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Biomedical subjects

G Warren

Publications and source records attributed to G Warren.

At least 55 records · Page 3Linked to original sources
PubMed1997

p47 is a cofactor for p97-mediated membrane fusion.

At least two distinct ATPases, NSF and p97, are known to be involved in the heterotypic fusion of transport vesicles with their target membranes and the homotypic fusion of membrane compartments. The NSF-mediated fusion pathway is the best characterized, many of the components having been identified and their functions analysed. In contrast, none of the accessory proteins for the p97-mediated fusion pathway has been identified. Now we have identified the first such component, a protein of relative molecular mass 47,000 (p47), which forms a tight, stoichiometric complex with cytosolic p97 (one trimer of p47 per hexamer of p97). It is essential for the p97-mediated regrowth of Golgi cisternae from mitotic Golgi fragments, a process restricted to animal cells. As a homologue of p47 exists in budding yeast, this indicates that it might also be involved in other membrane fusion reactions catalysed by p97, such as karyogamy.

Adenosine Triphosphatases↗
PubMed1997

Partitioning of the Golgi apparatus during mitosis in living HeLa cells.

The Golgi apparatus of HeLa cells was fluorescently tagged with a green fluorescent protein (GFP), localized by attachment to the NH2-terminal retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscopy using a variety of Golgi markers. The behavior of the fluorescent Golgi marker was observed in fixed and living mitotic cells using confocal microscopy. By metaphase, cells contained a constant number of Golgi fragments dispersed throughout the cytoplasm. Conventional and cryoimmunoelectron microscopy showed that the NAGT I-GFP chimera (NAGFP)-positive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly little effect on the polarity of Golgi membrane markers at the level of fluorescence microscopy. In living cells, there was little self-directed movement of the clusters in the period from metaphase to early telophase. In late telophase, the Golgi ribbon began to be reformed by a dynamic process of congregation and tubulation of the newly inherited Golgi fragments. The accuracy of partitioning the NAGFP-tagged Golgi was found to exceed that expected for a stochastic partitioning process. The results provide direct evidence for mitotic clusters as the unit of partitioning and suggest that precise regulation of the number, position, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus.

Cells, Cultured↗
PubMed1997

The vesicle docking protein p115 binds GM130, a cis-Golgi matrix protein, in a mitotically regulated manner.

The docking of transport vesicles with their target membrane is thought to be mediated by p115. We show here that GM130, a cis-Golgi matrix protein, interacts specifically with p115 and so could provide a membrane docking site. Deletion analysis showed that the N-terminus binds to p115, whereas the C-terminus binds to Golgi membranes. Mitotic phosphorylation of GM130 or a peptide derived from the N-terminus prevented binding to p115. The peptide also inhibited the NSF- but not the p97-dependent reassembly of Golgi cisternae from mitotic fragments, unless it was mitotically phosphorylated. Together, these data provide a molecular explanation for the COPI-mediated fragmentation of the Golgi apparatus at the onset of mitosis.

Amino Acid Sequence↗
PubMed1997

Conservative amputation of the neuropathic foot- The Pirogoff procedure.

INDICATIONS: Lesions limited to the forefoot when reconstruction of the forefoot is impossible. In situations where due to the unavailability of protheses a forefoot amputation has to be done. CONTRAINDICATIONS: Absolute: Badly scarred or destroyed calcaneum and/or heel pad. Osteomyelitis of lower tibia or calcaneum. Malignancy of the heel area. Relative: Inadequate circulation of the heel area. Uncontrolled diabetes mellitus. SURGICAL TECHNIQUE: Forefoot amputation with arthrodesis between tibia and os calcis. Specially applicable in patients with neuropathy because this simple procedure leaves a stable stump. Prosthetic fitting not mandatory. Negligible shortening of limb. POSTOPERATIVE MANAGEMENT: Bed rest, elevation of limb, and protective splinting for 3 postoperative days. Non weight bearing on crutches for the next 2 weeks, then well fitted contact cast with Böhler iron for walking. Application of prosthesis not before bony consolidation. POSSIBLE COMPLICATIONS: Infections. Treat very intensively. RESULTS: Fifty modified Pirogoff procedures were done over 30 years, mostly for leprosy or malignancy. Long-term follow-up available of 16 patients who suffered from leprosy (see Figure 11).

Journal Article↗
PubMed1997

An isoform of the Golgi t-SNARE, syntaxin 5, with an endoplasmic reticulum retrieval signal.

The early Golgi t-SNARE (target-membrane-associated soluble-N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 5 is thought to specify the docking site for both COPI and COPII coated vesicles originating from the endoplasmic reticulum (ER) and COPI vesicles on the retrograde pathway. We now show that there are two forms of syntaxin 5 that appear to be generated from the same mRNA by alternative initiation of translation. The short form (35 kDa) corresponds to the published sequence. The long form (42 kDa) has an N-terminal cytoplasmic extension containing a predicted type II ER retrieval signal. When grafted onto a reporter molecule, this signal localized the construct to the ER. Biochemical fractionation and immunofluorescence microscopy showed that there was less of the long form in the Golgi apparatus and more in peripheral punctate structures, some of which colocalized with markers of the intermediate compartment. The predicted absence of the long form in budding yeast points to a function unique to higher organisms.

Alternative Splicing↗
PubMed1996

Binding of the vesicle docking protein p115 to Golgi membranes is inhibited under mitotic conditions.

The vesicle docking protein p115 showed saturable, high affinity binding to interphase Golgi membranes. The affinity of binding was up to 20-fold lower using membranes preincubated with mitotic cytosol. In contrast, binding was not affected by mitotic pretreatment of p115. The reduction in p115 binding was mediated by phosphorylation, could be induced by a cyclin-dependent kinase, and was fully reversible. A shift of p115 from membranes to cytosol was also found after fractionating mitotic cells. The functional significance of the decreased binding was addressed by in vitro mitotic incubations which disassemble Golgi cisternae, predominantly producing transport vesicles. The addition of excess p115 decreased loss of membrane from cisternae, indicating that p115's action is limiting while transport vesicles accumulate. The cessation of intra-Golgi traffic in mitosis has been hypothesized to result from an inhibition of membrane fusion while budding of transport vesicles continues. This process also contributes to mitotic Golgi disassembly. Our results imply that there is a mitotic modification to Golgi membranes leading to a reduction in the affinity of the p115 receptor. Reduced p115 binding may play a part in the inhibition of membrane fusion by preventing prior vesicle docking.

Animals↗
PubMed1996

Physical and genetic localization of the gene encoding the AP-2 transcription factor to mouse chromosome 13.

Transcription factors are a major determinant of developmental fate. The chromosomal localization of the genes encoding these proteins provides important information that can link them to known genetic abnormalities. Here, we report the mapping of the mouse gene for transcription factor AP-2, a protein that has been implicated in human oncogenesis. Using FISH, we have mapped the gene encoding the transcription factor AP-2, Tcfap2, to mouse Chromosome 13A5-B1. We have also extended this analysis by placing Tcfap2 on the mouse genetic map, and we discuss the candidate mouse mutations that map in the vicinity of this transcription factor.

Animals↗
PubMed1996

Sorting by COP I-coated vesicles under interphase and mitotic conditions.

COP I-coated vesicles were analyzed for their content of resident Golgi enzymes (N-acetylgalactosaminyltransferase; N-acetylglucosaminyltransferase I; mannosidase II; galactosyltransferase), cargo (rat serum albumin; polyimmunoglobulin receptor), and recycling proteins (-KDEL receptor; ERGIC-53/p58) using biochemical and morphological techniques. The levels of these proteins were similar when the vesicles were prepared under interphase or mitotic conditions showing that sorting was unaffected. The average density relative to starting membranes for resident enzymes (14-30%), cargo (16-23%), and recycling proteins (81-125%) provides clues to the function of COP I vesicles in transport through the Golgi apparatus.

Amino Acid Sequence↗
PubMed1996

Isolation of mutations affecting the development of freezing tolerance in Arabidopsis thaliana (L.) Heynh.

We screened for mutations deleterious to the freezing tolerance of Arabidopsis thaliana (L.) Heynh. ecotype Columbia. Tolerance was assayed by the vigor and regrowth of intact plants after cold acclimation and freezing. From a chemically mutagenized population, we obtained 13 lines of mutants with highly penetrant phenotypes. In 5 of these, freezing sensitivity was attributable to chilling injury sustained during cold acclimation, but in the remaining 8 lines, the absence of injury prior to freezing suggested that they were affected specifically in the development of freezing tolerance. In backcrosses, freezing sensitivity from each line segregated as a single nuclear mutation. Complementation tests indicated that the 8 lines contained mutations in 7 different genes. The mutants' freezing sensitivity was also detectable in the leakage of electrolytes from frozen leaves. However, 1 mutant line that displayed a strong phenotype at the whole-plant level showed a relatively weak phenotype by the electrolyte leakage assay.

Adaptation, Physiological↗
PubMed1996

Primate homologues of rat TGN38: primary structure, expression and functional implications.

cDNAs encoding the human and macaque homologues of rat TGN38 have been cloned and sequenced. The proteins have a highly conserved N terminus (comprising the signal peptide) and C terminus (comprising part of the lumenal domain, the membrane spanning region and cytoplasmic tail) but vary in the other part of the lumenal domain, which contains the repeat region. Whereas rat TGN38 contains 6 tandem repeats of an 8mer, both primate proteins possess 14 tandem repeats of a 14mer sequence. The human protein, like rat TGN38, is localised primarily to the TGN but is present on the cell surface and returns via endosomes. This behaviour is consistent with conservation of the membrane spanning region and the cytoplasmic tail, which contain the retention and retrieval signals, respectively, for localisation in the TGN. The unexpected differences in the lumenal domain can best be rationalised by the fact that both types of repeat domains have most of the properties of mucins. We suggest that TGN38 homologues are mucin-like molecules that regulate membrane traffic to and from the TGN.

Amino Acid Sequence↗
PubMed1996

The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural maintenance of the Golgi apparatus in HeLa cells.

Using a series of chimeric and truncated N-acetylglucosaminyltransferase I (NAGT I) molecules we have shown that part of the lumenal stalk region is both necessary and sufficient for kin recognition of mannosidase II and retention in the Golgi stack. The membrane-spanning domain was not required for retention, but replacing part or all of this domain with leucine residues did have a dramatic effect on Golgi morphology. In stable cell lines, stacked cisternae were replaced by tubulo-vesicular clusters containing the mutated NAGT I. The loss of stacked cisternae was proportional to the number of leucines used to replace the membrane-spanning domain.

Amino Acid Sequence↗
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

Intracellular membrane morphology.

The membrane-bound organelles on the exocytic and endocytic pathways are linked by vesicles which bud from one membrane compartment, carrying selected cargo, and fuse with the next on the pathway. The principles underlying this vesicle-mediated traffic go a long way towards explaining the morphology of these intracellular organelles and their behaviour during mitosis.

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↗