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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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Evidence for direct Golgi disassembly in cells lacking p115. Cells were injected with anti-p115 antibodies, incubated for 6 h, and then costained directly for giantin with FITC-coupled antigiantin pAb (A) and indirectly for ERGIC-53 with a mAb (B). Cells were also treated with BFA to collapse the Golgi into the ER, injected with anti-p115, washed to remove the BFA, incubated in the absence of BFA for 4 h, and then costained directly for giantin (C) and indirectly for ERGIC-53 (D). All cells shown were injected. Note the lack of Golgi staining in the ERGIC after breakdown and its presence after BFA washout. Bar, 10 μm.
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fig5: Evidence for direct Golgi disassembly in cells lacking p115. Cells were injected with anti-p115 antibodies, incubated for 6 h, and then costained directly for giantin with FITC-coupled antigiantin pAb (A) and indirectly for ERGIC-53 with a mAb (B). Cells were also treated with BFA to collapse the Golgi into the ER, injected with anti-p115, washed to remove the BFA, incubated in the absence of BFA for 4 h, and then costained directly for giantin (C) and indirectly for ERGIC-53 (D). All cells shown were injected. Note the lack of Golgi staining in the ERGIC after breakdown and its presence after BFA washout. Bar, 10 μm.

Mentions: We also considered the possibility that p115 absence actually causes Golgi apparatus disassembly by recycling Golgi proteins through the ER, which leads to their accumulation in a post-ER compartment such as COPII vesicles or the ERGIC. Costaining of anti-p115–injected cells for the Golgi marker, giantin (Fig. 5 A), and the ERGIC marker, ERGIC-53 (Fig. 5 B), revealed that the disassembled Golgi apparatus was almost entirely distinct from the ERGIC and that the ERGIC maintained its normal punctate pattern. Because ERGIC-53 rapidly cycles between the ER and ERGIC, mostly bypassing the Golgi apparatus (Hauri et al., 2000, and references therein), and is redistributed to the ER under conditions of ER export blockade (Shima et al., 1998; Lee and Linstedt, 1999), the normal ERGIC pattern implies that ER export and ERGIC formation/maintenance continue in the absence of p115. If ER to ERGIC transport persists in the absence of p115, then the lack of abundant Golgi staining in the ERGIC argues against a disassembly pathway involving the ER. To directly demonstrate ER to ERGIC transport, we microinjected BFA-treated cells with anti-p115 antibodies and then performed a BFA washout. In this case, giantin (Fig. 5 C) moved from its redistributed position in the ER into structures that costained with ERGIC-53 (Fig. 5 D). Similar results were obtained for the Golgi marker GPP130. These observations indicate that anti-p115–induced Golgi breakdown is primarily direct.


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)

Evidence for direct Golgi disassembly in cells lacking p115. Cells were injected with anti-p115 antibodies, incubated for 6 h, and then costained directly for giantin with FITC-coupled antigiantin pAb (A) and indirectly for ERGIC-53 with a mAb (B). Cells were also treated with BFA to collapse the Golgi into the ER, injected with anti-p115, washed to remove the BFA, incubated in the absence of BFA for 4 h, and then costained directly for giantin (C) and indirectly for ERGIC-53 (D). All cells shown were injected. Note the lack of Golgi staining in the ERGIC after breakdown and its presence after BFA washout. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198842&req=5

fig5: Evidence for direct Golgi disassembly in cells lacking p115. Cells were injected with anti-p115 antibodies, incubated for 6 h, and then costained directly for giantin with FITC-coupled antigiantin pAb (A) and indirectly for ERGIC-53 with a mAb (B). Cells were also treated with BFA to collapse the Golgi into the ER, injected with anti-p115, washed to remove the BFA, incubated in the absence of BFA for 4 h, and then costained directly for giantin (C) and indirectly for ERGIC-53 (D). All cells shown were injected. Note the lack of Golgi staining in the ERGIC after breakdown and its presence after BFA washout. Bar, 10 μm.
Mentions: We also considered the possibility that p115 absence actually causes Golgi apparatus disassembly by recycling Golgi proteins through the ER, which leads to their accumulation in a post-ER compartment such as COPII vesicles or the ERGIC. Costaining of anti-p115–injected cells for the Golgi marker, giantin (Fig. 5 A), and the ERGIC marker, ERGIC-53 (Fig. 5 B), revealed that the disassembled Golgi apparatus was almost entirely distinct from the ERGIC and that the ERGIC maintained its normal punctate pattern. Because ERGIC-53 rapidly cycles between the ER and ERGIC, mostly bypassing the Golgi apparatus (Hauri et al., 2000, and references therein), and is redistributed to the ER under conditions of ER export blockade (Shima et al., 1998; Lee and Linstedt, 1999), the normal ERGIC pattern implies that ER export and ERGIC formation/maintenance continue in the absence of p115. If ER to ERGIC transport persists in the absence of p115, then the lack of abundant Golgi staining in the ERGIC argues against a disassembly pathway involving the ER. To directly demonstrate ER to ERGIC transport, we microinjected BFA-treated cells with anti-p115 antibodies and then performed a BFA washout. In this case, giantin (Fig. 5 C) moved from its redistributed position in the ER into structures that costained with ERGIC-53 (Fig. 5 D). Similar results were obtained for the Golgi marker GPP130. These observations indicate that anti-p115–induced Golgi breakdown is primarily direct.

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