RESUMO
Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20 degrees C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.
Assuntos
Compartimento Celular , Endossomos/metabolismo , Glicoproteínas , Proteínas de Membrana , Transporte Proteico/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Rede trans-Golgi/metabolismo , Células HeLa , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Melanoma Experimental , Glicoproteínas de Membrana/metabolismo , Mutação , Proteínas de Neoplasias/metabolismo , Receptor IGF Tipo 2/metabolismo , Receptores da Transferrina/metabolismo , Toxina Shiga/metabolismo , Sialiltransferases/metabolismoRESUMO
We recently identified a region of preferential replication initiation, oriGNAI3, near the 3' end of the Chinese hamster GNAI3 gene. oriGNAI3 is co-amplified in mutants selected for AMPD2 amplification, a process generating chromosomal rearrangements. In this report we have taken advantage of cell lines with truncated and translocated amplified units to show that these rearrangements do not alter the function of ori GNAI3. These results indicate that replication initiation at this locus relies essentially on local features. Interestingly, the study of one line in which a rearrangement has disrupted the GNAI3 gene shows that ongoing transcription of this gene is not required for initiation at oriGNAI3. In order to obtain further insight into the sequences and/or chromatin structures required for oriGNAI3 function, we have analyzed the DNase I sensitivity and nucleotide sequence of the region. The features important for replication initiation appear to cluster in a 7-12 kb region which includes oriGNAI3.
Assuntos
Cromatina/genética , Replicação do DNA , Origem de Replicação/genética , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , Cromatina/química , Cricetinae , Cricetulus , Primers do DNA , Hibridização in Situ FluorescenteRESUMO
The nature of mammalian origins of DNA replication remains controversial and this is primarily because two-dimensional gel replicon mapping techniques have identified broad zones of replication initiation whereas several other techniques, such as quantitative PCR, have disclosed more discrete sites of initiation at the same chromosomal loci. In this report we analyze the replication of an amplified genomic region encompassing the 3'-end of the GNAI3 gene, the entire GNAT2 gene and the intergenic region between them in exponentially growing Chinese hamster fibroblasts. These cells express GNAI3 but not GNAT2 . The replication pattern was first analyzed by two-dimensional neutral-alkaline gel electrophoresis. Surprisingly, the results revealed a small preferential zone of replication initiation, of at most 1.7 kb, located in a limited part of the GNAI3 - GNAT2 intergenic region. Mapping of this initiation zone was then confirmed by quantitative PCR. The agreement between the two techniques exploited here strengthens the hypothesis that preferred sites of replication initiation do exist in mammalian genomes.
Assuntos
Replicação do DNA/genética , Eletroforese em Gel Bidimensional/métodos , Proteínas de Ligação ao GTP/genética , Reação em Cadeia da Polimerase/métodos , Origem de Replicação/genética , AMP Desaminase/genética , Animais , Células CHO , Mapeamento Cromossômico , Cricetinae , Dados de Sequência Molecular , RepliconAssuntos
Cromossomos Humanos Par 1 , Cisteína Endopeptidases/genética , Complexos Multienzimáticos/genética , Animais , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Cricetinae , Cricetulus , Ligação Genética , Células HeLa , Humanos , Células Híbridas , Complexo de Endopeptidases do ProteassomaRESUMO
As a first step towards developing a set of compartment-specific probes for studying protein trafficking in the malaria-infected erythrocyte, we describe here a family of Plasmodium falciparum Rab proteins. We characterise in detail P. falciparum Rab6 (PfRab6) a marker which in other cells is specific for the Golgi/trans Golgi network. Although PfRab6 mRNA is expressed throughout the intraerythrocytic cycle, maximal expression occurs at the trophozoite stage. Immunofluorescence microscopy shows that the distribution of PfRab6 changes during the final stages of parasite maturation, coalescing into multiple foci, each of which is associated with the nucleus of a forming daughter parasite.
Assuntos
Proteínas de Transporte/análise , Proteínas de Transporte/genética , Genes de Protozoários , Plasmodium falciparum/genética , Proteínas rab de Ligação ao GTP , Proteínas ras/análise , Proteínas ras/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Núcleo Celular/química , Clonagem Molecular , DNA Complementar/genética , Eritrócitos/parasitologia , Expressão Gênica , Microscopia de Fluorescência , Dados de Sequência Molecular , Plasmodium falciparum/química , Plasmodium falciparum/crescimento & desenvolvimento , Reação em Cadeia da Polimerase , Proteínas de Protozoários/análise , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas ras/químicaRESUMO
Artificial fatty acylation of proteins has attracted significant attention during the last decade as a method for modification of protein specificity and efficacy of action on mammalian cells (A. V. Kabanov and V. Yu. Alakhov (1994) J. Contr. Release 28, 15-35). Horse radish peroxidase (HRP) is used in this work to study the interaction of a fatty acylated protein with various mammalian cells. The HRP is modified with the chloranhydride of the stearic acid in the reversed micelles of sodium bis-(2-ethylhexyl)sulfosuccinate (Aerosol OT) in octane, a convenient protocol allowing production of protein molecules with a controlled, low modification degree (A.V. Kabanov et al. (1987) Ann. N. Y. Acad. Sci. 501, 63-66). The influence of the hydrophobic group on the binding and internalization of HRP in MDCK, P3-X63-Ag8, CHO, and HepG2 cells is examined. The major results are as follows: (i) the fatty acylation of a protein significantly enhances its binding to all tested mammalian cell lines, with a line-specific efficiency; (ii) the binding efficiency can be modified by changing growth conditions in a defined medium; (iii) along with the enhancement of protein adsorption on the plasma membrane, fatty acylation increases internalization of the protein during incubations at 37 degrees C; (iv) internalized protein was observed in endocytic vesicles; no evidence was obtained for a cytoplasmic distribution. These results are discussed in connection with previously observed effects of the fatty acylated proteins on cell activity.
Assuntos
Proteínas/química , Proteínas/metabolismo , Acilação , Adsorção , Animais , Transporte Biológico Ativo , Células CHO , Linhagem Celular , Membrana Celular/metabolismo , Cricetinae , Cães , Ácidos Graxos/química , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Camundongos , Micelas , Modelos Biológicos , Processamento de Proteína Pós-Traducional , Frações Subcelulares/metabolismoRESUMO
The identity of a gene coamplified with the adenylate deaminase 2 gene (AMPD2) in coformycin-resistant cells was determined by analysis of its genomic sequence. Sequence comparisons reveal a significant homology with the 3' terminal part of the gene encoding the alpha i3 subunit of Gi proteins from several species (GNAI3). Identification of the gene was confirmed by Western blot analysis of its products. A precise sequence comparison was performed with the human genomic sequence. It showed that conservation remains important in noncoding exons as well as in introns. However, sequences corresponding to combined U6 snRNA and E protein pseudogene, previously identified inside intron 7 of the human gene, were not found in the Chinese hamster gene. GNAI3 is mapped to a region of conserved linkage between human chromosome 1 (locus 1p13) and mouse chromosome 3 (at 48.4 cM). The Chinese hamster GNAI3 gene maps to chromosome 1 within a 120-kb fragment that also comprises the AMPD2 and GSTM genes.
Assuntos
AMP Desaminase/genética , Sequência Conservada , Íntrons , Proteínas/genética , RNA Nuclear Pequeno/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 1 , Clonagem Molecular , Cricetinae , Cricetulus , DNA , Humanos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosAssuntos
Proteínas de Ligação ao GTP/metabolismo , Complexo de Golgi/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular , Exocitose , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/isolamento & purificação , Insetos , Neurônios/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Vesículas Sinápticas/metabolismo , TransfecçãoRESUMO
We have recently localized a small GTP-binding protein (Rab6p) thought to be involved in vesicular membrane transport, to the medial and trans-cisternae of the Golgi apparatus in NRK (normal rat kidney) cells. Here, we have localized and quantified Rab6p during the development in culture of embryonic neurons, up to synapse formation, and compared its subcellular distribution and level of expression to that of synaptophysin, a major integral membrane protein of small synaptic vesicles. Using immunocytochemistry (laser scanning confocal microscopy, immunoelectron microscopy), fractionation and immunoisolation methods, we show that during the early phase of synaptogenesis, Rab6p is associated with synaptophysin-containing membranes of a trans-Golgi subcompartment, post-Golgi vesicles and small synaptic vesicles or their precursors. Concomitantly, Rab6p undergoes translocation from cytosol to membranes and its level of expression increases. However, at late stages, the association of Rab6p to small synaptic vesicles sharply decreases and its level of expression plateaus. These findings suggest a role for Rab6p in the post-Golgi transport of synaptophysin, at an early step of the biogenesis of small synaptic vesicles.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Hipotálamo/metabolismo , Neurônios/metabolismo , Animais , Especificidade de Anticorpos , Axônios/metabolismo , Transporte Biológico Ativo , Células Cultivadas , Proteínas de Ligação ao GTP/imunologia , Complexo de Golgi/metabolismo , Hipotálamo/embriologia , Hipotálamo/ultraestrutura , Imuno-Histoquímica , Camundongos , Frações Subcelulares/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptofisina/metabolismoRESUMO
A key role in the regulation of membrane traffic is played by the rab proteins, members of a family of ras-related small GTP-binding proteins. This family comprises at least 25 identified members, the intracellular localization of only a few of which has been investigated. rab6p has been shown to be distributed along the exocytic pathway in association with the medial and trans regions of the Golgi apparatus. A confocal laser scanning microscopic (CLSM) approach coupled with image analysis was used to compare the localization of rab6p with selected reference Golgi markers by double immunofluorescence on culture cell lines. CLSM analysis shows that, under a set of well-defined conditions, one can investigate the possible colocalization of known markers of Golgi compartments and orientate a couple of labeled Golgi antigens with regard to the polarity of the Golgi apparatus. Thus, having validated the CLSM analysis, the localization of rab6p was studied and compared with some of these markers and the VSV-G protein in VSV (vesicular stomatitis virus)-infected cells blocked at 20 degrees C. rab6p is shown to be associated in all the cell lines used with the last cisternae of the Golgi apparatus and particularly with the trans-Golgi network (TGN), the site of protein sorting at the exit of the Golgi apparatus. These results were supported by an electron microscopic study using double-immunolabeled cryosections: rab6p was found in some flat cisternae of the Golgi stack and colocalized with the VSV-G protein in the TGN. Our results show that the small GTP-binding protein rab6p is distributed from medial Golgi to TGN along the exocytic pathway.
Assuntos
Proteínas de Ligação ao GTP/análise , Glicoproteínas , Complexo de Golgi/química , Proteínas de Membrana , Transporte Biológico , Linhagem Celular , Complexo de Golgi/ultraestrutura , Imuno-Histoquímica , Manosidases/análise , Glicoproteínas de Membrana/análise , Microscopia/instrumentação , Microscopia/métodosRESUMO
Rab6 protein (rab6p) belongs to a family of ras-like GTP-binding proteins thought to be involved in the regulation of intracellular transport in mammalian cells. We have constructed a recombinant baculovirus in order to express rab6p in insect cells. We report here the characterization of four forms of this protein which are found in cytosolic and membrane fractions of infected Sf9 cells. The two major forms are a cytosolic 24 kD protein which represents the unprocessed precursor form of rab6p and a membrane-bound isoprenylated 23 kD protein which represents the processed form. Two other minor forms were also detected: a cytosolic isoprenylated 23 kD protein which may represent a pool in equilibrium with the 23 kD membrane-bound form and a 24 kD non-isoprenylated membrane-bound form which may represent an intermediate in the processing of rab6p.
Assuntos
Proteínas de Ligação ao GTP/isolamento & purificação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas p21(ras)/isolamento & purificação , Animais , Baculoviridae/genética , Western Blotting , Linhagem Celular , Membrana Celular/metabolismo , Clonagem Molecular/métodos , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Insetos , Peso Molecular , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , TransfecçãoRESUMO
Alignment of published 16S rRNA sequences allowed the definition of a pair of oligonucleotides suitable for polymerase chain reaction (PCR). Using this pair of PCR primers, several mycoplasmas including the four human parasites Mycoplasma genitalium, M. hominis, M. salivarium and M. orale were detected. This DNA amplification was restricted to species of the genus Mycoplasma while no cross-reaction was observed with DNA from other bacteria and eukaryotic cells. Subsequent analysis of amplified products by either specific oligonucleotide hybridization or dideoxy sequencing specified the identity of the detected mycoplasmas. This method offers a highly discriminating and sensitive assay for the direct detection and identification of these microorganisms without the need for prior cultivation.