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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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Absence of detectable p115 induces Golgi breakdown. HeLa cells were microinjected with anti-p115 and stained for the Golgi apparatus using anti-GPP130 mAb at 10 min (A), 30 min (B), 2 h (C), and 4 h (D) after injection. Injected cells are outlined. Note the prominent diffuse cytoplasmic Golgi staining at the last time point. Images are representative of three experiments with at least 25 cells examined in each experiment. Uninjected cells showed a normal Golgi apparatus at all time points. Bar, 10 μm.
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fig2: Absence of detectable p115 induces Golgi breakdown. HeLa cells were microinjected with anti-p115 and stained for the Golgi apparatus using anti-GPP130 mAb at 10 min (A), 30 min (B), 2 h (C), and 4 h (D) after injection. Injected cells are outlined. Note the prominent diffuse cytoplasmic Golgi staining at the last time point. Images are representative of three experiments with at least 25 cells examined in each experiment. Uninjected cells showed a normal Golgi apparatus at all time points. Bar, 10 μm.

Mentions: To test the effect of p115 loss on Golgi structure, we stained the Golgi apparatus in cells that had been incubated for various times after microinjection of anti-p115. The staining was performed using a monoclonal antibody that recognizes Golgi phosphoprotein 130 (GPP130), an integral membrane component of the cis-Golgi apparatus (Linstedt et al., 1997). The Golgi pattern remained normal 10 min after injection (Fig. 2 A), but 30 min after injection, the Golgi apparatus was noticeably fragmented (Fig. 2 B). 2 h after injection, the Golgi apparatus was present as dispersed fragments and there was a noticeable increase in diffuse staining (Fig. 2 C). 4 h after injection, the Golgi apparatus was almost entirely present as diffuse cytoplasmic staining with a few larger structures, or remnants, still visible (Fig. 2 D). A similar pattern of Golgi breakdown was observed when giantin or GM130 was used to mark the Golgi apparatus, and also when cells were allowed to pass through mitosis after injection, as described below (unpublished data). Injection of control anti-GST antibodies had no detectable effect on the Golgi apparatus at similar time points (unpublished data). Thus, loss of p115 led to a dramatic breakdown of at least the early Golgi structure.


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)

Absence of detectable p115 induces Golgi breakdown. HeLa cells were microinjected with anti-p115 and stained for the Golgi apparatus using anti-GPP130 mAb at 10 min (A), 30 min (B), 2 h (C), and 4 h (D) after injection. Injected cells are outlined. Note the prominent diffuse cytoplasmic Golgi staining at the last time point. Images are representative of three experiments with at least 25 cells examined in each experiment. Uninjected cells showed a normal Golgi apparatus at all time points. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198842&req=5

fig2: Absence of detectable p115 induces Golgi breakdown. HeLa cells were microinjected with anti-p115 and stained for the Golgi apparatus using anti-GPP130 mAb at 10 min (A), 30 min (B), 2 h (C), and 4 h (D) after injection. Injected cells are outlined. Note the prominent diffuse cytoplasmic Golgi staining at the last time point. Images are representative of three experiments with at least 25 cells examined in each experiment. Uninjected cells showed a normal Golgi apparatus at all time points. Bar, 10 μm.
Mentions: To test the effect of p115 loss on Golgi structure, we stained the Golgi apparatus in cells that had been incubated for various times after microinjection of anti-p115. The staining was performed using a monoclonal antibody that recognizes Golgi phosphoprotein 130 (GPP130), an integral membrane component of the cis-Golgi apparatus (Linstedt et al., 1997). The Golgi pattern remained normal 10 min after injection (Fig. 2 A), but 30 min after injection, the Golgi apparatus was noticeably fragmented (Fig. 2 B). 2 h after injection, the Golgi apparatus was present as dispersed fragments and there was a noticeable increase in diffuse staining (Fig. 2 C). 4 h after injection, the Golgi apparatus was almost entirely present as diffuse cytoplasmic staining with a few larger structures, or remnants, still visible (Fig. 2 D). A similar pattern of Golgi breakdown was observed when giantin or GM130 was used to mark the Golgi apparatus, and also when cells were allowed to pass through mitosis after injection, as described below (unpublished data). Injection of control anti-GST antibodies had no detectable effect on the Golgi apparatus at similar time points (unpublished data). Thus, loss of p115 led to a dramatic breakdown of at least the early Golgi structure.

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