Budding vesicles in living cells.
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Biological Transport↗
Biomedical subjects
L Orci
Publications and source records attributed to L Orci.
A v-SNARE implicated in intra-Golgi transport.
We report the identification of a putative v-SNARE (GOS-28), localized primarily to transport vesicles at the terminal rims of Golgi stacks. In vitro, GOS-28, A Golgi SNARE of 28 kD, is efficiently packaged into Golgi-derived vesicles, which are most likely COPI coated. Antibodies directed against GOS-28 block its ability to bind alpha-SNAP, partially inhibit transport from the cis to the medial cisternae, and do not inhibit budding of COP-coated vesicles, but do accumulate docked uncoated vesicles.
Amino Acid Sequence↗
Architecture of coatomer: molecular characterization of delta-COP and protein interactions within the complex.
Coatomer is a cytosolic protein complex that forms the coat of COP I-coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.
Amino Acid Sequence↗
A major transmembrane protein of Golgi-derived COPI-coated vesicles involved in coatomer binding.
Formation of non-clathrin-coated vesicles requires the recruitment of several cytosolic factors to the Golgi membrane. To identify membrane proteins involved in this budding process, a highly abundant type I transmembrane protein (p23) was isolated from mammalian Golgi-derived COPI-coated vesicles, and its cDNA was cloned and sequenced. It belongs to the p24 family of proteins involved in the budding of transport vesicles (Stamnes, M.A., M.W. Craighead, M.H. Hoe, N. Lampen, S. Geromanos, P. Tempst, and J.E. Rothman. 1995. Proc. Natl. Acad. Sci. USA. 92:8011-8015). p23 consists of a large NH2-terminal luminal domain and a short COOH-terminal cytoplasmic tail (-LRRFFKAKKLIE-CO2-) that shows similarity, but not identity, with the sequence motif-KKXX-CO2-, known as a signal for retrieval of escaped ER-resident membrane proteins (Jackson, M.R., T. Nilsson, and P.A. Peterson. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:3153-3162; Nilsson, T., M. Jackson, and P.A. Peterson. 1989. Cell. 58:707-718). The cytoplasmic tail of p23 binds to coatomer with similar efficiency as known KKXX motifs. However, the p23 tail differs from the KKXX motif in having an additional motif needed for binding of coatomer. p23 is localized to Golgi cisternae and, during vesicle formation, it concentrates into COPI-coated buds and vesicles. Biochemical analysis revealed that p23 is enriched in vesicles by a factor of approximately 20, as compared with the donor Golgi fraction, and is present in amounts stoichiometric to the small GTP-binding protein ADP-ribosylation factor (ARF) and coatomer. From these data we conclude that p23 represents a Golgi-specific receptor for coatomer involved in the formation of COPI-coated vesicles.
Amino Acid Sequence↗
Cloning and functional characterization of mammalian homologues of the COPII component Sec23.
We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa. hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p. Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p. Cytosolic hSec23Ap migrated with an apparent molecular weight of 350 kDa on a gel filtration column, suggesting that it is part of a protein complex. By immunoelectron microscopy, hSec23Ap was found essentially in the ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles. hSec23Ap is a functional homologue of the yeast Sec23p as the hSec23A isoform complemented the temperature sensitivity of the Saccharomyces cerevisiae sec23-1 mutation at a restrictive temperature of 34 degrees C. RNase protection assays indicated that both hSec23 isoforms are coexpressed in various human tissues, although at a variable ratio. Our data demonstrate that hSec23Ap is the functional human counterpart of the yeast COPII component Sec23p and suggest that it plays a similar role in mammalian protein export from the ER. The exact function of hSec23Bp remains to be determined.
Animals↗
Angiogenesis: a paradigm for balanced extracellular proteolysis during cell migration and morphogenesis.
Extracellular proteolysis is required for matrix degradation and the regulation of cytokine activity during angiogenesis, and this is dependent on a cohort of proteases and protease inhibitors produced by endothelial and nonendothelial cells. The plasminogen activator (PA)/plasmin system has been extensively investigated in these processes, and descriptive studies have demonstrated that urokinase-type PA (uPA), uPA receptor (uPAR) and PA inhibitor-1 (PAI-1) are expressed by endothelial cells during angiogenesis in vivo. In vitro studies have led to the notion that normal capillary morphogenesis is dependent on a protease-antiprotease equilibrium. These findings are discussed in the context of recent observations on uPA-, uPAR-, PAI-1 and plaminogen-deficient mice, in which developmental and physiological angiogenesis appear to occur normally. This has led to a reevaluation of the role of the PA/plasmin system during angiogenesis. In particular, these observations raise the possibility that the role of this system may be limited to situations in which endothelial cells encounter and must degrade fibrin in order to form new capillary sprouts.
Animals↗
Synergistic effect of hyaluronan oligosaccharides and vascular endothelial growth factor on angiogenesis in vitro.
The aim of the present study was to determine whether hyaluronan (HA) degradation products, which have been shown to be angiogenic in vivo, influence endothelial cell invasion of a 3-dimensional matrix, an essential component of the neovascularization process. Using a previously described in vitro assay, we demonstrate that like the angiogenic cytokines basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), HA oligosaccharides (OHA) induce bovine microvascular endothelial cells to invade a 3-dimensional collagen gel within which they form capillary-like tubes, with an optimal effect at approximately 0.5 to 2 micrograms/ml. Strikingly, co-addition of OHA (0.5 - 2 micrograms/ml) and VEGF (30 ng/ml), but not co-addition of OHA and bFGF (10 ng/ml), induced an in vitro angiogenic response that was greater than the sum of the effects elicited by either agent separately. In contrast to OHA, native high molecular weight HA was consistently inactive, whether added alone or in combination with VEGF or bFGF. Because endothelial cell invasion is believed to require extracellular proteolytic activity, we also investigated the effect of OHA on the plasminogen activator (PA)-plasmin system. OHA (0.01 to 1 microgram/ml) but not native high molecular weight HA induced a dose-dependent increase in mRNA levels of urokinase type PA (uPA), urokinase type PA receptor and PA inhibitor type 1, and a parallel increase in the functional activity of urokinase type PA and PA inhibitor type 1, as determined by zymography and reverse zymography, respectively. The effects of OHA on proteolytic activity were additive with those of VEGF, but not with those of bFGF. Taken together, these results demonstrate that OHA modulate the invasive and proteolytic properties of bovine microvascular endothelial cells and synergize specifically with VEGF in the induction of angiogenesis in vitro. We suggest that the synergism between OHA and VEGF plays a role in the regulation of angiogenesis and that it may be exploited therapeutically in situations that would benefit from stimulation of new blood vessel growth.
Adrenal Cortex↗
COPI- and COPII-coated vesicles bud directly from the endoplasmic reticulum in yeast.
The cytosolic yeast proteins Sec13p-Sec31p, Sec23p-Sec24p, and the small GTP-binding protein Sar1p generate protein transport vesicles by forming the membrane coat termed COPII. We demonstrate by thin section and immunoelectron microscopy that purified COPII components form transport vesicles directly from the outer membrane of isolated yeast nuclei. Another set of yeast cytosolic proteins, coatomer and Arf1p (COPI), also form coated buds and vesicles from the nuclear envelope. Formation of COPI-coated, but not COPII-coated, buds and vesicles on the nuclear envelope is inhibited by the fungal metabolite brefeldin A. The two vesicle populations are distinct. However, both vesicle types are devoid of endoplasmic reticulum (ER) resident proteins, and each contains targeting proteins necessary for docking at the Golgi complex. Our data suggest that COPI and COPII mediate separate vesicular transport pathways from the ER.
Anti-Bacterial Agents↗
TGF-beta 1 overexpression in murine pancreas induces chronic pancreatitis and, together with TNF-alpha, triggers insulin-dependent diabetes.
We have generated transgenic mice overexpressing TGF-beta 1 in pancreatic beta cells. This resulted in massive fibrosis of the pancreas; in adult mice, most of the acini were replaced by fibrotic and adipose tissues. A conspicuous disorganization of the islets of Langerhans was also observed; however, the number of beta cells was not decreased and the mice were normoglycemic. Backcrossing to transgenic mice overexpressing TNF-alpha in their islet beta cells (which also remain normoglycemic, (1)) yielded double transgenics, most of which became diabetic by the age of 4 months; histological analysis revealed a dramatic decrease in insulin-containing beta cells.
Amylases↗
Non-clathrin-coat protein alpha is a conserved subunit of coatomer and in Saccharomyces cerevisiae is essential for growth.
To complete the molecular characterization of coatomer, the preformed cytosolic complex that is involved in the formation of biosynthetic transport vesicles, we have cloned and characterized the gene for non-clathrin-coat protein alpha (alpha-COP) from Saccharomyces cerevisiae. The derived protein, molecular weight of 135,500, contains four WD-40 repeated motifs (Trp/Asp-containing motifs of approximately 40 amino acids). Disruption of the yeast alpha-COP gene is lethal. Comparison of the DNA-derived primary structure with peptides from bovine alpha-COP shows a striking homology. alpha-COP is localized to coated transport vesicles and coated buds of Golgi membranes derived from CHO cells.
Amino Acid Sequence↗
Modulation of hepatocyte growth factor and c-met in the rat mammary gland during pregnancy, lactation, and involution.
Epithelial tubulogenesis is responsible for the exquisitely intricate organization of functional units of parenchymal organs. We have previously demonstrated that hepatocyte growth factor (HGF--also known as scatter factor) is a stroma-derived epithelial morphogen, which induces tubulogenesis by kidney-derived epithelial cells in vitro. The mammary gland provides a particularly attractive model for the study of epithelial morphogenesis, since its development in postnatal life involves elongation and branching of epithelial tubules. The aim of the present studies was to assess the expression and modulation of HGF and its receptor c-Met in the rat mammary gland during pregnancy, lactation, and involution. By ribonuclease protection assay, we demonstrate that levels of both HGF and c-met transcripts are progressively reduced during pregnancy, are virtually undetectable during lactation, and increase during the phase of involution to prepregnancy levels. The reduction in HGF and c-met expression corresponds to periods in which functions other than tubulogenesis predominate in the mammary gland, namely alveologenesis (mid to late pregnancy) and milk protein synthesis (lactation). Using a murine mammary gland-derived epithelial cell line, we demonstrate that levels of c-met mRNA are significantly reduced by exogenously added prolactin, providing a possible explanation for the reduction in c-met in the rat mammary gland during lactation. The potential significance of down-regulation of HGF/c-met during lactation is discussed.
Amino Acid Sequence↗
Human SEC13Rp functions in yeast and is located on transport vesicles budding from the endoplasmic reticulum.
In the yeast Saccharomyces cerevisiae, Sec13p is required for intracellular protein transport from the ER to the Golgi apparatus, and has also been identified as a component of the COPII vesicle coat structure. Recently, a human cDNA encoding a protein 53% identical to yeast Sec13p has been isolated. In this report, we apply the genetic assays of complementation and synthetic lethality to demonstrate the conservation of function between this human protein, designated SEC13Rp, and yeast Sec13p. We show that two reciprocal human/yeast fusion constructs, encoding the NH2-terminal half of one protein and the COOH-terminal half of the other, can each complement the secretion defect of a sec13-1 mutant at 36 degrees C. The chimera encoding the NH2-terminal half of the yeast protein and the COOH-terminal half of the human protein is also able to complement a SEC13 deletion. Overexpression of either the entire human SEC13Rp protein or the chimera encoding the NH2-terminal half of the human protein and the COOH-terminal half of the yeast protein inhibits the growth of a sec13-1 mutant at 24 degrees C; this growth inhibition is not seen in a wild-type strain nor in other sec mutants, suggesting that the NH2-terminal half of SEC13Rp may compete with Sec13-1p for a common target. We show by immunoelectronmicroscopy of mammalian cells that SEC13Rp (like the putative mammalian homologues of the COPII subunits Sar1p and Sec23p) resides in the region of the transitional ER. We also show that the distribution of SEC13Rp is not affected by brefeldin A treatment. This report presents the first demonstration of a putative mammalian COPII component functioning in yeast, and highlights a potentially useful approach for the study of conserved mammalian proteins in a genetically tractable system.
Amino Acid Sequence↗
Adequate connexin-mediated coupling is required for proper insulin production.
To assess whether connexin (Cx) expression contributes to insulin secretion, we have investigated normal and tumoral insulin-producing cells for connexins, gap junctions, and coupling. We have found that the glucose-sensitive cells of pancreatic islets and of a rat insulinoma are functionally coupled by gap junctions made of Cx43. In contrast, cells of several lines secreting insulin abnormally do not express Cx43, gap junctions, and coupling. After correction of these defects by stable transfection of Cx43 cDNA, cells expressing modest levels of Cx43 and coupling, as observed in native beta-cells, showed an expression of the insulin gene and an insulin content that were markedly elevated, compared with those observed in both wild-type (uncoupled) cells and in transfected cells overexpressing Cx43. These findings indicate that adequate levels of Cx-mediated coupling are required for proper insulin production and storage.
Animals↗
Coat proteins and selective protein packaging into transport vesicles.
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Amino Acid Sequence↗
Leukemia inhibitory factor (LIF) inhibits angiogenesis in vitro.
Using an in vitro model in which endothelial cells can be induced to invade a three-dimensional collagen gel to form capillary-like tubular structures, we demonstrate that leukemia inhibitory factor (LIF) inhibits angiogenesis in vitro. The inhibitory effect was observed on both bovine aortic endothelial (BAE) and bovine microvascular endothelial (BME) cell, and occurred irrespective of the angiogenic stimulus, which included basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), the synergistic effect of the two in combination, or the tumor promoter phorbol myristate acetate. LIF inhibited bFGF- and VEGF-induced proliferation in BAE and BME cells. In addition, LIF inhibited BAE but not BME cell migration in a conventional two-dimensional assay. Finally, LIF decreased the proteolytic activity of BAE and BME cells and increased their expression of plasminogen activator inhibitor-1. These results demonstrate that LIF inhibits angiogenesis in vitro, an effect that can be correlated with a LIF-mediated decrease in endothelial cell proliferation, migration and extracellular proteolysis.
Adrenal Cortex↗
Hepatocyte growth factor stimulates extensive development of branching duct-like structures by cloned mammary gland epithelial cells.
Although epithelial-mesenchymal (stromal) interactions are thought to play an important role in embryonic and postnatal development of the mammary gland, the underlying mechanisms are still poorly understood. To address this issue, we assessed the effect of fibroblast-derived diffusible factors on the growth and morphogenetic properties of a clonally derived subpopulation (clone TAC-2) of normal murine mammary gland (NMuMG) epithelial cells embedded in collagen gels. Under control conditions, TAC-2 mammary gland epithelial cells suspended within collagen gels formed either irregularly shaped cell aggregates or short branching cord-like structures. Addition of conditioned medium from Swiss 3T3 or MRC-5 fibroblasts dramatically stimulated cord formation by TAC-2 cells, resulting in the development of an extensive, highly arborized system of duct-like structures, which in appropriate sections were seen to contain a central lumen. The effect of fibroblast conditioned medium was completely abrogated by antibodies against hepatocyte growth factor (also known as scatter factor), a fibroblast-derived polypeptide that we have previously shown induces tubulogenesis by Madin-Darby canine kidney epithelial cells. Addition of exogenous recombinant human hepatocyte growth factor to collagen gel cultures of TAC-2 cells mimicked the tubulogenic activity of fibroblast conditioned medium by stimulating formation of branching duct-like structures in a dose-dependent manner, with a maximal 77-fold increase in cord length at 20 ng/ml. The effect of either fibroblast conditioned medium or hepatocyte growth factor was markedly potentiated by the simultaneous addition of hydrocortisone (1 microgram/ml), which also enhanced lumen formation. These results demonstrate that hepatocyte growth factor promotes the formation of branching duct-like structures by mammary gland epithelial cells in vitro, and suggest that it may act as a mediator of the inducing effect of mesenchyme (or stroma) on mammary gland development.
Animals↗
Ablation of islet endocrine cells by targeted expression of hormone-promoter-driven toxigenes.
Ontogenic relationships between the different types of endocrine cells in the islets of Langerhans were explored by generating transgenic mouse embryos in which cells transcribing the glucagon, insulin, or pancreatic polypeptide genes were destroyed through the promoter-targeted expression of the diphtheria toxin A chain. Embryos lacking glucagon- or insulin-containing cells did not exhibit alterations in the development of the nontargeted islet cell types, whereas embryos lacking pancreatic polypeptide gene-expressing cells also lacked pancreatic insulin- and somatostatin-containing cells. These results show that neither glucagon nor insulin gene-expressing cells are essential for the differentiation of the other islet endocrine-cell types. These results also suggest that pancreatic polypeptide gene-expressing cells are indispensable for the differentiation of islet beta and delta cells because the former produce a necessary paracrine or endocrine factor and/or operate through a cell-lineage relationship.
Animals↗
Coatomer-rich endoplasmic reticulum.
We identify in normal cells the existence of two distinct sites of the transitional endoplasmic reticulum (ER), one housing the Sec23p protein complex (the classical transitional element), the other the coatomer protein complex (the coatomer-rich ER). Experimental conditions that reduce transport from the ER to the Golgi complex lead to the overexpression of this newly defined coatomer-rich ER.
Animals↗