Limits...
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.

Show MeSH

Related in: MedlinePlus

Golgi maintenance and reassembly in the absence of detectable giantin. Cells injected with rabbit antigiantin antibodies were incubated for 2 h in the absence (A) or 3 h in the presence (B) of 50 μM MG132 before staining with mouse antigiantin antibodies. Injected cells (outlined) lacked detectable giantin in the absence, but not the presence, of the proteasome inhibitor. Antigiantin- injected cells were incubated for 16 h and stained with anti-GPP130 (C) or anti-p115 (D). Despite giantin absence, injected cells exhibited normal staining patterns. Isolated single cells were injected, incubated for 20 h to allow cell division, and then stained for giantin (E), GPP130 (F), or costained for tubulin (G) and GPP130 (H). Note that daughter cells exhibited normal Golgi morphology in the absence of detectable giantin. Each injected cell divided in this experiment (n = 20). Images in A, B, E, and F were acquired with a 20× objective. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2198842&req=5

fig6: Golgi maintenance and reassembly in the absence of detectable giantin. Cells injected with rabbit antigiantin antibodies were incubated for 2 h in the absence (A) or 3 h in the presence (B) of 50 μM MG132 before staining with mouse antigiantin antibodies. Injected cells (outlined) lacked detectable giantin in the absence, but not the presence, of the proteasome inhibitor. Antigiantin- injected cells were incubated for 16 h and stained with anti-GPP130 (C) or anti-p115 (D). Despite giantin absence, injected cells exhibited normal staining patterns. Isolated single cells were injected, incubated for 20 h to allow cell division, and then stained for giantin (E), GPP130 (F), or costained for tubulin (G) and GPP130 (H). Note that daughter cells exhibited normal Golgi morphology in the absence of detectable giantin. Each injected cell divided in this experiment (n = 20). Images in A, B, E, and F were acquired with a 20× objective. Bar, 10 μm.

Mentions: Our finding that loss of p115 led to Golgi breakdown confirms a previous report that p115 is required for maintenance of interphase Golgi structure (Alvarez et al., 1999). Further, because the breakdown involved an apparent direct vesiculation, it suggests that p115 plays a role in the docking of Golgi-derived vesicles. Therefore, we next tested whether the role of p115 in Golgi structure maintenance requires binding to either of the putative tether components, giantin or GM130. Polyclonal antibodies were generated against a peptide containing the mapped p115 binding site in the NH2 terminus of giantin. Upon microinjection into HeLa cells, the antigiantin antibodies induced degradation of giantin with a time course that was similar to the anti-p115–induced degradation of p115. Thus, early time points exhibited clustered giantin staining (unpublished data), and 2 h after injection giantin staining was undetectable (Fig. 6 A). The degradation was specific to giantin as other marker proteins did not exhibit any reductions in staining (see below), and giantin degradation did not occur upon injection of control anti-GST antibodies (unpublished data). The antigiantin-induced giantin degradation, but not the antibody-induced giantin clustering, was blocked in microinjected cells incubated in the presence of the proteasome inhibitor MG132 (Fig. 6 B). This result indicated that, similar to the anti-p115–induced p115 degradation, the giantin degradation was proteasome mediated. Further, the absence of detectable giantin persisted for at least 48 h after injection with no apparent loss in cell viability, which allowed us to perform relatively long experiments with cells lacking giantin.


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 the absence of detectable giantin. Cells injected with rabbit antigiantin antibodies were incubated for 2 h in the absence (A) or 3 h in the presence (B) of 50 μM MG132 before staining with mouse antigiantin antibodies. Injected cells (outlined) lacked detectable giantin in the absence, but not the presence, of the proteasome inhibitor. Antigiantin- injected cells were incubated for 16 h and stained with anti-GPP130 (C) or anti-p115 (D). Despite giantin absence, injected cells exhibited normal staining patterns. Isolated single cells were injected, incubated for 20 h to allow cell division, and then stained for giantin (E), GPP130 (F), or costained for tubulin (G) and GPP130 (H). Note that daughter cells exhibited normal Golgi morphology in the absence of detectable giantin. Each injected cell divided in this experiment (n = 20). Images in A, B, E, and F were acquired with a 20× objective. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Golgi maintenance and reassembly in the absence of detectable giantin. Cells injected with rabbit antigiantin antibodies were incubated for 2 h in the absence (A) or 3 h in the presence (B) of 50 μM MG132 before staining with mouse antigiantin antibodies. Injected cells (outlined) lacked detectable giantin in the absence, but not the presence, of the proteasome inhibitor. Antigiantin- injected cells were incubated for 16 h and stained with anti-GPP130 (C) or anti-p115 (D). Despite giantin absence, injected cells exhibited normal staining patterns. Isolated single cells were injected, incubated for 20 h to allow cell division, and then stained for giantin (E), GPP130 (F), or costained for tubulin (G) and GPP130 (H). Note that daughter cells exhibited normal Golgi morphology in the absence of detectable giantin. Each injected cell divided in this experiment (n = 20). Images in A, B, E, and F were acquired with a 20× objective. Bar, 10 μm.
Mentions: Our finding that loss of p115 led to Golgi breakdown confirms a previous report that p115 is required for maintenance of interphase Golgi structure (Alvarez et al., 1999). Further, because the breakdown involved an apparent direct vesiculation, it suggests that p115 plays a role in the docking of Golgi-derived vesicles. Therefore, we next tested whether the role of p115 in Golgi structure maintenance requires binding to either of the putative tether components, giantin or GM130. Polyclonal antibodies were generated against a peptide containing the mapped p115 binding site in the NH2 terminus of giantin. Upon microinjection into HeLa cells, the antigiantin antibodies induced degradation of giantin with a time course that was similar to the anti-p115–induced degradation of p115. Thus, early time points exhibited clustered giantin staining (unpublished data), and 2 h after injection giantin staining was undetectable (Fig. 6 A). The degradation was specific to giantin as other marker proteins did not exhibit any reductions in staining (see below), and giantin degradation did not occur upon injection of control anti-GST antibodies (unpublished data). The antigiantin-induced giantin degradation, but not the antibody-induced giantin clustering, was blocked in microinjected cells incubated in the presence of the proteasome inhibitor MG132 (Fig. 6 B). This result indicated that, similar to the anti-p115–induced p115 degradation, the giantin degradation was proteasome mediated. Further, the absence of detectable giantin persisted for at least 48 h after injection with no apparent loss in cell viability, which allowed us to perform relatively long experiments with cells lacking giantin.

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