The Mitotic Phosphorylation Cycle of the Cis-Golgi Matrix Protein Gm130

Martin Lowe; Nicholas K. Gonatas; Graham Warren

doi:10.1083/jcb.149.2.341

The Mitotic Phosphorylation Cycle of the Cis-Golgi Matrix Protein Gm130

The Journal of Cell Biology ◽

10.1083/jcb.149.2.341 ◽

2000 ◽

Vol 149 (2) ◽

pp. 341-356 ◽

Cited By ~ 108

Author(s):

Martin Lowe ◽

Nicholas K. Gonatas ◽

Graham Warren

Keyword(s):

Matrix Protein ◽

Active Species ◽

Regulatory Subunit ◽

Temporal Regulation ◽

Vesicle Tethering ◽

Golgi Membranes ◽

Golgi Fragmentation ◽

Mitotic Phosphorylation ◽

Golgi Matrix

The cis-Golgi matrix protein GM130 is phosphorylated in mitosis on serine 25. Phosphorylation inhibits binding to p115, a vesicle-tethering protein, and has been implicated as an important step in the mitotic Golgi fragmentation process. We have generated an antibody that specifically recognizes GM130 phosphorylated on serine 25, and used this antibody to study the temporal regulation of phosphorylation in vivo. GM130 is phosphorylated in prophase as the Golgi complex starts to break down, and remains phosphorylated during further breakdown and partitioning of the Golgi fragments in metaphase and anaphase. In telophase, GM130 is dephosphorylated as the Golgi fragments start to reassemble. The timing of phosphorylation and dephosphorylation correlates with the dissociation and reassociation of p115 with Golgi membranes. GM130 phosphorylation and p115 dissociation appear specific to mitosis, since they are not induced by several drugs that trigger nonmitotic Golgi fragmentation. The phosphatase responsible for dephosphorylation of mitotic GM130 was identified as PP2A. The active species was identified as heterotrimeric phosphatase containing the Bα regulatory subunit, suggesting a role for this isoform in the reassembly of mitotic Golgi membranes at the end of mitosis.

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A Specific Activation of the Mitogen-Activated Protein Kinase Kinase 1 (Mek1) Is Required for Golgi Fragmentation during Mitosis

The Journal of Cell Biology ◽

10.1083/jcb.149.2.331 ◽

2000 ◽

Vol 149 (2) ◽

pp. 331-340 ◽

Cited By ~ 74

Author(s):

Antonino Colanzi ◽

Thomas J. Deerinck ◽

Mark H. Ellisman ◽

Vivek Malhotra

Keyword(s):

Protein Kinase ◽

Rat Kidney ◽

Lethal Factor ◽

Mitogen Activated Protein Kinase ◽

Permeabilized Cells ◽

Golgi Membranes ◽

Mitogen Activated Protein ◽

Golgi Fragmentation ◽

Protein Kinase Kinase

Incubation of permeabilized cells with mitotic extracts results in extensive fragmentation of the pericentriolarly organized stacks of cisternae. The fragmented Golgi membranes are subsequently dispersed from the pericentriolar region. We have shown previously that this process requires the cytosolic protein mitogen-activated protein kinase kinase 1 (MEK1). Extracellular signal–regulated kinase (ERK) 1 and ERK2, the known downstream targets of MEK1, are not required for this fragmentation (Acharya et al. 1998). We now provide evidence that MEK1 is specifically phosphorylated during mitosis. The mitotically phosphorylated MEK1, upon partial proteolysis with trypsin, generates a different peptide population compared with interphase MEK1. MEK1 cleaved with the lethal factor of the anthrax toxin can still be activated by its upstream mitotic kinases, and this form is fully active in the Golgi fragmentation process. We believe that the mitotic phosphorylation induces a change in the conformation of MEK1 and that this form of MEK1 recognizes Golgi membranes as a target compartment. Immunoelectron microscopy analysis reveals that treatment of permeabilized normal rat kidney (NRK) cells with mitotic extracts, treated with or without lethal factor, converts stacks of pericentriolar Golgi membranes into smaller fragments composed predominantly of tubuloreticular elements. These fragments are similar in distribution, morphology, and size to the fragments observed in the prometaphase/metaphase stage of the cell cycle in vivo.

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Cdc2 Kinase Directly Phosphorylates the cis-Golgi Matrix Protein GM130 and Is Required for Golgi Fragmentation in Mitosis

Cell ◽

10.1016/s0092-8674(00)81737-7 ◽

1998 ◽

Vol 94 (6) ◽

pp. 783-793 ◽

Cited By ~ 224

Author(s):

Martin Lowe ◽

Catherine Rabouille ◽

Nobuhiro Nakamura ◽

Rose Watson ◽

Mark Jackman ◽

...

Keyword(s):

Matrix Protein ◽

Cdc2 Kinase ◽

Golgi Fragmentation ◽

Golgi Matrix

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A Role for Giantin in Docking COPI Vesicles to Golgi Membranes

The Journal of Cell Biology ◽

10.1083/jcb.140.5.1013 ◽

1998 ◽

Vol 140 (5) ◽

pp. 1013-1021 ◽

Cited By ~ 212

Author(s):

Birte Sönnichsen ◽

Martin Lowe ◽

Tim Levine ◽

Eija Jämsä ◽

Barbara Dirac-Svejstrup ◽

...

Keyword(s):

Membrane Protein ◽

Matrix Protein ◽

Membrane Receptor ◽

Golgi Membrane ◽

Vesicle Docking ◽

Golgi Membranes ◽

Docking Protein ◽

Golgi Matrix

We have previously shown that p115, a vesicle docking protein, binds to two proteins (p130 and p400) in detergent extracts of Golgi membranes. p130 was identified as GM130, a Golgi matrix protein, and was shown to act as a membrane receptor for p115. p400 has now been identified as giantin, a Golgi membrane protein with most of its mass projecting into the cytoplasm. Giantin is found on COPI vesicles and pretreatment with antibodies inhibits both the binding of p115 and the docking of these vesicles with Golgi membranes. In contrast, GM130 is depleted from COPI vesicles and inhibition of the GM130 on Golgi membranes, using either antibodies or an NH2-terminal GM130 peptide, inhibits p115 binding and vesicle docking. Together these results suggest that COPI vesicles are docked by giantin on the COPI vesicles and GM130 on Golgi membranes with p115 providing a bridge.

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The localization and phosphorylation of p47 are important for Golgi disassembly–assembly during the cell cycle

The Journal of Cell Biology ◽

10.1083/jcb.200303048 ◽

2003 ◽

Vol 161 (6) ◽

pp. 1067-1079 ◽

Cited By ~ 65

Author(s):

Keiji Uchiyama ◽

Eija Jokitalo ◽

Mervi Lindman ◽

Mark Jackman ◽

Fumi Kano ◽

...

Keyword(s):

Cell Cycle ◽

Mammalian Cells ◽

In Vitro Assay ◽

Inhibitory Mechanism ◽

Vitro Assay ◽

Daughter Cells ◽

Golgi Membranes ◽

Golgi Fragmentation ◽

Mitotic Phosphorylation

In mammalian cells, the Golgi apparatus is disassembled at the onset of mitosis and reassembled at the end of mitosis. This disassembly–reassembly is generally believed to be essential for the equal partitioning of Golgi into two daughter cells. For Golgi disassembly, membrane fusion, which is mediated by NSF and p97, needs to be blocked. For the NSF pathway, the tethering of p115-GM130 is disrupted by the mitotic phosphorylation of GM130, resulting in the inhibition of NSF-mediated fusion. In contrast, the p97/p47 pathway does not require p115-GM130 tethering, and its mitotic inhibitory mechanism has been unclear. Now, we have found that p47, which mainly localizes to the nucleus during interphase, is phosphorylated on Serine-140 by Cdc2 at mitosis. The phosphorylated p47 does not bind to Golgi membranes. An in vitro assay shows that this phosphorylation is required for Golgi disassembly. Microinjection of p47(S140A), which is unable to be phosphorylated, allows the cell to keep Golgi stacks during mitosis and has no effect on the equal partitioning of Golgi into two daughter cells, suggesting that Golgi fragmentation-dispersion may not be obligatory for equal partitioning even in mammalian cells.

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Evidence that Golgi structure depends on a p115 activity that is independent of the vesicle tether components giantin and GM130

The Journal of Cell Biology ◽

10.1083/jcb.200105005 ◽

2001 ◽

Vol 155 (2) ◽

pp. 227-238 ◽

Cited By ~ 81

Author(s):

Manojkumar A. Puthenveedu ◽

Adam D. Linstedt

Keyword(s):

Brefeldin A ◽

Detectable Effect ◽

Vesicle Tethering ◽

Culture Cells ◽

Binding Partners ◽

Golgi Fragmentation ◽

Copi Vesicle ◽

Golgi Structure

Inhibition of the putative coatomer protein I (COPI) vesicle tethering complex, giantin–p115–GM130, may contribute to mitotic Golgi breakdown. However, neither this, nor the role of the giantin–p115–GM130 complex in the maintenance of Golgi structure has been demonstrated in vivo. Therefore, we generated antibodies directed against the mapped binding sites in each protein of the complex and injected these into mammalian tissue culture cells. Surprisingly, the injected anti-p115 and antigiantin antibodies caused proteasome-mediated degradation of the corresponding antigens. Reduction of p115 levels below detection led to COPI-dependent Golgi fragmentation and apparent accumulation of Golgi-derived vesicles. In contrast, neither reduction of giantin below detectable levels, nor inhibition of p115 binding to GM130, had any detectable effect on Golgi structure or Golgi reassembly after cell division or brefeldin A washout. These observations indicate that inhibition of p115 can induce a mitotic-like Golgi disassembly, but its essential role in Golgi structure is independent of its Golgi-localized binding partners giantin and GM130.

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The Role of the Tethering Proteins p115 and GM130 in Transport through the Golgi Apparatus In Vivo

Molecular Biology of the Cell ◽

10.1091/mbc.11.2.635 ◽

2000 ◽

Vol 11 (2) ◽

pp. 635-645 ◽

Cited By ~ 134

Author(s):

Joachim Seemann ◽

Eija Jämsä Jokitalo ◽

Graham Warren

Keyword(s):

Intracellular Transport ◽

Microscopic Analysis ◽

Electron Microscopic ◽

Golgi Membranes ◽

Transport Vesicles ◽

Golgi Region ◽

Quantitative Immunofluorescence ◽

Target Membrane ◽

Mitotic Phosphorylation

Biochemical data have shown that COPI-coated vesicles are tethered to Golgi membranes by a complex of at least three proteins: p115, giantin, and GM130. p115 binds to giantin on the vesicles and to GM130 on the membrane. We now examine the function of this tethering complex in vivo. Microinjection of an N-terminal peptide of GM130 or overexpression of GM130 lacking this N-terminal peptide inhibits the binding of p115 to Golgi membranes. Electron microscopic analysis of single microinjected cells shows that the number of COP-sized transport vesicles in the Golgi region increases substantially, suggesting that transport vesicles continue to bud but are less able to fuse. This was corroborated by quantitative immunofluorescence analysis, which showed that the intracellular transport of the VSV-G protein was significantly inhibited. Together, these data suggest that this tethering complex increases the efficiency with which transport vesicles fuse with their target membrane. They also provide support for a model of mitotic Golgi fragmentation in which the tethering complex is disrupted by mitotic phosphorylation of GM130.

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In vivo characterisation of the Golgi matrix protein giantin: linking extracellular matrix secretion and cilia function

Cilia ◽

10.1186/2046-2530-4-s1-p38 ◽

2015 ◽

Vol 4 (S1) ◽

Author(s):

D Bergen ◽

D Asante ◽

N Stevenson ◽

P Verkade ◽

C Hammond ◽

...

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Golgi Matrix ◽

Extracellular Matrix Secretion

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A caspase cleavage fragment of p115 induces fragmentation of the Golgi apparatus and apoptosis

The Journal of Cell Biology ◽

10.1083/jcb.200208013 ◽

2002 ◽

Vol 159 (4) ◽

pp. 637-648 ◽

Cited By ~ 100

Author(s):

Raymond Chiu ◽

Leonid Novikov ◽

Shaeri Mukherjee ◽

Dennis Shields

Keyword(s):

Golgi Apparatus ◽

Mammalian Cells ◽

Resistant Mutant ◽

Apoptotic Response ◽

Caspase Cleavage ◽

Vesicle Tethering ◽

Cleavage Fragment ◽

Resistant Form ◽

Golgi Fragmentation

In mammalian cells, the Golgi apparatus undergoes extensive fragmentation during apoptosis. p115 is a key vesicle tethering protein required for maintaining the structural organization of the Golgi apparatus. Here, we demonstrate that p115 was cleaved during apoptosis by caspases 3 and 8. Compared with control cells expressing native p115, those expressing a cleavage-resistant form of p115 delayed Golgi fragmentation during apoptosis. Expression of cDNAs encoding full-length or an NH2-terminal caspase cleavage fragment of p115 had no effect on Golgi morphology. In contrast, expression of the COOH-terminal caspase cleavage product of p115 itself caused Golgi fragmentation. Furthermore, this fragment translocated to the nucleus and its expression was sufficient to induce apoptosis. Most significantly, in vivo expression of the COOH-terminal fragment in the presence of caspase inhibitors, or upon coexpression with a cleavage-resistant mutant of p115, showed that p115 degradation plays a key role in amplifying the apoptotic response independently of Golgi fragmentation.

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