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William J Brown - One of the best experts on this subject based on the ideXlab platform.

  • reconstitution of phospholipase a2 dependent Golgi Membrane tubules
    Methods of Molecular Biology, 2016
    Co-Authors: Edward B Cluett, Marie E. Bechler, Paul De Figueiredo, Kevin D Thorsen, William J Brown
    Abstract:

    The Golgi complex is the Grand Central Station of intracellular Membrane trafficking in the secretory and endocytic pathways. Anterograde and retrograde export of cargo from the Golgi complex involves a complex interplay between the formation of coated vesicles and Membrane tubules, although much less is known about tubule-mediated trafficking. Recent advances using in vitro assays have identified several cytoplasmic phospholipase A2 (PLA2) enzymes that are required for the biogenesis of Membrane tubules and their roles in the functional organization of the Golgi complex. In this chapter we describe methods for the cell-free reconstitution of PLA2-dependent Golgi Membrane tubule formation. These methods should facilitate the identification of other proteins that regulate this process.

  • Gβ1γ2 activates phospholipase A2-dependent Golgi Membrane tubule formation
    Frontiers in cell and developmental biology, 2014
    Co-Authors: Marie E. Bechler, William J Brown
    Abstract:

    Heterotrimeric G proteins transduce the ligand binding of transMembrane G protein coupled receptors into a variety of intracellular signaling pathways. Recently, heterotrimeric Gβγ subunit signaling at the Golgi complex has been shown to regulate the formation of vesicular transport carriers that deliver cargo from the Golgi to the plasma Membrane. In addition to vesicles, Membrane tubules have also been shown to mediate export from the Golgi complex, which requires the activity of cytoplasmic phospholipase A2 (PLA2) enzyme activity. Through the use of an in vitro reconstitution assay with isolated Golgi complexes, we provide evidence that Gβ1γ2 signaling also stimulates Golgi Membrane tubule formation. In addition, we show that an inhibitor of Gβγ activation of PLA2 enzymes inhibits in vitro Golgi Membrane tubule formation. Additionally, purified Gβγ protein stimulates Membrane tubules in the presence of low (sub-threshold) cytosol concentrations. Importantly, this Gβγ stimulation of Golgi Membrane tubule formation was inhibited by treatment with the PLA2 antagonist ONO-RS-082. These studies indicate that Gβ1γ2 signaling activates PLA2 enzymes required for Golgi Membrane tubule formation, thus establishing a new layer of regulation for this process.

  • inhibition of a Golgi complex lysophospholipid acyltransferase induces Membrane tubule formation and retrograde trafficking
    Molecular Biology of the Cell, 2003
    Co-Authors: Dan Drecktrah, Kimberly Chambers, Esther L Racoosin, Edward B Cluett, Amy Gucwa, Brian Jackson, William J Brown
    Abstract:

    Recent studies have suggested that formation of Golgi Membrane tubules involves the generation of Membrane-associated lysophospholipids by a cytoplasmic Ca2+-independent phospholipase A2 (PLA2). Herein, we provide additional support for this idea by showing that inhibition of lysophospholipid reacylation by a novel Golgi-associated lysophosphatidylcholine acyltransferase (LPAT) induces the rapid tubulation of Golgi Membranes, leading in their retrograde movement to the endoplasmic reticulum. Inhibition of the Golgi LPAT was achieved by 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976), a previously characterized antagonist of acyl-CoA cholesterol acyltransferase. The effect of CI-976 was similar to that of brefeldin A, except that the coatomer subunit β-COP remained on Golgi-derived Membrane tubules. CI-976 also enhanced the cytosol-dependent formation of tubules from Golgi complexes in vitro and increased the levels of lysophosphatidylcholine in Golgi Membranes. Moreover, preincubation of cells with PLA2 antagonists inhibited the ability of CI-976 to induce tubules. These results suggest that Golgi Membrane tubule formation can result from increasing the content of lysophospholipids in Membranes, either by stimulation of a PLA2 or by inhibition of an LPAT. These two opposing enzyme activities may help to coordinately regulate Golgi Membrane shape and tubule formation.

  • Stimulation of Golgi Membrane tubulation and retrograde trafficking to the ER by phospholipase A2 activating protein (PLAP) peptide
    Journal of cellular biochemistry, 1999
    Co-Authors: Renée S. Polizotto, Paul De Figueiredo, William J Brown
    Abstract:

    Recent pharmacological studies using specific antagonists of phospholipase A2 (PLA2) activity have suggested that the formation of Golgi Membrane tubules, 60–80 nm in diameter and up to several microns long, both in vivo and in a cell-free cytosol-dependent reconstitution system, requires the activity of a cytoplasmic Ca2+-independent PLA2. We confirm and extend these studies by demonstrating that the stimulators of PLA2, melittin and PLA2 activating protein peptide (PLAPp), enhance cytosol-dependent Golgi Membrane tubulation. Starting with preparations of bovine brain cytosol (BBC), or a fraction of BBC that is highly enriched in tubulation activity, called the gel filtration (GF) fraction, that are at subsaturating concentrations for inducing tubulation in vitro, we found that increasing concentrations of melittin or PLAPp produced a linear and saturable stimulation of Golgi Membrane tubulation. This stimulation was inhibited by cytosolic PLA2 antagonists, including the Ca2+-independent PLA2-specific antagonist, bromoenol lactone. The stimulatory effect of PLAPp, and its inhibition by PLA2 antagonists, was reproduced using a permeabilized cell system, which reconstitutes both cytosol-dependent Golgi Membrane tubulation and retrograde trafficking to the endoplasmic reticulum (ER). Taken together, these results are consistent with the idea that cytosolic PLA2 activity is involved in the formation of Golgi Membrane tubules, which can serve as trafficking intermediates in Golgi-to-ER retrograde movement. J. Cell. Biochem. 74:670–683, 1999. © 1999 Wiley-Liss, Inc.

Toshiaki Mitsui - One of the best experts on this subject based on the ideXlab platform.

  • Proteomic Analysis of Rice Golgi Membranes Isolated by Floating Through Discontinuous Sucrose Density Gradient.
    Methods in molecular biology (Clifton N.J.), 2017
    Co-Authors: Kazusato Oikawa, Takuya Inomata, Yoshitoshi Hirao, Tadashi Yamamoto, Marouane Baslam, Kentaro Kaneko, Toshiaki Mitsui
    Abstract:

    The Golgi apparatus is an endoMembrane system organelle and has roles in glycosylation, sorting, and secretion of proteins in the secretory pathway. It has a central function in living organism and is also essential for plant growth. Proteomic approaches to identify the Golgi Membrane proteins have been performed in cell suspension cultures and many Golgi Membrane-associated proteins were found, whereas it has well established in rice seedling yet. In this chapter, our recent improving published methods for isolated rice Golgi Membranes by floating through a discontinuous sucrose density gradient are provided in detail with proteomic analyses.

  • Purification and Characterization of Golgi Membrane-Bound Nucleoside Diphosphatase from Suspension-Cultured Cells of Sycamore (Acer Pseudoplatanus L.)
    Plant Biotechnology, 2001
    Co-Authors: Satoshi Mikami, Ryo Suganuma, Hidetaka Hori, Toshiaki Mitsui
    Abstract:

    Inosine diphosphatase (IDPase) isoforms associated with Golgi Membranes were studied in sycamore cell culture. These enzyme isoforms were solubilized with Triton X-100 and purified by chromatography using DEAE-Toyopearl and SOURCE-S columns. The isoforms were separated into two distinguishable fractions (peak 1 and 2) by SOURCE-S column chromatography. Furthermore the peak 1 contained at least two isoform bands detected by native-PAGE analysis. The apparent molecular sizes of these three isoforms were estimated by both gel filtration and SDS-PAGE to be 50 kDa, indicating that the Golgi Membrane-bound IDPase has a monomeric structure. These IDPase isoforms required divalent cations (Ca2+, Mg2+, Co2+, Mn2+) for their hydrolyzing activity, and were inhibited by ATP. IDP, UDP, and GDP were effective substrates for these enzymes. It is clearly indicated that the sycamore Golgi Membrane-bound IDPase is a nucleoside diphosphatase.

  • STRUCTURE AND FUNCTION OF THE Golgi COMPLEX IN RICE CELLS. II: PURIFICATION AND CHARACTERIZATION OF Golgi Membrane-BOUND NUCLEOSIDE DIPHOSPHATASE
    Plant physiology, 1994
    Co-Authors: Toshiaki Mitsui, M. Honma, T. Kondo, N. Hashimoto, S. Kimura, Ikuo Igaue
    Abstract:

    Inosine diphosphatase bound to Golgi Membranes was studied in rice (Oryza sativa L. cv Nipponkai) cells. The enzyme was solubilized with Triton X-100 from isolated rice Golgi Membranes and was highly purified employing a series of chromatography steps in the presence of 20% glycerol and 0.1% Triton X-100. The apparent molecular mass of the enzyme was estimated by gel filtration column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 200 and 55 kD, respectively. The isoelectric point of the enzyme was determined to be 7.5. The optimal pH for the enzyme activity was around 7 and the enzyme required Mg2+ for hydrolyzing activity. IDP, UDP, and GDP were effective substrate for the purified rice Golgi Membrane-bound inosine diphosphatase, whereas activity with ADP, CDP, and thymidine 5[prime]-diphosphate was 10 to 20% of IDP. The Km values for IDP, UDP, and GDP were 0.48, 0.50, and 0.67 mM, respectively, and Vmax values were 1.85, 1.54, and 1.67 [mu]mol min-1 mg-1, respectively. These results indicate that the rice Golgi enzyme is a nucleoside diphosphatase that is specific for IDP, UDP, and GDP. Furthermore, this rice Golgi nucleoside diphosphatase stimulated the activity of glucan synthase I also localized in rice Golgi Membranes. The results strongly support the view that this nucleoside diphosphatase is involved in regulation of [beta]-glucan synthesis in the plant Golgi complex.

Carlos B Hirschberg - One of the best experts on this subject based on the ideXlab platform.

  • reconstitution identification and purification of the rat liver Golgi Membrane gdp fucose transporter
    Journal of Biological Chemistry, 1999
    Co-Authors: Luigi Puglielli, Carlos B Hirschberg
    Abstract:

    Abstract Glycosylation of glycoproteins, proteoglycans, and glycolipids occurring in the Golgi apparatus requires the translocation of nucleotide sugars from the cytosol into the lumen of the Golgi. Translocation is mediated by specific nucleotide sugar transporters, integral Golgi Membrane proteins that regulate the above glycosylation reactions. A defect in GDP-fucose transport into the lumen of the Golgi apparatus has been recently identified in a patient affected by leukocyte adhesion deficiency type II syndrome (Lubke, T., Marquardt, T., von Figura, K., and Korner, C. (1999) J. Biol. Chem. 274, 25986–25989). We have now identified and purified the rat liver Golgi Membrane GDP-fucose transporter, a protein with an apparent molecular mass of 39 kDa, by a combination of column chromatography, native functional size determination on a glycerol gradient, and photoaffinity labeling with 8-azidoguanosine-5′-[α-32P] triphosphate, an analog of GDP-fucose. The purified transporter appears to exist as a homodimer within the Golgi Membrane. When reconstituted into phosphatidylcholine liposomes, it was active in GDP-fucose transport and was specifically photolabeled with 8-azidoguanosine-5′-[α-32P]triphosphate. Transport was also stimulated 2–3-fold after preloading proteoliposomes with GMP, the putative antiporter.

  • Identification, Purification, and Characterization of the Rat Liver Golgi Membrane ATP Transporter
    The Journal of biological chemistry, 1999
    Co-Authors: Luigi Puglielli, Elisabet C. Mandon, Carlos B Hirschberg
    Abstract:

    Abstract Phosphorylation of secretory and integral Membrane proteins and of proteoglycans also occurs in the lumen of the Golgi apparatus. ATP, the phosphate donor in these reactions, must first cross the Golgi Membrane before it can serve as substrate. The existence of a specific ATP transporter in the Golgi Membrane has been previously demonstrated in vitro using intact Golgi Membrane vesicles from rat liver and mammary gland. We have now identified and purified the rat liver Golgi Membrane ATP transporter. The transporter was purified to apparent homogeneity by a combination of conventional ion exchange, dye color, and affinity chromatography. An ∼70,000-fold purification (2% yield) was achieved starting from crude rat liver Golgi Membranes. A protein with an apparent molecular mass of 60 kDa was identified as the putative transporter by a combination of column chromatography, photoaffinity labeling with an analog of ATP, and native functional size determination on a glycerol gradient. The purified transporter appears to exist as a homodimer within the Golgi Membrane, and when reconstituted into phosphatidylcholine liposomes, was active in ATP but not nucleotide sugar or adenosine 3′-phosphate 5′-phosphosulfate transport. The transport activity was saturable with an apparentK m very similar to that of intact Golgi vesicles.

  • identification and purification of the rat liver Golgi Membrane udp n acetylgalactosamine transporter
    Journal of Biological Chemistry, 1999
    Co-Authors: Luigi Puglielli, David M Rancour, Elisabet C. Mandon, Anant K Menon, Carlos B Hirschberg
    Abstract:

    Abstract Glycosylation of glycoproteins, proteoglycans, and glycosphingolipids occurs mainly in the lumen of the endoplasmic reticulum and the Golgi apparatus. Nucleotide sugars, donors of all the sugars involved in Golgi glycosylation reactions, are synthesized in the cytoplasm and require specialized transporters to be translocated into the lumen of the Golgi apparatus. By controlling the supply of sugar nucleotides in the lumen of the Golgi apparatus, these transporters directly regulate the glycosylation of macromolecules transiting the Golgi. We have identified and purified the rat liver Golgi Membrane UDP-N-acetylgalactosamine transporter. The transporter was purified to apparent homogeneity by a combination of conventional and dye color chromatography. An ∼63,000-fold purification (6% yield) was achieved starting from crude rat liver Golgi Membranes and resulting in a protein with an apparent molecular mass of 43 kDa. The transporter was active when reconstituted into phosphatidylcholine vesicles and could be specifically photolabeled withP 3-(4-azidoanilido)-uridine-5′-[P 1-32P]triphosphate, an analog of UDP-N-acetylgalactosamine. Native functional size determination on a glycerol gradient suggested that the transporter exists as a homodimer within the Golgi Membrane.

  • purification of the Golgi adenosine 3 phosphate 5 phosphosulfate transporter a homodimer within the Membrane
    Proceedings of the National Academy of Sciences of the United States of America, 1994
    Co-Authors: Elisabet C. Mandon, Ellis S. Kempner, Marcos E Milla, Carlos B Hirschberg
    Abstract:

    Abstract Sulfation of proteoglycans, secretory and Membrane proteins, and glycolipids occurs in the lumen of the Golgi apparatus. Adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the sulfate donor in these reactions, must be transported from the cytosol, its site of synthesis, into the lumen of the Golgi apparatus. We have identified and purified to apparent homogeneity the rat liver Golgi Membrane PAPS transporter by a combination of conventional and affinity chromatography as well as photoaffinity radiolabeling with adenosine 3',5'-bisphosphate, a competitive inhibitor of PAPS transport. The transporter, a 75-kDa protein, was purified 70,000-fold over homogenate (6% yield) and transported PAPS into phosphatidylcholine liposomes selectively and in a saturable manner (apparent Km of 1.7 microM). Radiation target-inactivation analyses of the transport activity in rat liver Golgi vesicles, together with the above described biochemical approaches, demonstrate that the PAPS transporter within the Golgi Membrane is a homodimer.

  • A monomeric protein in the Golgi Membrane catalyzes both N-deacetylation and N-sulfation of heparan sulfate.
    The Journal of biological chemistry, 1994
    Co-Authors: Elisabet C. Mandon, Ellis S. Kempner, Masayuki Ishihara, Carlos B Hirschberg
    Abstract:

    Abstract Recent studies have shown that the rat liver heparan sulfate N-deacetylase/N-sulfotransferase is a glycoprotein encoded by a single polypeptide chain of 882 amino acids. Using radiation inactivation analyses, we have now determined that in rat liver Golgi vesicles the target size for the N-deacetylase is 88 +/- 14 kDa, whereas that of the N-sulfotransferase is 92 +/- 8 kDa. These results, together with previous biochemical and molecular cloning approaches, demonstrate that 1) in rat liver Golgi Membranes there exists only on population of molecules expressing both activities, 2) the active protein in the Golgi Membrane functions as a monomer, and 3) there is no evidence that a large independent protein acts as a regulator of either activity.

Yanzhuang Wang - One of the best experts on this subject based on the ideXlab platform.

  • monoubiquitination of syntaxin 5 regulates Golgi Membrane dynamics during the cell cycle
    Developmental Cell, 2016
    Co-Authors: Shijiao Huang, Danming Tang, Yanzhuang Wang
    Abstract:

    The Golgi apparatus undergoes a ubiquitin-dependent disassembly and reassembly process during each cycle of cell division. Here we report the identification of the Golgi t-SNARE syntaxin 5 (Syn5) as the ubiquitinated substrate. Syn5 is monoubiquitinated by the ubiquitin ligase HACE1 in early mitosis and deubiquitinated by the deubiquitinase VCIP135 in late mitosis. Syn5 ubiquitination on lysine 270 (K270) in the SNARE domain impairs the interaction between Syn5 and the cognate v-SNARE Bet1 but increases its binding to p47, the adaptor protein of p97. Expression of the Syn5 K270R mutant in cells impairs post-mitotic Golgi reassembly. Therefore, monoubiquitination of Syn5 in early mitosis disrupts SNARE complex formation. Subsequently, ubiquitinated Syn5 recruits p97/p47 to the mitotic Golgi fragments and promotes post-mitotic Golgi reassembly upon ubiquitin removal by VCIP135. Overall, this study reveals both the substrate and the mechanism of ubiquitin-mediated regulation of Golgi Membrane dynamics during the cell cycle.

  • cell cycle regulation of vcip135 deubiquitinase activity and function in p97 p47 mediated Golgi reassembly
    Molecular Biology of the Cell, 2015
    Co-Authors: Xiaoyan Zhang, Yanzhuang Wang
    Abstract:

    In mammalian cells, the inheritance of the Golgi apparatus into the daughter cells during each cycle of cell division is mediated by a disassembly and reassembly process, and this process is precisely controlled by phosphorylation and ubiquitination. VCIP135 (valosin-containing protein p97/p47 complex–interacting protein, p135), a deubiquitinating enzyme required for p97/p47-mediated postmitotic Golgi Membrane fusion, is phosphorylated at multiple sites during mitosis. However, whether phosphorylation directly regulates VCIP135 deubiquitinase activity and Golgi Membrane fusion in the cell cycle remains unknown. We show that, in early mitosis, phosphorylation of VCIP135 by Cdk1 at a single residue, S130, is sufficient to inactivate the enzyme and inhibit p97/p47-mediated Golgi Membrane fusion. At the end of mitosis, VCIP135 S130 is dephosphorylated, which is accompanied by the recovery of its deubiquitinase activity and Golgi reassembly. Our results demonstrate that phosphorylation and ubiquitination are coordinated via VCIP135 to control Golgi Membrane dynamics in the cell cycle.

  • Monoubiquitination Regulates Golgi Membrane Dynamics In The Cell Cycle
    The FASEB Journal, 2015
    Co-Authors: Yanzhuang Wang, Xiaoyan Zhang, Shijiao Huang
    Abstract:

    Partitioning of the Golgi Membrane into daughter cells during mammalian cell division occurs through a unique disassembly and reassembly process. Several converging lines of evidence have suggested that monoubiquitination plays an essential role in the regulation of post-mitotic Golgi Membrane fusion. Monoubiquitination, as a regulatory signal, occurs during mitotic Golgi disassembly and is required for subsequent Golgi reassembly. The AAA ATPase p97 and its adapter protein p47 are involved in Membrane fusion during post-mitotic Golgi reassembly. The p97/p47 complex binds to monoubiquitin through the UBA domain of p47, and this interaction is required for p97-mediated Golgi Membrane fusion. Proteasome activity is not involved in either Golgi disassembly or reassembly. We have identified a Golgi-localized ubiquitin E3 ligase HACE1 which is involved in mitotic Golgi disassembly; reduced HACE1 activity is human cancer such as Wilms' tumor results in fragmented Golgi. We have also discovered the p97/p47 bindi...

  • phosphorylation regulates vcip135 function in Golgi Membrane fusion during the cell cycle
    Journal of Cell Science, 2014
    Co-Authors: Xiaoyan Zhang, Honghao Zhang, Yanzhuang Wang
    Abstract:

    The Golgi apparatus in mammalian cells consists of stacks that are often laterally linked into a ribbon-like structure. During cell division, the Golgi disassembles into tubulovesicular structures in the early stages of mitosis and reforms in the two daughter cells by the end of mitosis. Valosin-containing protein p97–p47 complex-interacting protein, p135 (VCIP135), an essential factor involved in p97-mediated Membrane fusion pathways, is required for postmitotic Golgi cisternae regrowth and Golgi structure maintenance in interphase. However, how VCIP135 function is regulated in the cell cycle remains unclear. Here, we report that VCIP135 depletion by RNA interference results in Golgi fragmentation. VCIP135 function requires Membrane association and p97 interaction, both of which are inhibited in mitosis by VCIP135 phosphorylation. We found that wild-type VCIP135, but not its phosphomimetic mutants, rescues Golgi structure in VCIP135-depleted cells. Our results demonstrate that VCIP135 phosphorylation regulates its Golgi Membrane association and p97 interaction, and thus contributes to the tight control of the Golgi disassembly and reassembly process during the cell cycle.

  • cell cycle regulation of Golgi Membrane dynamics
    Trends in Cell Biology, 2013
    Co-Authors: Danming Tang, Yanzhuang Wang
    Abstract:

    The Golgi apparatus is a membranous organelle in the cell that plays essential roles in protein and lipid trafficking, sorting, processing, and modification. Its basic structure is a stack of closely aligned flattened cisternae. In mammalian cells, dozens of Golgi stacks are often laterally linked into a ribbon-like structure. Biogenesis of the Golgi during cell division occurs through a sophisticated disassembly and reassembly process that can be divided into three distinct but cooperative steps, including the deformation and reformation of the Golgi cisternae, stacks, and ribbon. Here, we review our current understanding of the protein machineries that control these three steps in the cycle of mammalian cell division: GRASP65 and GRASP55 in Golgi stack and ribbon formation; ubiquitin and AAA ATPases in postmitotic Golgi Membrane fusion; and Golgins and cytoskeleton in Golgi ribbon formation.

Vytas A. Bankaitis - One of the best experts on this subject based on the ideXlab platform.

  • The Cirque du Soleil of Golgi Membrane dynamics.
    The Journal of cell biology, 2009
    Co-Authors: Vytas A. Bankaitis
    Abstract:

    The role of lipid metabolic enzymes in Golgi Membrane remodeling is a subject of intense interest. Now, in this issue, Schmidt and Brown (2009. J. Cell Biol. doi:10.1083/jcb.200904147) report that lysophosphatidic acid–specific acyltransferase, LPAAT3, contributes to Golgi Membrane dynamics by suppressing tubule formation.

  • A phosphatidylinositol transfer protein controls the phosphatidylcholine content of yeast Golgi Membranes
    The Journal of cell biology, 1994
    Co-Authors: Todd P. Mcgee, Henry B. Skinner, Eric A. Whitters, Susan A. Henry, Vytas A. Bankaitis
    Abstract:

    SEC14p is required for protein transport from the yeast Golgi complex. We describe a quantitative analysis of yeast bulk Membrane and Golgi Membrane phospholipid composition under conditions where Golgi secretory function has been uncoupled from its usual SEC14p requirement. The data demonstrate that SEC14p specifically functions to maintain a reduced phosphatidylcholine content in Golgi Membranes and indicate that overproduction of SEC14p markedly reduces the apparent rate of phosphatidylcholine biosynthesis via the CDP-choline pathway in vivo. We suggest that SEC14p serves as a sensor of Golgi Membrane phospholipid composition through which the activity of the CDP-choline pathway in Golgi Membranes is regulated such that a phosphatidylcholine content that is compatible with the essential secretory function of these Membranes is maintained.