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Biomedical subjects

G Warren

Publications and source records attributed to G Warren.

At least 19 recordsLinked to original sources
PubMed1995

Evidence for recycling of the resident medial/trans Golgi enzyme, N-acetylglucosaminyltransferase I, in ldlD cells.

ldlD cells, which lack the UDP-Gal/UDP-GalNAc 4-epimerase, were stably transfected with a Myc-tagged version of N-acetylglucosaminyltransferase I (Myc-Glc-NAc-T I). In the absence of GalNAc and Gal, newly synthesized GlcNAc-T I did not acquire O-linked oligosaccharides but was catalytically active and was transported to the Golgi region as defined using both immunofluorescence and immunoelectron microscopy. After addition of cycloheximide to prevent further synthesis, GalNAc and Gal were added, and the unglycosylated GlcNAc-T I was found to acquire mature, O-linked oligosaccharides with a half-time of about 150 min. The addition of these sugars was sensitive to N-ethylmaleimide and okadaic acid, both inhibitors of vesicle-mediated traffic. Together, these results suggest that Myc-Glc-NAc-T I undergoes retrograde transport to the early part of the Golgi apparatus where the first O-linked sugar, GalNAc, is added followed by anterograde transport back to the Golgi stack, where addition of Gal and sialic acid occurs.

Animals↗
PubMed1995

An NSF-like ATPase, p97, and NSF mediate cisternal regrowth from mitotic Golgi fragments.

Golgi cisternae regrew in a cell-free system from mitotic Golgi fragments incubated with buffer alone. Pretreatment with NEM or salt washing inhibited regrowth, but this could be restored either by p97, an NSF-like ATPase, or by NSF together with SNAPs and p115, a vesicle docking protein. The morphology of cisternae regrown with p97 and NSF-SNAPs-p115 differed, suggesting that they play distinct roles in rebuilding Golgi cisternae after mitosis.

Adenosine Triphosphatases↗
PubMed1995

Restoration of the Epstein-Barr virus ZEBRA protein's capacity to disrupt latency by the addition of heterologous activation regions.

The ZEBRA protein has a unique biological function among herpesviral proteins. It is responsible for the disruption of Epstein-Barr virus (EBV) latency and the induction of the lytic cycle. ZEBRA is a bZIP transcriptional activator which binds as a dimer to 7-bp response elements within EBV promoters and is directly involved in the stimulation of virus replication at the EBV lytic origin. We have employed the ZEBRA/EBV biological system to test whether a heterologous activation domain can substitute for another activation domain (the ZEBRA domain). The ZEBRA activation region was replaced with the potent acid activation region from the herpes simplex virus VP16 protein or with the activation region of the EBV R protein. Both chimeras were found to transactivate model and native promoters at equivalent or better levels than ZEBRA itself. Activation was not target- or cell-type dependent, nor was it dependent on the presence of virus. These activation domains restored ZEBRA's ability to induce early antigen and to stimulate origin replication to levels that were equal to or greater than those of wild type. These studies suggest that the specificities of some of the known biological functions of ZEBRA are not dependent upon the nature of the activation domain present within ZEBRA.

B-Lymphocytes↗
PubMed1995

Male mice defective in the DNA mismatch repair gene PMS2 exhibit abnormal chromosome synapsis in meiosis.

Using gene targeting in embryonic stem cells, we have derived mice with a null mutation in a DNA mismatch repair gene homolog, PMS2. We observed microsatellite instability in the male germline, in tail, and in tumor DNA of PMS2-deficient animals. We therefore conclude that PMS2 is involved in DNA mismatch repair in a variety of tissues. PMS2-deficient animals appear prone to sarcomas and lymphomas. PMS2-deficient males are infertile, producing only abnormal spermatozoa. Analysis of axial element and synaptonemal complex formation during prophase of meiosis I indicates abnormalities in chromosome synapsis. These observations suggest links among mismatch repair, genetic recombination, and chromosome synapsis in meiosis.

Adenosine Triphosphatases↗
PubMed1995

A role for tubular networks and a COP I-independent pathway in the mitotic fragmentation of Golgi stacks in a cell-free system.

Golgi stacks were previously shown to be converted into tubular networks when incubated in mitotic cytosol depleted of the coatomer subunit of COP I coats (Misteli and Warren, 1994). Similar, though smaller, networks are now shown to be an early intermediate on the Golgi fragmentation pathway both in vitro and in vivo. Their appearance mirrors the disappearance of Golgi cisternae and at their peak they constitute 35% of total Golgi membrane. They are consumed by two pathways, the first involving the budding of COP I-coated vesicles described previously (Misteli and Warren, 1994). The second involves a COP I-independent mechanism that leads eventually to a vesicle fraction that is larger in size and more heterogeneous than that produced by the COP I-mechanism. We suggest that both pathways operate concurrently at the onset of mitotic fragmentation. The COP I-independent pathway converts cisternae into tubular networks that then fragment. The COP I-dependent pathway partially consumes first the cisternae at the beginning of the incubation and then the tubular networks that form from them.

Animals↗
PubMed1995

Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides.

The distribution of beta 1,2 N-acetylglucosaminyltransferase I (NAGT I), alpha 1,3-1,6 mannosidase II (Mann II), beta 1,4 galactosyltransferase (GalT), alpha 2,6 sialyltransferase (SialylT) was determined by immuno-labelling of cryo-sections from HeLa cell lines. Antibody labelling in the HeLa cell line was made possible by stable expression of epitope-tagged forms of these proteins or forms from species to which specific antibodies were available. NAGT I and Mann II had the same distribution occupying the medial and trans cisternae of the stack. GalT and SialylT also had the same distribution but they occupied the trans cisterna and the trans-Golgi network (TGN). These results generalise our earlier observations on the overlapping distribution of Golgi enzymes and show that each of the trans compartments of the Golgi apparatus in HeLa cells contains unique mixtures of those Golgi enzymes involved in the construction of complex, N-linked oligosaccharides.

Animals↗
PubMed1995

Mitotic disassembly of the Golgi apparatus in vivo.

Populations enriched in prophase cells were obtained either by using a cell line with a temperature-sensitive mutation in the mitotic kinase, p34cdc2, or by treating cells with olomoucine, an inhibitor of this kinase. Both methods resulted in efficient and reversible block of the cells at the G2/M boundary. After cells were released from the cell cycle block, the morphological changes to the Golgi apparatus were characterised using both quantitative conventional electron microscopy and immuno-gold microscopy. The early mitotic phases were divided into six stages (G2 to pro-metaphase) based on the morphology of the nucleus. During prophase the cross-sectional length of Golgi stacks decreased prior to unstacking. At the same time, small vesicular profiles, typically 50-70 nm in diameter, accumulated in the vicinity of the stacks. The disappearance of Golgi stacks was accompanied by the transient appearance of tubular networks. By the time cells entered prometaphase, the stacks had completely disassembled and only clusters consisting of Golgi vesicles and short tubular elements were left. When cells were released from the G2/M boundary and pulsed briefly with [AlF4]- to prevent uncoating of transport vesicles, vesicular profiles with a morphology reminiscent of COP-coated vesicles appeared. These vesicular profiles were either associated with Golgi stacks or, at later stages, with clusters, but were formed at all stages of disassembly. Together these results provide further support for our model that continued budding of vesicles from the rims of Golgi cisternae is at least partly responsible for the disassembly of the Golgi apparatus.

Animals↗
PubMed1994

Localization of an alpha 1,2 galactosyltransferase activity to the Golgi apparatus of Schizosaccharomyces pombe.

We have cloned a gene encoding an alpha 1,2 galactosyltransferase activity from Schizosaccharomyces pombe. The open reading frame of the gene (gma12 for galactomannan, alpha 1,2), combined with the previous protein purification (Chappell and Warren, 1989), predicts an O-linked glycoprotein with type II transmembrane topology. By homologous gene disruption, we have demonstrated that the gma12 gene product (gma12p) is nonessential. The deletion strain (gma12-D10::ura4) has a significantly reduced level of galactosyltransferase activity relative to the parental strain, but both in situ lectin binding and in vitro biochemical assays demonstrate the presence of further galactosyltransferase activity in addition to gma12p. Although gma12p is not the only galactosyltransferase in S. pombe, it produces a unique carbohydrate structure on the surface of the yeast cells. We have generated a polyclonal antiserum against this carbohydrate epitope and shown that gma12p is capable of synthesizing the epitope both in vitro and in vivo. Electron microscopic localization of the gma12+ specific epitope in gma12+ cells revealed that gma12p synthesizes the carbohydrate structure in the Golgi apparatus, and subsequent intracellular transport distributes the epitope to later stages of the secretory pathway. The immunolocalization studies confirm the presence of one or more galactosyltransferase activities in the Golgi apparatus in fission yeast.

Amino Acid Sequence↗
PubMed1992

Sphingolipid transport in mitotic HeLa cells.

Mitotic and interphase HeLa cells were labeled with [3H]serine. Ceramide and its derivatives, lactosylceramide and sphingomyelin, were biosynthetically labeled under both conditions. Only in the absence of nocodazole, as the cells entered telophase, was an additional glycosphingolipid synthesized which was identified as GA2 (GalNAc(beta 1,4)Gal(beta 1,4)Glc(beta 1,1)Cer). Ceramide, the basic sphingolipid precursor, is synthesized in the endoplasmic reticulum, whereas its immediate derivatives are synthesized in early Golgi compartments. Transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi is inhibited in mitotic cells while ceramide acquires early Golgi modifications under the same conditions, suggesting that ceramide can be delivered to the Golgi by a different route. Since GA2 is synthesized in late Golgi, its absence in mitotic cells strongly argues for an in vivo inhibition of intra-Golgi transport, an observation with important implications for the mechanism of Golgi division.

Biological Transport↗
PubMed1992

Mitosis and inhibition of intracellular transport stimulate palmitoylation of a 62-kD protein.

Recent studies suggest that a cycle of acylation/deacylation is involved in the vesicular transport of proteins between intracellular compartments at both the budding and the fusion stage (Glick, B. S., and J. E. Rothman. 1987. Nature (Lond.). 326:309-312). Since a number of cellular processes requiring vesicular transport are inhibited during mitosis, we examined the fatty acylation of proteins in interphase and mitotic cells. We have identified a major palmitoylated protein with an apparent molecular weight of 62,000 (p62), whose level of acylation increases 5-10-fold during mitosis. Acylation was reversible and p62 was no longer palmitoylated in cells that have exited mitosis and entered G1. p62 is tightly bound to the cytoplasmic side of membranes, since it was sensitive to digestion with proteases in the absence of detergent and was not removed by treatment with 1 M KCl. p62 is removed from membranes by nonionic detergents or concentrations of urea greater than 4 M. The localization of p62 by subcellular fractionation is consistent with it being in the cis-Golgi or the cis-Golgi network. A palmitoylated protein of the same molecular weight was also observed in interphase cells treated with inhibitors of intracellular transport, such as brefeldin A, monensin, carbonylcyanide m-chlorophenylhydrazone, or aluminum fluoride. The protein palmitoylated in the presence of brefeldin A was shown to be the same as that palmitoylated during mitosis using partial proteolysis. Digestion with two enzymes, alkaline protease and endoprotease lys-C, generated the same 3H-palmitate-labeled peptide fragments from p62 from mitotic or brefeldin A-treated cells. We suggest that the acylation and deacylation of p62 may be important in vesicular transport and that this process may be regulated during mitosis.

Acylation↗
PubMed1992

Cell-free fusion of endocytic vesicles is regulated by phosphorylation.

Okadaic acid and microcystin-LR, both potent inhibitors of protein phosphatases (PP), blocked vesicle fusion in a cell-free system. The effect of okadaic acid was reversed by the purified catalytic subunit of PP2A, but not PP1. Inhibition was gradual, required Mg-ATP, and was reduced by protein kinase inhibitors, indicating that it was mediated via protein phosphorylation. A candidate protein kinase would be cdc2 kinase, which normally is active in mitotic extracts and has been shown to inhibit endocytic vesicle fusion (Tuomikoski, T., M.-A. Felix, M. Dorée, and J. Gruenberg. 1989. Nature (Lond.). 342:942-945). However, it would appear that cdc2 kinase is not responsible for inhibition by okadaic acid. When compared to cytosol prepared from mitotic cells, okadaic acid did not increase cdc2 kinase activity sufficiently to account for the inhibition. In addition, inhibition was maintained when cdc2 protein was depleted from cytosol.

CDC2 Protein Kinase↗
PubMed1991

The membrane spanning domain of beta-1,4-galactosyltransferase specifies trans Golgi localization.

Chimeric cDNAs were constructed so as to generate hybrid proteins in which different parts of the N-terminal domain of the human invariant chain were replaced by equivalent sequences from the trans Golgi resident enzyme, beta-1,4-galactosyltransferase. The cytoplasmic and membrane spanning domains of galactosyltransferase were found to be sufficient to retain all of the hybrid invariant chain in trans Golgi cisternae as judged by indirect immunofluorescence, treatment with brefeldin A and immuno-electron microscopy. As few as ten amino acids corresponding to the lumenal half of the membrane spanning domain of the Golgi enzyme sufficed to localize most of the hybrid invariant chain to the trans cisternae. A cytoplasmic domain was necessary for complete retention as assessed by flow cytofluorometry but could be provided either by galactosyltransferase or by invariant chain. This suggests that the cytoplasmic domain plays a role accessory to the membrane spanning domain, the latter mediating compartmental specificity.

Amino Acid Sequence↗
PubMed1991

Isolation of functional, coated, endocytic vesicles.

Brief internalization of [125I]transferrin was used to label coated endocytic vesicles, which were then purified using a combination of 2H2O and 2H2O/Ficoll density gradients. Purification was monitored using an assay measuring fusion of endocytic organelles, so as to isolate functional vesicles. Isolated vesicles had all the properties of clathrin-coated vesicles, being enriched for the major components of clathrin coats and uncoated by either 1 M Tris-HCl or an uncoating ATPase. Nearly half of the labeled vesicles were able to participate in subsequent fusion events, as measured by the cell-free assay. Fusion was specific, requiring energy and cytosol, and being sensitive to N-ethyl maleimide.

Cell Fractionation↗
PubMed1991

Mitotic cytosol inhibits invagination of coated pits in broken mitotic cells.

Receptor-mediated endocytosis is inhibited during mitosis in mammalian cells and earlier work on A431 cells suggested that one of the sites inhibited was the invagination of coated pits (Pypaert, M., J. M. Lucocq, and G. Warren. 1987. Eur. J. Cell Biol. 45: 23-29). To explore this inhibition further, we have reproduced it in broken HeLa cells. Mitotic or interphase cells were broken by freeze-thawing in liquid nitrogen and warmed in the presence of mitotic or interphase cytosol. Using a morphological assay, we found invagination to be inhibited only when mitotic cells were incubated in mitotic cytosol. This inhibition was reversed by diluting the cytosol during the incubation. Reversal was sensitive to okadaic acid, a potent phosphatase inhibitor, showing that phosphorylation was involved in the inhibition of invagination. This was confirmed using purified cdc2 kinase which alone could partially substitute for mitotic cytosol.

CDC2 Protein Kinase↗