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

Anti-GM130 blocks p115–GM130 complex formation without displacing membrane-associated p115. (A) NRK cell lysates were added to bead-attached GST-GM130 that was preincubated with the amount of anti-GM130 (lanes 1–3) or anti-GST (lane 4) indicated. The amount of cellular p115 recovered on the beads was determined by immunoblotting. (B) NRK membrane fractions were pretreated in absence (lane 1) or the presence of the indicated amounts of anti-GM130 (lanes 2–4) or antigiantin (lane 5) before coimmunoprecipitation with anti-p115 covalently attached to beads. The amount of cellular GM130 and p115 recovered was determined by immunoblotting. (C) NRK membrane fractions were also pretreated without antibody (lane 1), with 5 μl anti-GM130 antibody (lane 2), or with 5 μl antigiantin (lane 3) and then collected by centrifugation. The recovery of membrane-associated p115 and GM130 was then determined by immunoblotting. Note that although anti-GM130 blocked p115 binding to GST-GM130, and it blocked recovery of p115–GM130 complexes from membranes; it did not block p115 membrane association. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2198842&req=5

fig8: Anti-GM130 blocks p115–GM130 complex formation without displacing membrane-associated p115. (A) NRK cell lysates were added to bead-attached GST-GM130 that was preincubated with the amount of anti-GM130 (lanes 1–3) or anti-GST (lane 4) indicated. The amount of cellular p115 recovered on the beads was determined by immunoblotting. (B) NRK membrane fractions were pretreated in absence (lane 1) or the presence of the indicated amounts of anti-GM130 (lanes 2–4) or antigiantin (lane 5) before coimmunoprecipitation with anti-p115 covalently attached to beads. The amount of cellular GM130 and p115 recovered was determined by immunoblotting. (C) NRK membrane fractions were also pretreated without antibody (lane 1), with 5 μl anti-GM130 antibody (lane 2), or with 5 μl antigiantin (lane 3) and then collected by centrifugation. The recovery of membrane-associated p115 and GM130 was then determined by immunoblotting. Note that although anti-GM130 blocked p115 binding to GST-GM130, and it blocked recovery of p115–GM130 complexes from membranes; it did not block p115 membrane association. Bar, 10 μm.

Mentions: In the first experiment, an immobilized GST–GM130 fusion protein containing its p115 binding site (Linstedt et al., 2000) was incubated with p115-containing cell extracts in the presence of anti-GM130 or control antibodies. Despite the relatively high concentration of GM130 present in the assay, the anti-GM130 antibody showed a concentration-dependent inhibition of p115 binding, whereas control anti-GST antibodies did not (Fig. 8 A). Thus, the anti-GM130 antibody had inhibitory activity against p115–GM130 complex formation, yet its microinjection did not displace p115 from the Golgi complex (Fig. 7 B). This observation is actually consistent with previous reports indicating that versions of p115 lacking the GM130 binding domain are still Golgi apparatus targeted (Nelson et al., 1998); that is, p115 appears to have at least one other Golgi-localized receptor. The receptor in question is not giantin because the giantin binding site in p115 maps to the same position as the GM130 binding site (Linstedt et al., 2000). Furthermore, co-injection of both antigiantin and anti-GM130 also did not displace p115 or cause any detectable Golgi abnormality (unpublished data). Therefore, to confirm that anti-GM130 antibodies disrupt p115–GM130 interaction without blocking p115 membrane binding, we performed a second binding experiment.


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)

Anti-GM130 blocks p115–GM130 complex formation without displacing membrane-associated p115. (A) NRK cell lysates were added to bead-attached GST-GM130 that was preincubated with the amount of anti-GM130 (lanes 1–3) or anti-GST (lane 4) indicated. The amount of cellular p115 recovered on the beads was determined by immunoblotting. (B) NRK membrane fractions were pretreated in absence (lane 1) or the presence of the indicated amounts of anti-GM130 (lanes 2–4) or antigiantin (lane 5) before coimmunoprecipitation with anti-p115 covalently attached to beads. The amount of cellular GM130 and p115 recovered was determined by immunoblotting. (C) NRK membrane fractions were also pretreated without antibody (lane 1), with 5 μl anti-GM130 antibody (lane 2), or with 5 μl antigiantin (lane 3) and then collected by centrifugation. The recovery of membrane-associated p115 and GM130 was then determined by immunoblotting. Note that although anti-GM130 blocked p115 binding to GST-GM130, and it blocked recovery of p115–GM130 complexes from membranes; it did not block p115 membrane association. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Anti-GM130 blocks p115–GM130 complex formation without displacing membrane-associated p115. (A) NRK cell lysates were added to bead-attached GST-GM130 that was preincubated with the amount of anti-GM130 (lanes 1–3) or anti-GST (lane 4) indicated. The amount of cellular p115 recovered on the beads was determined by immunoblotting. (B) NRK membrane fractions were pretreated in absence (lane 1) or the presence of the indicated amounts of anti-GM130 (lanes 2–4) or antigiantin (lane 5) before coimmunoprecipitation with anti-p115 covalently attached to beads. The amount of cellular GM130 and p115 recovered was determined by immunoblotting. (C) NRK membrane fractions were also pretreated without antibody (lane 1), with 5 μl anti-GM130 antibody (lane 2), or with 5 μl antigiantin (lane 3) and then collected by centrifugation. The recovery of membrane-associated p115 and GM130 was then determined by immunoblotting. Note that although anti-GM130 blocked p115 binding to GST-GM130, and it blocked recovery of p115–GM130 complexes from membranes; it did not block p115 membrane association. Bar, 10 μm.
Mentions: In the first experiment, an immobilized GST–GM130 fusion protein containing its p115 binding site (Linstedt et al., 2000) was incubated with p115-containing cell extracts in the presence of anti-GM130 or control antibodies. Despite the relatively high concentration of GM130 present in the assay, the anti-GM130 antibody showed a concentration-dependent inhibition of p115 binding, whereas control anti-GST antibodies did not (Fig. 8 A). Thus, the anti-GM130 antibody had inhibitory activity against p115–GM130 complex formation, yet its microinjection did not displace p115 from the Golgi complex (Fig. 7 B). This observation is actually consistent with previous reports indicating that versions of p115 lacking the GM130 binding domain are still Golgi apparatus targeted (Nelson et al., 1998); that is, p115 appears to have at least one other Golgi-localized receptor. The receptor in question is not giantin because the giantin binding site in p115 maps to the same position as the GM130 binding site (Linstedt et al., 2000). Furthermore, co-injection of both antigiantin and anti-GM130 also did not displace p115 or cause any detectable Golgi abnormality (unpublished data). Therefore, to confirm that anti-GM130 antibodies disrupt p115–GM130 interaction without blocking p115 membrane binding, we performed a second binding experiment.

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