Search PubMed⌕ Search

Biomedical subjects

G Banting

Publications and source records attributed to G Banting.

At least 55 records · Page 3Linked to original sources
PubMed1994

Okadaic acid treatment leads to a fragmentation of the trans-Golgi network and an increase in expression of TGN38 at the cell surface.

Okadaic acid (OA) is a protein phosphatase inhibitor which has, among other properties, previously been shown to induce a fragmentation of the cisternae of the Golgi stack [for review, see Lucocq (1992) J. Cell Sci. 103, 875-880]. The effects of OA an reversible and mimic intracellular events which occur during mitosis. To date, due to a lack of endogenous marker proteins, the effects of OA on the trans-Golgi network (TGN) has not been studied. Certain drugs, e.g. Brefeldin A (BFA), have different effects on the morphology of the Golgi stack and the TGN; it is therefore relevant to ask what effect(s) OA has on the TGN. We now present data from a study in which we have used antibodies to TGN38, an integral membrane protein predominantly localized to the TGN of rat NRK cells [Luzio, Brake, Banting, Howell, Braghetta and Stanley (1990) Biochem. J. 270, 97-102], to investigate the effects of OA on this organelle. OA induces a reversible fragmentation of the TGN. This fragmentation occurs with similar kinetics to that observed within the Golgi stack, and is independent of protein synthesis. The sensitivity of the TGN to OA is similar to that of the Golgi stack. The fragmentation of the TGN induced by OA also leads to a 10-fold increase in the level of TGN38 expressed at the plasma membrane.

Amino Acid Sequence↗
PubMed1994

Analysis of the co-localization of the insulin-responsive glucose transporter (GLUT4) and the trans Golgi network marker TGN38 within 3T3-L1 adipocytes.

The exposure of isolated adipocytes to insulin results in an approximately 20-fold increase in the rate of glucose transport into the cell. This increase is mediated by the movement of a pool of intracellular vesicles containing the so-called insulin-responsive glucose transporter (GLUT4) to the cell surface. In the resting state, most of the GLUT4 molecules are sequestered inside the adipocyte in an as yet unidentified intracellular compartment. TGN38 is an integral membrane protein which has been shown to be predominantly localized to the trans Golgi network [Luzio, Brake, Banting, Howell, Braghetta and Stanley (1990) Biochem. J. 270, 97-102]. Here we investigate whether GLUT4 and TGN38 are co-localized in the murine 3T3-L1 adipocyte cell line. Immuno-adsorption of intracellular vesicles containing GLUT4 with an anti-peptide antibody specific for this isoform did not deplete the low-density microsomal fraction of TGN38 in these cells; moreover, no TGN38 was detected in the GLUT4-containing vesicles by immunoblotting with a TGN38-specific antiserum. Immuno-adsorption of TGN38-containing vesicles and subsequent analysis of the proteins in these vesicles revealed that a detectable amount of GLUT4 (5-10%) did co-localise with TGN38. The amount of GLUT4 in the TGN38-containing vesicles did not change in response to insulin. Immunofluorescence analysis of TGN38 and GLUT4 in these cells revealed markedly different staining patterns. Reversal of insulin-stimulated glucose transport and subsequent analysis of the TGN38-containing vesicles demonstrated that during the re-cycling of GLUT4 to the intracellular storage site there was no increase in the amount of GLUT4 co-localized with TGN38. Taken together, these results suggest that the trans Golgi network is not the major site of the intracellular GLUT4 pool within 3T3-L1 adipocytes.

3T3 Cells↗
PubMed1994

Vacuolar ATPase inactivation blocks recycling to the trans-Golgi network from the plasma membrane.

TGN38/41 is an integral membrane protein which recycles between the trans-Golgi network (TGN) and the cell surface but is predominantly located in the TGN of rat (NRK) cells at steady state. As part of our studies on the mechanism and route of recycling between the TGN and the cell surface we have used chloroquine or Bafilomycin A1 to modulate the lumenal pH of endocytic organelles. The data we present demonstrate that inactivation of the proton pump which maintains the acidic environment within the lumen of endocytic organelles leads to an accumulation of TGN38/41 in early endosomes. These data confirm the observation that TGN38/41 recycles between the plasma membrane and the TGN and identifies a specific block in that recycling pathway.

Adenosine Triphosphatases↗
PubMed1994

The tyrosine-containing internalization motif in the cytoplasmic domain of TGN38/41 lies within a nascent helix.

TGN38 and TGN41 are isoforms of an integral membrane protein (TGN38/41) which is predominantly located in the trans-Golgi network of mammalian cells, but which constitutively recycles between the TGN and the plasma membrane. The cytoplasmic domain tetrapeptide sequence "YQRL" is responsible for the internalization of TGN38/41 from the plasma membrane. This sequence conforms to the tyrosine containing internalization motif ("YXXhydro," where X is any amino acid and hydro is any large bulky hydrophobic amino acid) found in other integral membrane proteins which are internalized from the plasma membrane via clathrin-coated vesicles. Structural predictions have suggested that the YXXhydro motif might adopt a tight turn structure in solution, a prediction supported previously by nuclear magnetic resonance (NMR) studies on short synthetic peptides corresponding to variants of the generic YXXhydro motif. We have synthesized a 21-amino acid peptide which encompasses the TGN38/41 internalization motif and used it as template for two-dimensional NMR analysis. The data from these experiments demonstrate that the internalization motif in the cytoplasmic domain of TGN38/41 lies within a nascent helix, not a tight turn. This is the first study to show that tyrosine containing internalization motifs do not necessarily adopt a beta-turn conformation.

Amino Acid Sequence↗
PubMed1994

Isolation and sequence of a full length cDNA encoding a novel rat inositol 1,4,5-trisphosphate 3-kinase.

Immunoscreening a rat liver cDNA expression library has led to the isolation of a full-length cDNA clone encoding a novel isoform of rat inositol 1,4,5-trisphosphate 3-kinase (IP3 3-kinase). Sequence comparison shows it (i) to be 93% identical to human hippocampus IP3 3-kinase B over 468 residues at the protein level, and (ii) to encode a protein 204 amino acids larger than the published sequence of its human homologue.

Amino Acid Sequence↗
PubMed1994

PBDX is the XG blood group gene.

We have identified the Xga antigen, encoded by the XG blood group gene, by employing rabbit polyclonal and mouse monoclonal antibodies raised against a peptide derived from the N-terminal domain of a candidate gene, referred to earlier as PBDX. In indirect haemagglutination assays, these anti-peptide antibodies react with Xg(a+) but not Xg(a-) erythrocytes. In antibody-specific immobilization of antigen (ASIA) and immunoblot assays, the anti-peptide antibodies react with the same molecule as does human anti-Xga. Therefore, by its identity with PBDX, Xga is identified as a cell-surface protein that is 48% homologous to CD99 (previously designated the 12E7 antigen), the product of MIC2 which is tightly linked to XG. PBDX is renamed here XG.

12E7 Antigen↗
PubMed1993

Eukaryotic membrane traffic: retrieval and retention mechanisms to achieve organelle residence.

The localization of integral membrane proteins to specific organelles is necessary to maintain the functional integrity of eukaryotic cells. Recent studies indicate firstly, that retrieval and retention mechanisms, requiring specific primary sequence motifs, are used to ensure that proteins reside in specific membranes of the secretory and endocytic pathways and secondly, that these membranes exist as components of two juxtaposed systems separated by the cisternae of the Golgi stack.

Amino Acid Sequence↗
PubMed1993

TGN38/41 recycles between the cell surface and the TGN: brefeldin A affects its rate of return to the TGN.

TGN38 and TGN41 are isoforms of an integral membrane protein (TGN38/41) that is predominantly localized to the trans-Golgi network (TGN) of normal rat kidney cells. Polyclonal antisera to TGN38/41 have been used to monitor its appearance at, and removal from, the surface of control and Brefeldin A (BFA)-treated cells. Antibodies that recognize the lumenal domain of TGN38/41 are capable of specific binding to the surface of both control and BFA-treated cells. In both control and BFA-treated cells internalized TGN38/41 is targeted to the TGN; however, there are differences in 1) the morphology of the intracellular structures through which TGN38/41 passes and 2) the kinetics of internalization. These data demonstrate that TGN38/41 cycles between the plasma membrane and the TGN in control and BFA-treated cells and suggest that recycling pathways between the plasma membrane and the TGN exist for predominantly TGN proteins as well as those that normally cycle to other intracellular compartments. They also demonstrate that addition of BFA not only alters the morphology and localization of the TGN but also the kinetics of endocytosis.

Animals↗
PubMed1992

Epitope mapping of two isoforms of a trans Golgi network specific integral membrane protein TGN38/41.

TGN38/41 is an integral membrane protein predominantly located in the trans Golgi network (TGN) of rat (NRK) cells. We have used a cDNA expression system to map the epitopes recognised by a panel of antibodies raised to TGN38/41 as a preliminary step in the accurate identification of the region(s) of the molecule responsible for its correct intracellular location. These studies have confirmed the predicted topology of the molecule, and have identified a region in the cytoplasmic domain which is immunologically (and hence potentially functionally) conserved between species.

Amino Acid Sequence↗
PubMed1992

Identification, molecular characterization and immunolocalization of an isoform of the trans-Golgi-network (TGN)-specific integral membrane protein TGN38.

TGN38 is an integral membrane protein previously shown to be predominantly localized to the trans-Golgi network (TGN) of cells by virtue of a signal contained within its cytoplasmic 'tail' [Luzio, Brake, Banting, Howell, Braghetta & Stanley (1990) Biochem. J. 270, 97-102]. We now (i) describe the isolation of cDNA clones encoding an isoform of TGN38, (ii) present the sequence of that isoform and (iii) describe the production and use of antibodies which specifically recognize the new isoform. This isoform, designated TGN41, is also predominantly localized to the TGN. The only sequence differences between the protein coding regions of cDNA clones encoding TGN38 and those encoding TGN41 occur within the region specifying the cytoplasmic tails of the two proteins. The TGN localization signal is shown to be within the sequence common to both proteins.

Amino Acid Sequence↗
PubMed1992

Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: redistribution of a TGN-specific integral membrane protein, TGN38.

Brefeldin A (BFA) has a dramatic effect on the morphology of the Golgi apparatus and induces a rapid redistribution of Golgi proteins into the ER (Lippincott-Schwartz, J., L. C. Yuan, J. S. Bonifacino, and R. D. Klausner. 1989. Cell. 56:801-813). To date, no evidence that BFA affects the morphology of the trans-Golgi network (TGN) has been presented. We describe the results of experiments, using a polyclonal antiserum to a TGN specific integral membrane protein (TGN38) (Luzio, J.P., B. Brake, G. Banting, K. E. Howell, P. Braghetta, and K. K. Stanley. 1990. Biochem. J. 270:97-102), which demonstrate that incubation of cells with BFA does induce morphological changes to the TGN. However, rather than redistributing to the ER, the majority of the TGN collapses around the microtubule organizing center (MTOC). The effect of BFA upon the TGN is (a) independent of protein synthesis, (b) fully reversible (c) microtubule dependent (as shown in nocodazole-treated cells), and (d) relies upon the hydrolysis of GTP (as shown by performing experiments in the presence of GTP gamma S). ATP depletion reduces the ability of BFA to induce a redistribution of Golgi proteins into the ER; however, it has no effect upon the BFA-induced relocalizations of the TGN. These data confirm that the TGN is an organelle which is independent of the Golgi, and suggest a dynamic interaction between the TGN and microtubules which is centered around the MTOC.

Animals↗
PubMed1991

pUBEX/pUBSEX: a versatile expression vector system for production of fusion and nonfusion proteins in Escherichia coli.

Despite the large number of expression vectors now available, none provide the facility of allowing fusion and nonfusion protein production from the same vector system. In some situations it is preferable to obtain an insoluble fusion protein, in others a soluble nonfusion protein may be required. We have designed, constructed and tested a modification of the pEX vectors, in which it is possible to express the product of a suitably inserted cDNA either as part of a Cro-beta-galactosidase (Cro-beta Gal) fusion or as a delta Cro fusion which contains only nine noninsert-encoded amino acids at its N terminus. The conversion from Cro-beta Gal to delta Cro fusion protein production is achieved by a simple intramolecular deletion of lacZ sequence from the pUBEX vector, to create the pUBSEX variant. Plasmid pUBEX can be induced to produce large amounts of insoluble Cro-beta Gal fusion proteins, whereas pUBSEX will produce predominantly soluble delta Cro fusion proteins.

Animals↗
PubMed1990

Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38).

Organelle-specific integral membrane proteins were identified by a novel strategy which gives rise to monospecific antibodies to these proteins as well as to the cDNA clones encoding them. A cDNA expression library was screened with a polyclonal antiserum raised against Triton X-114-extracted organelle proteins and clones were then grouped using antibodies affinity-purified on individual fusion proteins. The identification, molecular cloning and sequencing are described of a type 1 membrane protein (TGN38) which is located specifically in the trans-Golgi network.

Amino Acid Sequence↗
PubMed1990

A new recombinant DNA strategy for the molecular cloning of rare membrane proteins.

We have constructed a cDNA library in the plasmid expression vector pUEX enriched in sequences encoding membrane proteins. The procedure involved positive selection of sequences common to two different rat tissues (thus excluding tissue-specific mRNA) followed by positive selection between this material and RNA extracted from membrane bound polysomes (thus excluding cytoplasmic proteins). The resultant library prepared from rat kidney cDNA hybridized with rat liver poly(A)+ RNA, contained 30,000 clones and was shown to be enriched in cDNAs encoding membrane proteins. Seventeen clones selected because they encode large fusion proteins were shown to be single copy in the library, and not present in nucleotide data banks. Thus the strategy is particularly suitable for cloning low abundance cDNAs encoding membrane proteins.

Animals↗
PubMed1989

Intracellular targetting signals of polymeric immunoglobulin receptors are highly conserved between species.

A rat liver cDNA library, constructed in the plasmid expression vector pUEX, was immunoscreened using a rabbit polyclonal antiserum raised against rat liver Golgi membrane proteins. A sub-set of isolated clones were shown to encode the rat polymeric immunoglobulin receptor (pIgR). DNA sequence analysis of these clones provided the complete coding sequence of rat pIgR. Subsequent alignment of rat, rabbit and human predicted amino acid sequences demonstrated that the greatest degree of homology between the three pIgRs lies in their cytoplasmic tails; a region previously shown to be important for correct targetting and trancytosis of rabbit pIgR [(1984) Nature 308, 37-43].

Amino Acid Sequence↗