Inhibition of secretion by 1,3-Cyclohexanebis(methylamine), a dibasic compound that interferes with coatomer function.
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We noted previously that certain aminoglycoside antibiotics inhibit the binding of coatomer to Golgi membranes in vitro. The inhibition is mediated in part by two primary amino groups present at the 1 and 3 positions of the 2-deoxystreptamine moiety of the antibiotics. These two amines appear to mimic the epsilon-amino groups present in the two lysine residues of the KKXX motif that is known to bind coatomer. Here we report the effects of 1, 3-cyclohexanebis(methylamine) (CBM) on secretion in vivo, a compound chosen for study because it contains primary amino groups that resemble those in 2-deoxystreptamine and it should penetrate lipid bilayers more readily than antibiotics. CBM inhibited coatomer binding to Golgi membranes in vitro and in vivo and inhibited secretion by intact cells. Despite depressed binding of coatomer in vivo, the Golgi complex retained its characteristic perinuclear location in the presence of CBM and did not fuse with the endoplasmic reticulum (ER). Transport from the ER to the Golgi was also not blocked by CBM. These data suggest that a full complement of coat protein I (COPI) on membranes is not critical for maintenance of Golgi integrity or for traffic from the ER to the Golgi but is necessary for transport through the Golgi to the plasma membrane. (+info)
Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins.
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Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for "delayed GPI-anchored protein transport"), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Delta dgt1Delta cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non-GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Delta dgt1Delta cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Delta dgt1Delta cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins. (+info)
Selective localization of the polytopic membrane protein prominin in microvilli of epithelial cells - a combination of apical sorting and retention in plasma membrane protrusions.
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Prominin is a recently identified polytopic membrane protein expressed in various epithelial cells, where it is selectively associated with microvilli. When expressed in non-epithelial cells, prominin is enriched in plasma membrane protrusions. This raises the question of whether the selective association of prominin with microvilli in epithelial cells is solely due to its preference for, and stabilization in, plasma membrane protrusions, or is due to both sorting to the apical plasma membrane domain and subsequent enrichment in plasma membrane protrusions. To investigate this question, we have generated stably transfected MDCK cells expressing either full-length or C-terminally truncated forms of mouse prominin. Confocal immunofluorescence and domain-selective cell surface biotinylation experiments on transfected MDCK cells grown on permeable supports demonstrated the virtually exclusive apical localization of prominin at steady state. Pulse-chase experiments in combination with domain-selective cell surface biotinylation showed that newly synthesized prominin was directly targeted to the apical plasma membrane domain. Immunoelectron microscopy revealed that prominin was confined to microvilli rather than the planar region of the apical plasma membrane. Truncation of the cytoplasmic C-terminal tail of prominin impaired neither its apical cell surface expression nor its selective retention in microvilli. Both the apical-specific localization of prominin and its selective retention in microvilli were maintained when MDCK cells were cultured in low-calcium medium, i.e. in the absence of tight junctions. Taken together, our results show that: (i) prominin contains dual targeting information, for direct delivery to the apical plasma membrane domain and for the enrichment in the microvillar subdomain; and (ii) this dual targeting does not require the cytoplasmic C-terminal tail of prominin and still occurs in the absence of tight junctions. The latter observation suggests that entry into, and retention in, plasma membrane protrusions may play an important role in the establishment and maintenance of the apical-basal polarity of epithelial cells. (+info)
Thyroid hormone, T3-dependent phosphorylation and translocation of Trip230 from the Golgi complex to the nucleus.
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Trip230 is a novel coactivator of the thyroid hormone receptor that is negatively regulated by the retinoblastoma tumor-suppressor protein. In an examination of its subcellular distribution, Trip230 localized predominantly to the vicinity of the Golgi instead of the nucleus, as other nuclear hormone receptor coactivators. Using a series of deletion mutants, a critical region identified for Golgi area targeting coincided with a previously defined thyroid hormone receptor-binding domain of Trip230. During cell cycle progression, the expression level of Trip230 is constant and a significant portion is imported into the nucleus at S phase. Within an hour of treating cells with T3, Trip230 immunofluorescence transiently colocalized with TR in prominent subnuclear structures. T3-dependent nuclear import of Trip230 does not require new protein synthesis. Coincident with T3 treatment and nuclear import, newly phosphorylated residue(s) appeared in Trip230, suggesting that phosphorylation may be involved in its nuclear import. These findings provided a novel mechanism for the regulation of nuclear hormone transcription factors by hormone-responsive phosphorylation and nuclear import of cytoplasmically located coactivators. (+info)
Evectins: vesicular proteins that carry a pleckstrin homology domain and localize to post-Golgi membranes.
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We have identified two vesicular proteins, designated evectin (evt)-1 and -2. These proteins are approximately 25 kDa in molecular mass, lack a cleaved N-terminal signal sequence, and appear to be inserted into membranes through a C-terminal hydrophobic anchor. They also carry a pleckstrin homology domain at their N termini, which potentially couples them to signal transduction pathways that result in the production of lipid second messengers. evt-1 is specific to the nervous system, where it is expressed in photoreceptors and myelinating glia, polarized cell types in which plasma membrane biosynthesis is prodigious and regulated; in contrast, evt-2 is widely expressed in both neural and nonneural tissues. In photoreceptors, evt-1 localizes to rhodopsin-bearing membranes of the post-Golgi, an important transport compartment for which specific molecular markers have heretofore been lacking. The structure and subcellular distribution of evt-1 strongly implicate this protein as a mediator of post-Golgi trafficking in cells that produce large membrane-rich organelles. Its restricted cellular distribution and genetic locus make it a candidate gene for the inherited human retinopathy autosomal dominant familial exudative vitreoretinopathy and suggest that it also may be a susceptibility gene for multiple sclerosis. (+info)
Stable expression of human beta1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns.
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beta1,4-Galactosyltransferase (UDP galactose: beta-N-acetylglucosaminide: beta1,4-galactosyltransferase; EC 2.4.1. 22) catalyzes the transfer of galactose from UDP-Gal to N-acetylglucosamine in the penultimate stages of the terminal glycosylation of N-linked complex oligosaccharides in mammalian cells. Tobacco BY2 cells lack this Golgi enzyme. To determine to what extent the production of a mammalian glycosyltransferase can alter the glycosylation pathway of plant cells, tobacco BY2 suspension-cultured cells were stably transformed with the full-length human galactosyltransferase gene placed under the control of the cauliflower mosaic virus 35S promoter. The expression was confirmed by assaying enzymatic activity as well as by Southern and Western blotting. The transformant with the highest level of enzymatic activity has glycans with galactose residues at the terminal nonreducing ends, indicating the successful modification of the plant cell N-glycosylation pathway. Analysis of the oligosaccharide structures shows that the galactosylated N-glycans account for 47.3% of the total sugar chains. In addition, the absence of the dominant xylosidated- and fucosylated-type sugar chains confirms that the transformed cells can be used to produce glycoproteins without the highly immunogenic glycans typically found in plants. These results demonstrate the synthesis in plants of N-linked glycans with modified and defined sugar chain structures similar to mammalian glycoproteins. (+info)
Aberrant death in dark chondrocytes of the avian growth plate.
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Growth plate chondrocytes of embryonic chick femurs were examined by electron microscopy, cytophotometry and autoradiography. Apart from the well-described 'light' chondrocyte, a different 'dark' type of chondrocyte was present, comprising 10 - 35% of the cell population. They were found at all stages of chondrocyte differentiation and in all ages of the femurs studied. Well developed rough endoplasmatic reticulum and Golgi complex, many secretory vesicles, energetically active mitochondria and a lot of glycogen, indicating high activity of the cytoplasm, were combined with low RNA synthesis, gentle margination and scattered compaction of the chromatin. DNA cytometry revealed that most of dark cells were diploid, but 15 - 30% were tetraploid, with the absence of an S-phase. Substantial loss of DNA was found in about 10% of dark chondrocytes. The TUNEL reaction demonstrated a limited number of DNA strand breaks. Advanced dark cells possessed the nuclear features of both apoptosis and necrosis. Besides chromomeric-chromonemic compaction, a chromatin arrangement similar to that of prometaphase and metaphase, as well as amitotic nuclear segregation, all of them degenerative, were found. Our interpretation is that the dark chondrocytes undergo an aberrant type of cell death which may be combined with aberrant cell cycle. Cell death of dark chondrocytes is preceded by a pre-mortal burst of secretion. (+info)
Type 1 and type 2 cytokine regulation of macrophage endocytosis: differential activation by IL-4/IL-13 as opposed to IFN-gamma or IL-10.
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Cytokine regulation of endocytic activity in primary human macrophages was studied to define ultrastructural changes and mechanisms of pinocytic regulation associated with cytokines secreted by activated T cells. The effects of IFN-gamma (type 1) and IL-4/IL-13 and IL-10 (type 2) cytokines on fluid phase and mannose receptor-mediated endocytosis were assessed by horseradish peroxidase and colloidal gold-BSA uptake and computer-assisted morphometric analysis. IL-4 and IL-13 enhanced fluid phase pinocytosis and mannose receptor-mediated uptake by activation of phosphatidylinositol 3-kinase. Inhibition of actin assembly showed that both cytokines exerted actin-dependent and -independent effects. Ultrastructurally, IL-4 and IL-13 increased tubular vesicle formation underneath the plasma membrane and at pericentriolar sites, concurrent with decreased particle sorting to lysosomes. By contrast, IL-10 or IFN-gamma decreased both fluid phase pinocytosis and mannose receptor-mediated uptake. IFN-gamma stimulated increased particle sorting to perinuclear lysosomes, while IL-10 decreased this activity. In summary, our data document differential effects on macrophage endocytic functions by type 1 or type 2 cytokines associated with induction and effector pathways in immunity. (+info)