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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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Apparent release of Golgi vesicles upon digitonin permeabilization. HeLa cells were injected with anti-p115 antibodies and were either fixed after 4 h (A) or were permeabilized with 0.04 mg/ml digitonin before fixation (B). Cells were single stained using anti-GPP130 to exclude any possibility of bleedthrough. Interphase cells that exhibited fragmented Golgi staining were only present in microinjected areas of the coverslips. Representative examples of these are marked with asterisks. (C) The level of cytoplasmic GPP130 fluorescence excluding the Golgi region was quantified as described in Materials and methods before (open bars) and after (filled bars) digitonin permeabilization for anti-p115–injected (INJ, n = 65), uninjected (UNINJ, n = 23), and BFA-treated cells (BFA, n = 34). The analysis was also performed for the ER marker p63 (INJ, n = 28; UNINJ, n = 30). Fluorescence of the dispersed Golgi complex, but not the ER, was reduced to background levels after digitonin permeabilization. Bar, 10 μm.
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fig3: Apparent release of Golgi vesicles upon digitonin permeabilization. HeLa cells were injected with anti-p115 antibodies and were either fixed after 4 h (A) or were permeabilized with 0.04 mg/ml digitonin before fixation (B). Cells were single stained using anti-GPP130 to exclude any possibility of bleedthrough. Interphase cells that exhibited fragmented Golgi staining were only present in microinjected areas of the coverslips. Representative examples of these are marked with asterisks. (C) The level of cytoplasmic GPP130 fluorescence excluding the Golgi region was quantified as described in Materials and methods before (open bars) and after (filled bars) digitonin permeabilization for anti-p115–injected (INJ, n = 65), uninjected (UNINJ, n = 23), and BFA-treated cells (BFA, n = 34). The analysis was also performed for the ER marker p63 (INJ, n = 28; UNINJ, n = 30). Fluorescence of the dispersed Golgi complex, but not the ER, was reduced to background levels after digitonin permeabilization. Bar, 10 μm.

Mentions: Because Golgi breakdown in cells lacking detectable p115 resulted in diffuse Golgi staining, it was possible the anti-p115 microinjection induced Golgi vesiculation. However, it was also possible that the diffuse staining reflected redistribution of the Golgi apparatus into the ER membrane network, similar to that induced by brefeldin A (BFA) treatment. To distinguish between these possibilities, we performed a digitonin permeabilization of the injected cells. Because digitonin selectively permeabilizes the plasma membrane (Eilenberg et al., 1989), this treatment allows the release of small vesicles but not the large anchored ER network. The diffuse Golgi staining evident in cells 3 h after injection of anti-p115 (Fig. 3 A) was essentially lost if the cells were digitonin permeabilized before fixation (Fig. 3 B). Note that the Golgi apparatus present in adjacent, uninjected cells was unaffected by the permeabilization. In a quantitative analysis of these experiments, it was found that upon permeabilization the dispersed cytoplasmic fluorescence of the Golgi marker GPP130 in anti-p115–injected cells was reduced to the background level of cytoplasmic fluorescence in uninjected cells (Fig. 3 C). Two findings indicate that the ER was not released in these experiments. First, p63, an integral ER protein, was not released from uninjected or injected cells by digitonin treatment (Fig. 3 C). Second, after BFA treatment, ER-localized GPP130 was not released by digitonin treatment (Fig. 3 C). Thus, the Golgi breakdown pattern induced by the absence of detectable p115 did not reflect redistribution to the ER, but rather the accumulation of releasable structures likely to be small vesicles. This is consistent with the hypothesis that p115 mediates docking of Golgi-derived vesicles.


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)

Apparent release of Golgi vesicles upon digitonin permeabilization. HeLa cells were injected with anti-p115 antibodies and were either fixed after 4 h (A) or were permeabilized with 0.04 mg/ml digitonin before fixation (B). Cells were single stained using anti-GPP130 to exclude any possibility of bleedthrough. Interphase cells that exhibited fragmented Golgi staining were only present in microinjected areas of the coverslips. Representative examples of these are marked with asterisks. (C) The level of cytoplasmic GPP130 fluorescence excluding the Golgi region was quantified as described in Materials and methods before (open bars) and after (filled bars) digitonin permeabilization for anti-p115–injected (INJ, n = 65), uninjected (UNINJ, n = 23), and BFA-treated cells (BFA, n = 34). The analysis was also performed for the ER marker p63 (INJ, n = 28; UNINJ, n = 30). Fluorescence of the dispersed Golgi complex, but not the ER, was reduced to background levels after digitonin permeabilization. Bar, 10 μm.
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Related In: Results  -  Collection

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fig3: Apparent release of Golgi vesicles upon digitonin permeabilization. HeLa cells were injected with anti-p115 antibodies and were either fixed after 4 h (A) or were permeabilized with 0.04 mg/ml digitonin before fixation (B). Cells were single stained using anti-GPP130 to exclude any possibility of bleedthrough. Interphase cells that exhibited fragmented Golgi staining were only present in microinjected areas of the coverslips. Representative examples of these are marked with asterisks. (C) The level of cytoplasmic GPP130 fluorescence excluding the Golgi region was quantified as described in Materials and methods before (open bars) and after (filled bars) digitonin permeabilization for anti-p115–injected (INJ, n = 65), uninjected (UNINJ, n = 23), and BFA-treated cells (BFA, n = 34). The analysis was also performed for the ER marker p63 (INJ, n = 28; UNINJ, n = 30). Fluorescence of the dispersed Golgi complex, but not the ER, was reduced to background levels after digitonin permeabilization. Bar, 10 μm.
Mentions: Because Golgi breakdown in cells lacking detectable p115 resulted in diffuse Golgi staining, it was possible the anti-p115 microinjection induced Golgi vesiculation. However, it was also possible that the diffuse staining reflected redistribution of the Golgi apparatus into the ER membrane network, similar to that induced by brefeldin A (BFA) treatment. To distinguish between these possibilities, we performed a digitonin permeabilization of the injected cells. Because digitonin selectively permeabilizes the plasma membrane (Eilenberg et al., 1989), this treatment allows the release of small vesicles but not the large anchored ER network. The diffuse Golgi staining evident in cells 3 h after injection of anti-p115 (Fig. 3 A) was essentially lost if the cells were digitonin permeabilized before fixation (Fig. 3 B). Note that the Golgi apparatus present in adjacent, uninjected cells was unaffected by the permeabilization. In a quantitative analysis of these experiments, it was found that upon permeabilization the dispersed cytoplasmic fluorescence of the Golgi marker GPP130 in anti-p115–injected cells was reduced to the background level of cytoplasmic fluorescence in uninjected cells (Fig. 3 C). Two findings indicate that the ER was not released in these experiments. First, p63, an integral ER protein, was not released from uninjected or injected cells by digitonin treatment (Fig. 3 C). Second, after BFA treatment, ER-localized GPP130 was not released by digitonin treatment (Fig. 3 C). Thus, the Golgi breakdown pattern induced by the absence of detectable p115 did not reflect redistribution to the ER, but rather the accumulation of releasable structures likely to be small vesicles. This is consistent with the hypothesis that p115 mediates docking of Golgi-derived vesicles.

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