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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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Temporal sensitivity of stacking reaction to soluble GRASP65 and N73pep. MGF isolated through a 0.5-M sucrose cushion were incubated for the indicated time (time of addition) at 37°C with p97 (70 ng/μl), p47 (37.5 ng/μl), p115 (30 ng/μl), NSF (100 ng/μl), α-SNAP (25 ng/μl), and γ-SNAP (25 ng/μl). At this time, the reactions were transferred to ice and either fixed with 2% glutaraldehyde and processed for EM (A) and the percentage total membrane present as stacked regions of cisternae ± SEM  was determined, or the amount of p115 bound to the membranes at each time point was determined (shown below graph in A). Alternatively, reactions were transferred to ice and supplemented with buffer (B), 80 μM N73pep (C), or 75 ng/μl GRASP65 (D). After 15 min on ice, buffer-, N73pep-, and GRASP65-treated samples were transferred to 37°C and incubated for a total time of 60 min. Samples were fixed and processed for EM, quantitated, and the percentage total membrane present at the end of the incubation as stacked regions of cisternae ± SEM  was determined. The amount of p115 bound to Golgi membranes at the end of the incubation was also determined by Western blotting (shown below each graph).
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Figure 8: Temporal sensitivity of stacking reaction to soluble GRASP65 and N73pep. MGF isolated through a 0.5-M sucrose cushion were incubated for the indicated time (time of addition) at 37°C with p97 (70 ng/μl), p47 (37.5 ng/μl), p115 (30 ng/μl), NSF (100 ng/μl), α-SNAP (25 ng/μl), and γ-SNAP (25 ng/μl). At this time, the reactions were transferred to ice and either fixed with 2% glutaraldehyde and processed for EM (A) and the percentage total membrane present as stacked regions of cisternae ± SEM was determined, or the amount of p115 bound to the membranes at each time point was determined (shown below graph in A). Alternatively, reactions were transferred to ice and supplemented with buffer (B), 80 μM N73pep (C), or 75 ng/μl GRASP65 (D). After 15 min on ice, buffer-, N73pep-, and GRASP65-treated samples were transferred to 37°C and incubated for a total time of 60 min. Samples were fixed and processed for EM, quantitated, and the percentage total membrane present at the end of the incubation as stacked regions of cisternae ± SEM was determined. The amount of p115 bound to Golgi membranes at the end of the incubation was also determined by Western blotting (shown below each graph).

Mentions: To assess the temporal sensitivity of the stacking reaction to these two inhibitors, MGF were incubated in the NSF/p97-purified reaction for increasing time at 37°C. At various time points (time of addition, t; Fig. 8, A–D) the reaction was transferred to ice and either fixed with 2% glutaraldehyde and processed for EM, or treated with buffer (KHM, the GRASP65 and N73pep solvent), N73pep, or soluble GRASP65, and then reincubated at 37°C for a total time of 60 min.


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)

Temporal sensitivity of stacking reaction to soluble GRASP65 and N73pep. MGF isolated through a 0.5-M sucrose cushion were incubated for the indicated time (time of addition) at 37°C with p97 (70 ng/μl), p47 (37.5 ng/μl), p115 (30 ng/μl), NSF (100 ng/μl), α-SNAP (25 ng/μl), and γ-SNAP (25 ng/μl). At this time, the reactions were transferred to ice and either fixed with 2% glutaraldehyde and processed for EM (A) and the percentage total membrane present as stacked regions of cisternae ± SEM  was determined, or the amount of p115 bound to the membranes at each time point was determined (shown below graph in A). Alternatively, reactions were transferred to ice and supplemented with buffer (B), 80 μM N73pep (C), or 75 ng/μl GRASP65 (D). After 15 min on ice, buffer-, N73pep-, and GRASP65-treated samples were transferred to 37°C and incubated for a total time of 60 min. Samples were fixed and processed for EM, quantitated, and the percentage total membrane present at the end of the incubation as stacked regions of cisternae ± SEM  was determined. The amount of p115 bound to Golgi membranes at the end of the incubation was also determined by Western blotting (shown below each graph).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199741&req=5

Figure 8: Temporal sensitivity of stacking reaction to soluble GRASP65 and N73pep. MGF isolated through a 0.5-M sucrose cushion were incubated for the indicated time (time of addition) at 37°C with p97 (70 ng/μl), p47 (37.5 ng/μl), p115 (30 ng/μl), NSF (100 ng/μl), α-SNAP (25 ng/μl), and γ-SNAP (25 ng/μl). At this time, the reactions were transferred to ice and either fixed with 2% glutaraldehyde and processed for EM (A) and the percentage total membrane present as stacked regions of cisternae ± SEM was determined, or the amount of p115 bound to the membranes at each time point was determined (shown below graph in A). Alternatively, reactions were transferred to ice and supplemented with buffer (B), 80 μM N73pep (C), or 75 ng/μl GRASP65 (D). After 15 min on ice, buffer-, N73pep-, and GRASP65-treated samples were transferred to 37°C and incubated for a total time of 60 min. Samples were fixed and processed for EM, quantitated, and the percentage total membrane present at the end of the incubation as stacked regions of cisternae ± SEM was determined. The amount of p115 bound to Golgi membranes at the end of the incubation was also determined by Western blotting (shown below each graph).
Mentions: To assess the temporal sensitivity of the stacking reaction to these two inhibitors, MGF were incubated in the NSF/p97-purified reaction for increasing time at 37°C. At various time points (time of addition, t; Fig. 8, A–D) the reaction was transferred to ice and either fixed with 2% glutaraldehyde and processed for EM, or treated with buffer (KHM, the GRASP65 and N73pep solvent), N73pep, or soluble GRASP65, and then reincubated at 37°C for a total time of 60 min.

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