersaturation with respect to struvite and calcium phosphate; and (ii) formation of struvite and apatite crystals. Growth of Proteus in urea-free urine or in urine which contained a urease inhibitor did not cause alkalinization, supersaturation, or crystallization of struvite and apatite. Growth of Klebsiella, Escherichia coli, or Pseudomonas was not associated with significant alkalinization, supersaturation, or crystallization. Struvite and apatite crystals dissolved in Proteus-infected urine in which undersaturation was maintained by urease inhibition. Similar results in all experiments were obtained using human urine and a synthetic urine which was devoid of matrix, pyrophosphate, or other undefined solutes. Urease-induced supersaturation appears to be the primary cause of infection-induced urinary stones.


Assuntos
Bactérias/enzimologia , Infecções Bacterianas/urina , Urease/urina , Cálculos Urinários/urina , Infecções Urinárias/urina , Infecções Bacterianas/complicações , Cristalização , Humanos , Klebsiella/enzimologia , Masculino , Infecções por Proteus/urina , Pseudomonas aeruginosa/enzimologia , Urease/metabolismo , Cálculos Urinários/etiologia , Infecções Urinárias/complicações , Urina
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...