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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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Effect of soluble GRASP65 and N73pep on the reassembly process using pure components. MGF isolated through a 0.5-M sucrose cushion were preincubated for 15 min on ice with increasing amounts of soluble GRASP65 (A) or N73pep (B). The pretreated MGF were then incubated for 60 min 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). Samples were either fixed and processed for EM and quantitated, with the percentage total membrane present as cisternae ± SEM  and as stacked regions of cisternae ± SEM  shown; or the membranes were recovered and fractionated by SDS-PAGE using a 7.5% gel, transferred to nitrocellulose, and probed for GM130 and p115 using specific antibodies (shown below graphs in A and B).
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Figure 7: Effect of soluble GRASP65 and N73pep on the reassembly process using pure components. MGF isolated through a 0.5-M sucrose cushion were preincubated for 15 min on ice with increasing amounts of soluble GRASP65 (A) or N73pep (B). The pretreated MGF were then incubated for 60 min 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). Samples were either fixed and processed for EM and quantitated, with the percentage total membrane present as cisternae ± SEM and as stacked regions of cisternae ± SEM shown; or the membranes were recovered and fractionated by SDS-PAGE using a 7.5% gel, transferred to nitrocellulose, and probed for GM130 and p115 using specific antibodies (shown below graphs in A and B).

Mentions: An NEM-sensitive membrane-bound component of MGF is essential for the stacking reaction during reassembly. This factor was identified as GRASP65, a highly conserved, N-myristoylated protein that exists as a tight complex with GM130 on the membrane (Barr et al. 1997). A soluble, nonmyristoylated recombinant form of GRASP65 inhibited stacking, but not cisternal regrowth, in the cytosol based reassembly (Barr et al. 1997). This soluble GRASP65 was titrated into the NSF/p97 combined pathway of reassembly and was able to inhibit stacking potently without affecting cisternal regrowth (Fig. 7 A). This was also true in the p97-catalyzed and NSF-catalyzed reactions (data not shown). NEM-treated soluble GRASP65 had no effect on stacking or cisternal regrowth. Soluble GRASP65 prequenched with anti-GRASP65 antibodies also had no effect (data not shown). Soluble GRASP65 may then inhibit the stacking process by interacting with a Golgi membrane-bound factor.


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)

Effect of soluble GRASP65 and N73pep on the reassembly process using pure components. MGF isolated through a 0.5-M sucrose cushion were preincubated for 15 min on ice with increasing amounts of soluble GRASP65 (A) or N73pep (B). The pretreated MGF were then incubated for 60 min 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). Samples were either fixed and processed for EM and quantitated, with the percentage total membrane present as cisternae ± SEM  and as stacked regions of cisternae ± SEM  shown; or the membranes were recovered and fractionated by SDS-PAGE using a 7.5% gel, transferred to nitrocellulose, and probed for GM130 and p115 using specific antibodies (shown below graphs in A and B).
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Related In: Results  -  Collection

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Figure 7: Effect of soluble GRASP65 and N73pep on the reassembly process using pure components. MGF isolated through a 0.5-M sucrose cushion were preincubated for 15 min on ice with increasing amounts of soluble GRASP65 (A) or N73pep (B). The pretreated MGF were then incubated for 60 min 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). Samples were either fixed and processed for EM and quantitated, with the percentage total membrane present as cisternae ± SEM and as stacked regions of cisternae ± SEM shown; or the membranes were recovered and fractionated by SDS-PAGE using a 7.5% gel, transferred to nitrocellulose, and probed for GM130 and p115 using specific antibodies (shown below graphs in A and B).
Mentions: An NEM-sensitive membrane-bound component of MGF is essential for the stacking reaction during reassembly. This factor was identified as GRASP65, a highly conserved, N-myristoylated protein that exists as a tight complex with GM130 on the membrane (Barr et al. 1997). A soluble, nonmyristoylated recombinant form of GRASP65 inhibited stacking, but not cisternal regrowth, in the cytosol based reassembly (Barr et al. 1997). This soluble GRASP65 was titrated into the NSF/p97 combined pathway of reassembly and was able to inhibit stacking potently without affecting cisternal regrowth (Fig. 7 A). This was also true in the p97-catalyzed and NSF-catalyzed reactions (data not shown). NEM-treated soluble GRASP65 had no effect on stacking or cisternal regrowth. Soluble GRASP65 prequenched with anti-GRASP65 antibodies also had no effect (data not shown). Soluble GRASP65 may then inhibit the stacking process by interacting with a Golgi membrane-bound factor.

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