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G Warren

Publications and source records attributed to G Warren.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1994

Expression of mammalian protein kinase C in Schizosaccharomyces pombe: isotype-specific induction of growth arrest, vesicle formation, and endocytosis.

Mammalian protein kinase C (PKC) isotypes elicit a number of effects on expression in Schizosaccharomyces pombe. A small decrease in growth rate results from PKC-gamma expression, and treatment of these cells with phorbol esters leads to marked growth inhibition and vesicle formation. PKC-delta and -eta expression causes growth inhibition and vesiculation, and the magnitude of both of these effects is increased by phorbol esters. In contrast, PKC-epsilon expression produces growth inhibition but no vesicle accumulation, and this effect is not responsive to phorbol ester. Finally, PKC-zeta has no observable effect. Thus, isotype-specific biological effects are observed. The accumulation of vesicles correlates with phorbol ester-dependent growth inhibition and occurs only with expression of those isotypes that down-regulate in response to phorbol esters in these cells. Antibodies against mammalian clathrin light chain 1a identified clathrin-coated vesicles and up-regulation of clathrin expression in those cells where vesicles accumulate; the increased vesicular traffic includes an element of endocytosis. Thus expression of specific mammalian PKC isotypes up-regulates endocytosis in S. pombe, providing a likely explanation for PKC-mediated receptor internalization in higher eukaryotes.

Animals↗
PubMed1993

Cyclin A-mediated inhibition of intra-Golgi transport requires p34cdc2.

An in vitro assay was used to study the role of p34cdc2 in cyclin A-mediated vesicular transport inhibition. It was shown that the S-phase kinase p33cdk2 reduced the effect of cyclin A on transport assays performed with sHeLa cytosol, even though histone kinase was strongly activated. Also, transport with FT210 cytosol (which is temperature-sensitive for p34cdc2) was inhibited by cyclin A only at the permissive temperature. However, the phosphatase inhibitor microcystin inhibited transport without any requirement for p34cdc2 activity. These results show that transport is inhibited by cyclin A via p34cdc2, and also by another kinase, possibly downstream of p34cdc2.

Alkaloids↗
PubMed1993

Inhibition of intra-Golgi transport in vitro by mitotic kinase.

It has previously been shown that exocytic and endocytic membrane traffic are inhibited in mitotic mammalian cells. Here we have used a cell-free intra-Golgi transport assay supplemented with heterologous cytosols to mimic this effect in vitro. Cytosols with high histone kinase activity, made either from mitotic cells or by cyclin A treatment of interphase cells, inhibited intra-Golgi transport by up to 75%. Inhibition of transport was reversed by the kinase inhibitor staurosporine or by reduction in ATP levels leading to inactivation of histone kinase. The data indicate that cell cycle control of intra-Golgi transport is due to a reversible modification of cytosol, and this assay system may be used to study the molecular mechanism of mitotic transport inhibition in mammalian cells.

Alkaloids↗
PubMed1993

Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells.

Thin, frozen sections of a HeLa cell line were double labeled with specific antibodies to localize the trans-Golgi enzyme, beta 1,4 galactosyltransferase (GalT) and the medial enzyme, N-acetylglucosaminyltransferase I (NAGT I). The latter was detected by generating a HeLa cell line stably expressing a myc-tagged version of the endogenous protein. GalT was found in the trans-cisterna and trans-Golgi network but, contrary to expectation, NAGT I was found both in the medial- and trans-cisternae, overlapping the distribution of GalT. About one third of the NAGT I and half of the GalT were found in the shared, trans-cisterna. These data show that the differences between cisternae are determined not by different sets of enzymes but by different mixtures.

Base Sequence↗
PubMed1993

The Golgi stack reassembles during telophase before arrival of proteins transported from the endoplasmic reticulum.

HeLa cells arrested in prometaphase were pulse-labeled with [35S]methionine and chased in the absence of nocodazole to allow passage through mitosis and into G1. Transport of histocompatibility antigen (HLA) molecules to the medial- and trans-Golgi cisternae was measured by monitoring the resistance to endoglycosidase H and the acquisition of sialic acid residues, respectively. Transport to the plasma membrane was measured using neuraminidase to remove sialic acid residues on surface HLA molecules. The half-time for transport to each of these compartments was about 65-min longer in cells progressing out of mitosis than in G1 cells. This delay was only 5-min longer than the half-time for the fall in histone H1 kinase activity suggesting that inactivation of the mitotic kinase triggers the resumption of protein transport. The half-time for reassembly of the Golgi stack, measured using stereological procedures, was also 65 min, suggesting that both transport and reassembly are triggered at the same time. However, since reassembly was complete within 5 min, whereas HLA took 25 min to reach the medial-cisterna, we can conclude that the Golgi stack has reassembled by the time HLA reaches it.

Biological Transport↗
PubMed1993

In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment.

CDC2 Protein Kinase↗
PubMed1993

Mitotic Golgi clusters are not tubular endosomes.

HeLa cells were incubated with 15 nm BSA-gold for 1 or 2 hours to mark the endocytic pathway and mitotic cells were then isolated by shake-off. Thin, frozen sections were labelled with antibodies against two resident Golgi markers, beta-(1,4)-galactosyltransferase and N-acetylglucosaminyltransferase I. Detection of the latter was aided by the use of a HeLa cell line stably expressing a myc-tagged version of the endogenous protein. The secondary antibodies were coupled to either 5 or 10 nm gold so that the distribution of each of the three markers could be followed. Qualitative and quantitative studies showed that there were two populations of clusters, those described by us earlier and termed Golgi clusters (Lucocq et al. (1987) J. Cell Biol. 104, 865-874), containing either or both Golgi markers, and clusters of tubular endosomes containing BSA-gold. There was very little overlap showing that Golgi clusters cannot be tubular endosomes as concluded by Tooze and Hollinshead (1992) Eur. J. Cell Biol. 58, 228-242.

Antibodies, Monoclonal↗
PubMed1993

Stacking of Golgi cisternae in Schizosaccharomyces pombe requires intact microtubules.

Fission yeast was treated with the anti-microtubule agent, thiabendazole. Cytoplasmic microtubules broke down with a half-time of less than 10 minutes followed closely by the unstacking of Golgi cisternae. The final product appeared to be single Golgi cisternae. No other organelle seemed to be affected by this treatment, which was completely reversible. The nda3 mutant strain has an altered beta-tubulin and its cytoplasmic microtubules are resistant to thiabendazole. The Golgi in this cold-sensitive mutant was unaffected by treatment at the permissive temperature but unstacked at the non-permissive temperature even in the absence of thiabendazole. Taken together these data show that disruption of the microtubular network can cause dissociation of Golgi cisternae. Newly synthesised acid phosphatase was transported and secreted to the same extent and with the same kinetics whether or not the Golgi was unstacked. The possible role of microtubules in Golgi stacking and the lack of effect on secretion are discussed.

Acid Phosphatase↗
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↗