Biochemistry and Pharmacology - Research Publications
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ItemNo Preview AvailableThe yeast inositol polyphosphate 5-phosphatases Inp52p and Inp53p translocate to actin patches following hyperosmotic stress: Mechanism for regulating phosphatidylinositol 4,5-bisphosphate at plasma membrane invaginations(AMER SOC MICROBIOLOGY, 2000-12)The Saccharomyces cerevisiae inositol polyphosphate 5-phosphatases (Inp51p, Inp52p, and Inp53p) each contain an N-terminal Sac1 domain, followed by a 5-phosphatase domain and a C-terminal proline-rich domain. Disruption of any two of these 5-phosphatases results in abnormal vacuolar and plasma membrane morphology. We have cloned and characterized the Sac1-containing 5-phosphatases Inp52p and Inp53p. Purified recombinant Inp52p lacking the Sac1 domain hydrolyzed phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] and PtdIns(3, 5)P(2). Inp52p and Inp53p were expressed in yeast as N-terminal fusion proteins with green fluorescent protein (GFP). In resting cells recombinant GFP-tagged 5-phosphatases were expressed diffusely throughout the cell but were excluded from the nucleus. Following hyperosmotic stress the GFP-tagged 5-phosphatases rapidly and transiently associated with actin patches, independent of actin, in both the mother and daughter cells of budding yeast as demonstrated by colocalization with rhodamine phalloidin. Both the Sac1 domain and proline-rich domains were able to independently mediate translocation of Inp52p to actin patches, following hyperosmotic stress, while the Inp53p proline-rich domain alone was sufficient for stress-mediated localization. Overexpression of Inp52p or Inp53p, but not catalytically inactive Inp52p, which lacked PtdIns(4,5)P(2) 5-phosphatase activity, resulted in a dramatic reduction in the repolarization time of actin patches following hyperosmotic stress. We propose that the osmotic-stress-induced translocation of Inp52p and Inp53p results in the localized regulation of PtdIns(3,5)P(2) and PtdIns(4,5)P(2) at actin patches and associated plasma membrane invaginations. This may provide a mechanism for regulating actin polymerization and cell growth as an acute adaptive response to hyperosmotic stress.
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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)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)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)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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ItemExpression of HIV-1 Vpu Leads to Loss of the Viral Restriction Factor CD317/Tetherin from Lipid Rafts and Its Enhanced Lysosomal Degradation(PUBLIC LIBRARY SCIENCE, 2013-09-24)CD317/tetherin (aka BST2 or HM1.24 antigen) is an interferon inducible membrane protein present in regions of the lipid bilayer enriched in sphingolipids and cholesterol (often termed lipid rafts). It has been implicated in an eclectic mix of cellular processes including, most notably, the retention of fully formed viral particles at the surface of cells infected with HIV and other enveloped viruses. Expression of the HIV viral accessory protein Vpu has been shown to lead to intracellular sequestration and degradation of tetherin, thereby counteracting the inhibition of viral release. There is evidence that tetherin interacts directly with Vpu, but it remains unclear where in the cell this interaction occurs or if Vpu expression affects the lipid raft localisation of tetherin. We have addressed these points using biochemical and cell imaging approaches focused on endogenous rather than ectopically over-expressed tetherin. We find i) no evidence for an interaction between Vpu and endogenous tetherin at the cell surface, ii) the vast majority of endogenous tetherin that is at the cell surface in control cells is in lipid rafts, iii) internalised tetherin is present in non-raft fractions, iv) expression of Vpu in cells expressing endogenous tetherin leads to the loss of tetherin from lipid rafts, v) internalised tetherin enters early endosomes, and late endosomes, in both control cells and cells expressing Vpu, but the proportion of tetherin molecules destined for degradation rather than recycling is increased in cells expressing Vpu vi) lysosomes are the primary site for degradation of endogenous tetherin in cells expressing Vpu. Our studies underlie the importance of studying endogenous tetherin and let us propose a model in which Vpu intercepts newly internalised tetherin and diverts it for lysosomal destruction rather than recycling to the cell surface.
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ItemRab6a/a′ Are Important Golgi Regulators of Pro-Inflammatory TNF Secretion in Macrophages(PUBLIC LIBRARY SCIENCE, 2013-02-21)Lipopolysaccharide (LPS)-activated macrophages secrete pro-inflammatory cytokines, including tumor necrosis factor (TNF) to elicit innate immune responses. Secretion of these cytokines is also a major contributing factor in chronic inflammatory disease. In previous studies we have begun to elucidate the pathways and molecules that mediate the intracellular trafficking and secretion of TNF. Rab6a and Rab6a' (collectively Rab6) are trans-Golgi-localized GTPases known for roles in maintaining Golgi structure and Golgi-associated trafficking. We found that induction of TNF secretion by LPS promoted the selective increase of Rab6 expression. Depletion of Rab6 (via siRNA and shRNA) resulted in reorganization of the Golgi ribbon into more compact structures that at the resolution of electron microcopy consisted of elongated Golgi stacks that likely arose from fusion of smaller Golgi elements. Concomitantly, the delivery of TNF to the cell surface and subsequent release into the media was reduced. Dominant negative mutants of Rab6 had similar effects in disrupting TNF secretion. In live cells, Rab6-GFP were localized on trans-Golgi network (TGN)-derived tubular carriers demarked by the golgin p230. Rab6 depletion and inactive mutants altered carrier egress and partially reduced p230 membrane association. Our results show that Rab6 acts on TNF trafficking at the level of TGN exit in tubular carriers and our findings suggest Rab6 may stabilize p230 on the tubules to facilitate TNF transport. Both Rab6 isoforms are needed in macrophages for Golgi stack organization and for the efficient post-Golgi transport of TNF. This work provides new insights into Rab6 function and into the role of the Golgi complex in cytokine secretion in inflammatory macrophages.
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ItemSNX5 is essential for efficient macropinocytosis and antigen processing in primary macrophages(COMPANY OF BIOLOGISTS LTD, 2012-09-15)Macropinocytosis mediates the bulk endocytosis of solute molecules, nutrients and antigens. As this endocytic pathway is considered important in functions associated with immune responses, the molecular mechanisms regulating this pathway in immune cells is of particular significance. However, the regulators of macropinocytosis in primary cells remain poorly defined. Members of the sorting nexin (SNX) family have been implicated in macropinosome biogenesis in cultured cells and here we have analyzed the role of two SNX family members, SNX1 and its binding partner SNX5, in macropinocytosis of mouse primary macrophages. We show that endogenous SNX1 and SNX5 are localised to newly-formed macropinosomes in primary mouse macrophages and, moreover, demonstrate that SNX5 plays an essential role in macropinosome biogenesis. Depletion of SNX5 in bone marrow-derived macrophages dramatically decreased both the number and size of macropinosomes. Depletion of SNX5 also resulted in dramatic reduction in uptake and processing of soluble ovalbumin in macrophages, indicating that the majority of antigen uptake and delivery to late endosomes is via macropinocytosis. By contrast, the absence of SNX1 had no effect on endogenous SNX5 localisation and macropinosome biogenesis using macrophages from SNX1 knockout mice. Therefore, SNX5 can function independently of SNX1 and is a modulator of macropinocytosis that influences the uptake and processing of soluble antigen in primary mouse macrophages.
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ItemA Convenient Model of Severe, High Incidence Autoimmune Gastritis Caused by Polyclonal Effector T Cells and without Perturbation of Regulatory T Cells(PUBLIC LIBRARY SCIENCE, 2011-11-09)Autoimmune gastritis results from the breakdown of T cell tolerance to the gastric H(+)/K(+) ATPase. The gastric H(+)/K(+) ATPase is responsible for the acidification of gastric juice and consists of an α subunit (H/Kα) and a β subunit (H/Kβ). Here we show that CD4(+) T cells from H/Kα-deficient mice (H/Kα(-/-)) are highly pathogenic and autoimmune gastritis can be induced in sublethally irradiated wildtype mice by adoptive transfer of unfractionated CD4(+) T cells from H/Kα(-/-) mice. All recipient mice consistently developed the most severe form of autoimmune gastritis 8 weeks after the transfer, featuring hypertrophy of the gastric mucosa, complete depletion of the parietal and zymogenic cells, and presence of autoantibodies to H(+)/K(+) ATPase in the serum. Furthermore, we demonstrated that the disease significantly affected stomach weight and stomach pH of recipient mice. Depletion of parietal cells in this disease model required the presence of both H/Kα and H/Kβ since transfer of H/Kα(-/-) CD4(+) T cells did not result in depletion of parietal cells in H/Kα(-/-) or H/Kβ(-/-) recipient mice. The consistency of disease severity, the use of polyclonal T cells and a specific T cell response to the gastric autoantigen make this an ideal disease model for the study of many aspects of organ-specific autoimmunity including prevention and treatment of the disease.
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Itemc-Rel Controls Multiple Discrete Steps in the Thymic Development of Foxp3+ CD4 Regulatory T Cells(PUBLIC LIBRARY SCIENCE, 2011-10-31)The development of natural Foxp3(+) CD4 regulatory T cells (nTregs) proceeds via two steps that involve the initial antigen dependent generation of CD25(+)GITR(hi)Foxp3(-)CD4(+) nTreg precursors followed by the cytokine induction of Foxp3. Using mutant mouse models that lack c-Rel, the critical NF-κB transcription factor required for nTreg differentiation, we establish that c-Rel regulates both of these developmental steps. c-Rel controls the generation of nTreg precursors via a haplo-insufficient mechanism, indicating that this step is highly sensitive to c-Rel levels. However, maintenance of c-Rel in an inactive state in nTreg precursors demonstrates that it is not required for a constitutive function in these cells. While the subsequent IL-2 induction of Foxp3 in nTreg precursors requires c-Rel, this developmental transition does not coincide with the nuclear expression of c-Rel. Collectively, our results support a model of nTreg differentiation in which c-Rel generates a permissive state for foxp3 transcription during the development of nTreg precursors that influences the subsequent IL-2 dependent induction of Foxp3 without a need for c-Rel reactivation.
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ItemThe Golgi apparatus in the endomembrane-rich gastric parietal cells exist as functional stable mini-stacks dispersed throughout the cytoplasm(PORTLAND PRESS LTD, 2011-12)BACKGROUND INFORMATION: Acid-secreting gastric parietal cells are polarized epithelial cells that harbour highly abundant and specialized, H+,K+ ATPase-containing, tubulovesicular membranes in the apical cytoplasm. The Golgi apparatus has been implicated in the biogenesis of the tubulovesicular membranes; however, an unanswered question is how a typical Golgi organization could regulate normal membrane transport within the membrane-dense cytoplasm of parietal cells. RESULTS: Here, we demonstrate that the Golgi apparatus of parietal cells is not the typical juxta-nuclear ribbon of stacks, but rather individual Golgi units are scattered throughout the cytoplasm. The Golgi membrane structures labelled with markers of both cis- and trans-Golgi membrane, indicating the presence of intact Golgi stacks. The parietal cell Golgi stacks were closely aligned with the microtubule network and were shown to participate in both anterograde and retrograde transport pathways. Dispersed Golgi stacks were also observed in parietal cells from H+,K+ ATPase-deficient mice that lack tubulovesicular membranes. CONCLUSIONS: These results indicate that the unusual organization of individual Golgi stacks dispersed throughout the cytoplasm of these terminally differentiated cells is likely to be a developmentally regulated event.