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

Puthenveedu MA, Linstedt AD - J. Cell Biol. (2001)

Bottom Line: However, neither this, nor the role of the giantin-p115-GM130 complex in the maintenance of Golgi structure has been demonstrated in vivo.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

ABSTRACT
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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Golgi maintenance and reassembly in anti-GM130– injected cells. Cells injected with rabbit anti-GM130 were incubated for 16 h and stained using mouse antigiantin (A), or mouse anti-p115 (B). Isolated single cells were also injected, incubated for 20 h to allow cell division, and then stained for GPP130 (C). Note that injected cells (outlined) exhibited normal patterns. Bar, 10 μm.
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fig7: Golgi maintenance and reassembly in anti-GM130– injected cells. Cells injected with rabbit anti-GM130 were incubated for 16 h and stained using mouse antigiantin (A), or mouse anti-p115 (B). Isolated single cells were also injected, incubated for 20 h to allow cell division, and then stained for GPP130 (C). Note that injected cells (outlined) exhibited normal patterns. Bar, 10 μm.

Mentions: Polyclonal antibodies were also generated against the NH2-terminal domain of GM130, which contains its p115 binding site. Microinjection of these antibodies with subsequent incubations up to 16 h had no apparent effect on the staining patterns for giantin (Fig. 7 A), p115 (Fig. 7 B), GPP130 (unpublished data), and KDEL receptor (unpublished data). Not only was the interphase Golgi structure normal in anti-GM130–injected cells, but BFA-induced Golgi–ER fusion and Golgi reemergence upon BFA washout was indistinguishable in microinjected and adjacent, uninjected cells (unpublished data). Also, when isolated single cells on each coverslip were injected and incubated for 20 h, the cells divided normally and the daughter cells reassembled a normal Golgi apparatus as indicated by staining for GPP130 (Fig. 7 C).


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

Puthenveedu MA, Linstedt AD - J. Cell Biol. (2001)

Golgi maintenance and reassembly in anti-GM130– injected cells. Cells injected with rabbit anti-GM130 were incubated for 16 h and stained using mouse antigiantin (A), or mouse anti-p115 (B). Isolated single cells were also injected, incubated for 20 h to allow cell division, and then stained for GPP130 (C). Note that injected cells (outlined) exhibited normal patterns. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198842&req=5

fig7: Golgi maintenance and reassembly in anti-GM130– injected cells. Cells injected with rabbit anti-GM130 were incubated for 16 h and stained using mouse antigiantin (A), or mouse anti-p115 (B). Isolated single cells were also injected, incubated for 20 h to allow cell division, and then stained for GPP130 (C). Note that injected cells (outlined) exhibited normal patterns. Bar, 10 μm.
Mentions: Polyclonal antibodies were also generated against the NH2-terminal domain of GM130, which contains its p115 binding site. Microinjection of these antibodies with subsequent incubations up to 16 h had no apparent effect on the staining patterns for giantin (Fig. 7 A), p115 (Fig. 7 B), GPP130 (unpublished data), and KDEL receptor (unpublished data). Not only was the interphase Golgi structure normal in anti-GM130–injected cells, but BFA-induced Golgi–ER fusion and Golgi reemergence upon BFA washout was indistinguishable in microinjected and adjacent, uninjected cells (unpublished data). Also, when isolated single cells on each coverslip were injected and incubated for 20 h, the cells divided normally and the daughter cells reassembled a normal Golgi apparatus as indicated by staining for GPP130 (Fig. 7 C).

Bottom Line: However, neither this, nor the role of the giantin-p115-GM130 complex in the maintenance of Golgi structure has been demonstrated in vivo.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus