Biochemistry and Pharmacology - Research Publications
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ItemNo Preview AvailablePOSTTRANSLATIONAL MODIFICATIONS DISTINGUISH CELL-SURFACE FROM GOLGI-RETAINED BETA-1,4 GALACTOSYLTRANSFERASE MOLECULES - GOLGI LOCALIZATION INVOLVES ACTIVE RETENTION(OXFORD UNIV PRESS UNITED KINGDOM, 1994-12-01)beta 1,4 Galactosyltransferase (GalT) is a membrane-bound enzyme localized predominantly to the trans-Golgi cisternae. Our previous studies have shown that the transmembrane domain of bovine GalT plays a critical role in Golgi localization (Teasdale, R.D., D'Agostaro, G. and Gleeson, P.A., J. Biol. Chem., 267, 4084-4096, 1992). Here we have compared the localization and post-translational modifications of full-length bovine GalT with a GalT/hybrid molecule where the transmembrane domain of GalT was replaced with that of the transferrin receptor. GalT/hybrid molecules were expressed on the surface of transfected cells; however, differences were observed in the distribution of the hybrid molecules between transfected COS and murine L cells. In transfected COS cells, the GalT/hybrid protein was expressed efficiently at the cell surface, with little Golgi-localized material, whereas in stable murine L cells, which expressed lower levels of the construct, hybrid molecules were detected both at the cell surface and within the Golgi apparatus. Expression of the GalT constructs in either COS or L cells produced two glycoprotein products which differed in molecular mass by 7 kDa. The difference in size between the two products is due to post-translational modifications which are inhibited by brefeldin A and are therefore likely to occur in the trans-Golgi network (TGN). Very little of the high-molecular-weight species was detected for full-length GalT, whereas it was a major product for the GalT/hybrid protein. Only the higher molecular weight species was expressed at the cell surface. Thus, this additional 7 kDa post-translational modification distinguishes molecules retained within the Golgi apparatus (lower M(r) species) from those transported through the TGN to the cell surface. These studies indicate that (i) the level of expression influences the intracellular distribution of GalT/hybrid molecules and (ii) the localization of full-length GalT involves active retention within the Golgi stack, and not retrieval from later compartments. After treatment of membrane preparations from stable L cell clones with a heterobifunctional cross-linking agent, full-length bovine GalT molecules were found almost exclusively as high-molecular-weight aggregates, suggesting that GalT exists as an oligomer or aggregate. This ability to oligomerize may be a requirement for Golgi retention.
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ItemNo Preview AvailableTargeting of proteins to the Golgi apparatus(SPRINGER, 1998-05-01)The proteins that reside in the Golgi carry out functions associated with post-translational modifications, including glycosylation and proteolytic processing, membrane transport, recycling of endoplasmic reticulum proteins and maintenance of the structural organisation of the organelle itself. The latter includes Golgi stacking, interconnections between stacks and the microtubule-dependent positioning of the organelle within the cell. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recycling trans-Golgi network (TGN) proteins, peripheral membrane proteins and receptors. Considerable effort has been directed at understanding the basis of the localisation of Golgi glycosyltransferases and recycling TGN proteins; in both cases there is increasing evidence that multiple signals may be involved in their specific localisation. A number of models for the Golgi retention of glycosyltransferases have been proposed including oligomerisation, lipid-mediated sorting and intra-Golgi retrograde transport. More information is required to determine the contribution of each of these potential mechanisms in the targeting of different glycosyltransferases. Future work is also likely to focus on the relationship between the localisation of resident Golgi proteins and the maintenance of Golgi structure.
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ItemNo Preview AvailableTARGETING OF PROTEINS TO THE GOLGI-APPARATUS(SPRINGER, 1994-10-01)The Golgi apparatus maintains a highly organized structure in spite of the intense membrane traffic which flows into and out of this organelle. Resident Golgi proteins must have localization signals to ensure that they are targeted to the correct Golgi compartment and not swept further along the secretory pathway. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recycling trans-Golgi network proteins, peripheral membrane proteins, receptors and viral glycoproteins. Recent studies indicate that there are a number of different Golgi localization signals and mechanisms for retaining proteins to the Golgi apparatus. This review focuses on the current knowledge in this field.
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ItemAN AUTOIMMUNE-DISEASE WITH MULTIPLE MOLECULAR TARGETS ABROGATED BY THE TRANSGENIC EXPRESSION OF A SINGLE AUTOANTIGEN IN THE THYMUS(ROCKEFELLER UNIV PRESS, 1993-08-01)Many autoimmune diseases are characterized by autoantibody reactivities to multiple cellular antigens. Autoantigens are commonly defined as targets of the autoimmune B cell response, but the role, if any, of these autoantigens in T cell-mediated autoimmune diseases is generally unknown. Murine experimental autoimmune gastritis is a CD4+ T cell-mediated organ-specific autoimmune disease induced by neonatal thymectomy of BALB/c mice. The murine disease is similar to human autoimmune gastritis and pernicious anemia, and is characterized by parietal and chief cell loss, submucosal mononuclear cell infiltrates, and autoantibodies to the alpha and beta subunits of the gastric H/K ATPase. However, the specificity of T cells that cause the disease is not known. To examine the role of the H/K ATPase in this T cell-mediated disease, transgenic mice were generated that express the beta subunit of the H/K ATPase under the control of the major histocompatibility complex class II I-Ek alpha promoter. We show that transgenic expression of the gastric H/K ATPase beta subunit specifically prevents the onset of autoimmune gastritis after neonatal thymectomy. In addition, thymocyte transfer experiments suggest that tolerance of pathogenic autoreactive T cells is induced within the thymus of the transgenic mice. We conclude that the beta subunit of the gastric H/K ATPase is a major T cell target in autoimmune gastritis and that thymic expression of a single autoantigen can abrogate an autoimmune response to multiple autoantigens.