A novel Golgi-localisation domain shared by a class of coiled-coil peripheral membrane proteins

Kjer‐Nielsen, Lars; Teasdale, Rohan D.; Vliet, Catherine Julia van; Gleeson, Paul A.

doi:10.1016/s0960-9822(99)80168-7

Cited by 141 publications

(152 citation statements)

References 14 publications

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“…Targeting eNOS to the transand post-Golgi vesicles via the GRIP domain resulted in an enzyme that produced very low amounts of basal NO and that was hyporesponsive to both thapsigargin and coexpression of Akt compared with the WT enzyme. The GRIP domain comprises ϳ42 amino acids and is shared by a family of resident peripheral Golgi coiled-coil membrane proteins, including p230, golgin-97, and golgin-245 (34). The GRIP docking site has been identified on a specific subset of Golgi membranes in the trans-Golgi network (55).…”

Section: Discussionmentioning

confidence: 99%

“…This domain is shared by a family of coiled-coil peripheral membrane proteins, including p230, golgin-97, and golgin-245 (34). A region of golgin-97 containing the GRIP domain (87 amino acids) was isolated from human umbilical vein endothelial cell mRNA; fused in-frame to G2A eNOS using PCR overlap extension with primers 5Ј-TGT GCC TCG AGC GGG GCC ACA TGT T-3Ј (sense), 5Ј-CCA GAC ACC CCC GGC CCC GTC ACG AAT AAC ACT GAC-3Ј (middle sense), 5Ј-GTC AGT GTT ATT CGT GAC GGG GCC GGG GGT GTC TGG-3Ј (middle sense), and 5Ј-ACT AGT TGA GTC TAG ACT AGG ACC ATG GTA TCC GAG GG TT-3Ј (antisense); and encodes the GRIP domain (VTNNTDLTDAREI-NFEYLKHVVLKFMSCRESEAFHLIKAVSVLLNFSQEEENMLKETL-EYKMSWFGSKPAPKGSIRPSISNPRIPWS).…”

Section: Golgi Complex-targeted Constructsmentioning

confidence: 99%

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Targeting of Endothelial Nitric-oxide Synthase to the Cytoplasmic Face of the Golgi Complex or Plasma Membrane Regulates Akt- Versus Calcium-dependent Mechanisms for Nitric Oxide Release

Fulton

Babbitt

Zoellner

et al. 2004

Journal of Biological Chemistry

126

125

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The heterogeneous localization of endothelial nitricoxide synthase (eNOS) on the Golgi complex versus the plasma membrane has made it difficult to dissect the regulation of each pool of enzyme. Here, we generated fusion proteins that specifically target the plasma membrane or cytoplasmic aspects of the Golgi complex and have assessed eNOS activation. Plasma membrane-targeted eNOS constructs were constitutively active, phosphorylated, and responsive to transmembrane calcium fluxes, yet were insensitive to further activation by Aktmediated phosphorylation. In contrast, cis-Golgi complex-targeted eNOS behaved similarly to wild-type eNOS and was less sensitive to calcium-dependent activation and highly responsive to Akt-dependent phosphorylation compared with plasma membrane versions. In plasma membrane-and Golgi complex-targeted constructs, Ser 1179 is critical for NO production. This study provides clear evidence for functional roles of plasma membrane-and Golgi complex-localized eNOS and supports the concept that proteins thought to be regulated and to function exclusively in the plasma membrane of cells can indeed signal and be regulated in internal Golgi membranes.Within the vascular endothelium, nitric oxide (NO) is generated via the enzyme endothelial nitric-oxide synthase (eNOS). 1

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Section: Discussionmentioning

confidence: 99%

Section: Golgi Complex-targeted Constructsmentioning

confidence: 99%

Targeting of Endothelial Nitric-oxide Synthase to the Cytoplasmic Face of the Golgi Complex or Plasma Membrane Regulates Akt- Versus Calcium-dependent Mechanisms for Nitric Oxide Release

Fulton

Babbitt

Zoellner

et al. 2004

Journal of Biological Chemistry

126

125

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“…Fixed cells were permeabilized with 0.1% Triton X-100, PBS for 4 min and then rinsed three times in PBS. Monolayers were incubated in primary antibodies as described (26), and bound antibodies were detected with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies. Images were acquired using a Bio-Rad MRC 1024 confocal imaging system.…”

Section: Immunofluorescencementioning

confidence: 99%

Medial Golgi but Not Late Golgi Glycosyltransferases Exist as High Molecular Weight Complexes

Opat¹,

Houghton

Gleeson

2000

Journal of Biological Chemistry

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To investigate the organization of Golgi glycosyltransferases and their mechanism of localization, we have compared the properties of a number of medial and late acting Golgi enzymes. The medial Golgi enzymes, Nacetylglucosaminyltransferase I and II (GnTI and Gn-TII) required high salt for solubilization and migrated as high molecular weight complexes on sucrose density gradients. In contrast, the late acting Golgi enzymes, ␤1,4-galactosyltransferase and ␣1,2-fucosyltransferase, were readily solubilized in low salt and migrated as monomers/dimers by sucrose density gradient centrifugation. Analysis of membrane-bound GnTI chimeras indicates that the formation of high molecular weight complexes does not require the transmembrane domain and cytoplasmic tail sequences of GnTI. Furthermore, a soluble form of GnTI, containing the stem region and catalytic domain, accumulated in the Golgi prior to secretion, in contrast to ␤1,4-galactosyltransferase. Soluble GnTI, which also associated with high molecular weight complexes, was comparable with membranebound GnTI in its ability to glycosylate newly synthesized glycoproteins in vivo. Mutation of charged residues within the stem region of GnTI, known to be important for "kin recognition", had no effect on the efficiency of Golgi localization, the inclusion into high molecular weight complexes, nor functional activity in vivo. The differences in behavior between the medial and late acting Golgi enzymes may contribute to their differential localization and their ability to glycosylate efficiently in the correct Golgi subcompartment.

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“…There are four human TGN golgins, namely p230/golgin-245, golgin-97, GCC185 and GCC88 (8)(9)(10)(11)(12)(13), which all belong to a subfamily of golgins characterized by a C-terminal GRIP domain (14)(15)(16). Recruitment of both p230/ golgin-245 (p230) and golgin-97 to the TGN is mediated through an interaction with the small G protein Arl1 (17)(18)(19)(20); however, these two golgins are localized to distinct membrane domains of the TGN (21).…”

mentioning

confidence: 99%

A trans -Golgi network golgin is required for the regulated secretion of TNF in activated macrophages in vivo

Lieu

Lock

Hammond

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The transmembrane precursor of tumor necrosis factor-␣ (TNF) exits the trans-Golgi network (TGN) in tubular carriers for subsequent trafficking and delivery to the cell surface; however, the molecular machinery responsible for Golgi export is unknown. We previously reported that members of the TGN golgin family are associated with subdomains and tubules of the TGN. Here, we show that the TGN golgin, p230/golgin-245 (p230), is essential for intracellular trafficking and cell surface delivery of TNF in transfected HeLa cells and activated macrophages. Live-cell imaging revealed that TNF transport from the TGN is mediated selectively by tubules and carriers marked by p230. Significantly, LPS activation of macrophages resulted in a dramatic increase of p230-labeled tubules and carriers emerging from the TGN, indicating that macrophages up-regulate the transport pathway for TNF export. Depletion of p230 in LPS-stimulated macrophages reduced cell surface delivery of TNF by >10-fold compared with control cells. To determine whether p230 depletion blocked TNF secretion in vivo, we generated retrogenic mice expressing a microRNAvector to silence p230. Bone-marrow stem cells were transduced with recombinant retrovirus containing microRNA constructs and transplanted into irradiated recipients. LPS-activated peritoneal macrophages from p230 miRNA retrogenic mice were depleted of p230 and had dramatically reduced levels of cell surface TNF. Overall, these studies have identified p230 as a key regulator of TNF secretion and have shown that LPS activation of macrophages results in increased Golgi carriers for export. Also, we have demonstrated a previously undescribed approach to control cytokine secretion by the specific silencing of trafficking machinery. intracellular trafficking ͉ RNAi transgenic mice ͉ TNF secretion ͉ inflammation ͉ membrane transport T umor necrosis factor-␣ (TNF) is the main proinflammatory cytokine made and secreted by inflammatory macrophages. Early release of TNF in response to LPS or other inflammatory signals enhances the activation and recruitment of T cells and ensures robust innate and acquired immune responses (1). The excessive secretion of TNF is also a prevalent and clinically significant problem in acute inflammation and in chronic inflammatory disease (2). Anti-TNF treatments have shown success in the treatment of rheumatoid arthritis, inflammatory bowel disease, and other conditions (3). Now improved antiTNF strategies that may offer more constrained or cell type specific control of TNF secretion are being sought. This requires identification of molecular mediators of TNF secretion, particularly those that can be targeted to block TNF release.We have characterized the secretory pathway for TNF in activated macrophages and identified key organelles and components of the trafficking machinery whose expression or function is up-regulated by LPS to support cytokine secretion (4-6). Among these are members of the SNARE family of membrane fusion proteins required at different points along this ...

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A novel Golgi-localisation domain shared by a class of coiled-coil peripheral membrane proteins

Cited by 141 publications

References 14 publications

Targeting of Endothelial Nitric-oxide Synthase to the Cytoplasmic Face of the Golgi Complex or Plasma Membrane Regulates Akt- Versus Calcium-dependent Mechanisms for Nitric Oxide Release

Targeting of Endothelial Nitric-oxide Synthase to the Cytoplasmic Face of the Golgi Complex or Plasma Membrane Regulates Akt- Versus Calcium-dependent Mechanisms for Nitric Oxide Release

Medial Golgi but Not Late Golgi Glycosyltransferases Exist as High Molecular Weight Complexes

A trans -Golgi network golgin is required for the regulated secretion of TNF in activated macrophages in vivo

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