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A role for the vesicle tethering protein, p115, in the post-mitotic stacking of reassembling Golgi cisternae in a cell-free system.

Shorter J, Warren G - J. Cell Biol. (1999)

Bottom Line: Golgi reassembly stacking protein 65 (GRASP65), an NEM-sensitive membrane-bound component, is required for the stacking process.Temporal analysis suggests that p115 plays a transient role in stacking that may be upstream of GRASP65-mediated stacking.These results implicate p115 and its receptors in the initial alignment and docking of single cisternae that may be an important prerequisite for stack formation.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom. shorter@icrf.icnet.uk

ABSTRACT
During telophase, Golgi cisternae are regenerated and stacked from a heterogeneous population of tubulovesicular clusters. A cell-free system that reconstructs these events has revealed that cisternal regrowth requires interplay between soluble factors and soluble N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) attachment protein receptors (SNAREs) via two intersecting pathways controlled by the ATPases, p97 and NSF. Golgi reassembly stacking protein 65 (GRASP65), an NEM-sensitive membrane-bound component, is required for the stacking process. NSF-mediated cisternal regrowth requires a vesicle tethering protein, p115, which we now show operates through its two Golgi receptors, GM130 and giantin. p97-mediated cisternal regrowth is p115-independent, but we now demonstrate a role for p115, in conjunction with its receptors, in stacking p97 generated cisternae. Temporal analysis suggests that p115 plays a transient role in stacking that may be upstream of GRASP65-mediated stacking. These results implicate p115 and its receptors in the initial alignment and docking of single cisternae that may be an important prerequisite for stack formation.

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Quantitation of Golgi membrane reassembly in rat liver cytosol, p115-depleted cytosol, and p115-depleted cytosol supplemented with p115. MGF isolated through a 0.5-M sucrose cushion were incubated for 60 min at 37°C with increasing concentrations of either rat liver cytosol, p115-depleted cytosol, or p115-depleted cytosol supplemented with purified rat liver p115, fixed and processed for EM, and quantitated as described in Materials and Methods. (A) The percentage cisternal regrowth ± SEM  for each cytosol concentration tested. (B) The percentage total membrane present as stacked regions of cisternae ± SEM  for each cytosol concentration tested.
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Figure 3: Quantitation of Golgi membrane reassembly in rat liver cytosol, p115-depleted cytosol, and p115-depleted cytosol supplemented with p115. MGF isolated through a 0.5-M sucrose cushion were incubated for 60 min at 37°C with increasing concentrations of either rat liver cytosol, p115-depleted cytosol, or p115-depleted cytosol supplemented with purified rat liver p115, fixed and processed for EM, and quantitated as described in Materials and Methods. (A) The percentage cisternal regrowth ± SEM for each cytosol concentration tested. (B) The percentage total membrane present as stacked regions of cisternae ± SEM for each cytosol concentration tested.

Mentions: In rat liver cytosol, cisternal regrowth was near maximal at 1 mg/ml (Fig. 2, B–D, and 3 A) and the same was true for the mock depleted cytosols (data not shown). At cytosol concentrations below 1 mg/ml, the p115-depleted cytosol supported threefold less cisternal regrowth (Fig. 2 E and 3 A). This inhibition was reversed by adding purified p115 back to the depleted cytosol (Fig. 2 H and 3 A). Therefore, this loss of activity was due to p115 activity and not the activity of another factor that may have been codepleted from the cytosol by an interaction with p115. However, at cytosol concentrations of 1 mg/ml and above, p115-depleted cytosol supported full cisternal regrowth (Fig. 2F and Fig. G, and Fig. 3 A). Therefore, p115 is not essential for this process, or a p115-independent pathway of cisternal regrowth is operating. We favor the latter explanation because two nonadditive pathways of cisternal regrowth controlled by NSF and p97 have been described previously (Rabouille et al. 1995b, Rabouille et al. 1998). The p97 pathway has no requirement for p115 for cisternal regrowth, and is presumably responsible for the complete cisternal regrowth activity of p115-depleted cytosol. A hint that this may be true comes from the morphology of the cisternae reassembled in p115-depleted cytosol, in that they are often blunt-ended with few associated vesicles (compare asterisks in Fig. 2F and Fig. I). This is the characteristic phenotype for p97 reassembled cisternae (Rabouille et al. 1995b).


A role for the vesicle tethering protein, p115, in the post-mitotic stacking of reassembling Golgi cisternae in a cell-free system.

Shorter J, Warren G - J. Cell Biol. (1999)

Quantitation of Golgi membrane reassembly in rat liver cytosol, p115-depleted cytosol, and p115-depleted cytosol supplemented with p115. MGF isolated through a 0.5-M sucrose cushion were incubated for 60 min at 37°C with increasing concentrations of either rat liver cytosol, p115-depleted cytosol, or p115-depleted cytosol supplemented with purified rat liver p115, fixed and processed for EM, and quantitated as described in Materials and Methods. (A) The percentage cisternal regrowth ± SEM  for each cytosol concentration tested. (B) The percentage total membrane present as stacked regions of cisternae ± SEM  for each cytosol concentration tested.
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Related In: Results  -  Collection

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Figure 3: Quantitation of Golgi membrane reassembly in rat liver cytosol, p115-depleted cytosol, and p115-depleted cytosol supplemented with p115. MGF isolated through a 0.5-M sucrose cushion were incubated for 60 min at 37°C with increasing concentrations of either rat liver cytosol, p115-depleted cytosol, or p115-depleted cytosol supplemented with purified rat liver p115, fixed and processed for EM, and quantitated as described in Materials and Methods. (A) The percentage cisternal regrowth ± SEM for each cytosol concentration tested. (B) The percentage total membrane present as stacked regions of cisternae ± SEM for each cytosol concentration tested.
Mentions: In rat liver cytosol, cisternal regrowth was near maximal at 1 mg/ml (Fig. 2, B–D, and 3 A) and the same was true for the mock depleted cytosols (data not shown). At cytosol concentrations below 1 mg/ml, the p115-depleted cytosol supported threefold less cisternal regrowth (Fig. 2 E and 3 A). This inhibition was reversed by adding purified p115 back to the depleted cytosol (Fig. 2 H and 3 A). Therefore, this loss of activity was due to p115 activity and not the activity of another factor that may have been codepleted from the cytosol by an interaction with p115. However, at cytosol concentrations of 1 mg/ml and above, p115-depleted cytosol supported full cisternal regrowth (Fig. 2F and Fig. G, and Fig. 3 A). Therefore, p115 is not essential for this process, or a p115-independent pathway of cisternal regrowth is operating. We favor the latter explanation because two nonadditive pathways of cisternal regrowth controlled by NSF and p97 have been described previously (Rabouille et al. 1995b, Rabouille et al. 1998). The p97 pathway has no requirement for p115 for cisternal regrowth, and is presumably responsible for the complete cisternal regrowth activity of p115-depleted cytosol. A hint that this may be true comes from the morphology of the cisternae reassembled in p115-depleted cytosol, in that they are often blunt-ended with few associated vesicles (compare asterisks in Fig. 2F and Fig. I). This is the characteristic phenotype for p97 reassembled cisternae (Rabouille et al. 1995b).

Bottom Line: Golgi reassembly stacking protein 65 (GRASP65), an NEM-sensitive membrane-bound component, is required for the stacking process.Temporal analysis suggests that p115 plays a transient role in stacking that may be upstream of GRASP65-mediated stacking.These results implicate p115 and its receptors in the initial alignment and docking of single cisternae that may be an important prerequisite for stack formation.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom. shorter@icrf.icnet.uk

ABSTRACT
During telophase, Golgi cisternae are regenerated and stacked from a heterogeneous population of tubulovesicular clusters. A cell-free system that reconstructs these events has revealed that cisternal regrowth requires interplay between soluble factors and soluble N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) attachment protein receptors (SNAREs) via two intersecting pathways controlled by the ATPases, p97 and NSF. Golgi reassembly stacking protein 65 (GRASP65), an NEM-sensitive membrane-bound component, is required for the stacking process. NSF-mediated cisternal regrowth requires a vesicle tethering protein, p115, which we now show operates through its two Golgi receptors, GM130 and giantin. p97-mediated cisternal regrowth is p115-independent, but we now demonstrate a role for p115, in conjunction with its receptors, in stacking p97 generated cisternae. Temporal analysis suggests that p115 plays a transient role in stacking that may be upstream of GRASP65-mediated stacking. These results implicate p115 and its receptors in the initial alignment and docking of single cisternae that may be an important prerequisite for stack formation.

Show MeSH
Related in: MedlinePlus