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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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Titration of p115 into the p97, NSF, and NSF/p97 catalyzed reassembly reactions. MGF isolated through a 0.5-M sucrose cushion were incubated for 60 min at 37°C with: A, p97 (70 ng/μl) and p47 (37.5 ng/μl); B, NSF (100 ng/μl), α-SNAP (25 ng/μl) and γ-SNAP (25 ng/μl); or C, all these components combined with from 0–30 ng/μl p115. Samples were fixed and processed for EM, and then quantitated for percentage total membrane as cisternae ± SEM  and percentage total membrane present as stacked regions of cisternae ± SEM . Results are displayed in graphic form at the left and representative fields of the two extreme p115 concentrations are positioned adjacent. Note the long single, wrinkled cisternae formed by the p97 pathway in the absence of p115 (arrow in A) and the long stacks of two cisternae with maximum p115 (arrowheads in A). Note the virtual absence of cisternae for the NSF pathway in the absence of p115 (B) and the short stacks of three or more cisternae with maximum p115 (arrowheads in B). Note the long, wrinkled single cisternae formed by the NSF/p97 pathway in the absence of p115 (arrow in C) and the stacks of two or three cisternae with maximum p115 (arrowheads in C). Bar, 0.5 μm.
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Figure 5: Titration of p115 into the p97, NSF, and NSF/p97 catalyzed reassembly reactions. MGF isolated through a 0.5-M sucrose cushion were incubated for 60 min at 37°C with: A, p97 (70 ng/μl) and p47 (37.5 ng/μl); B, NSF (100 ng/μl), α-SNAP (25 ng/μl) and γ-SNAP (25 ng/μl); or C, all these components combined with from 0–30 ng/μl p115. Samples were fixed and processed for EM, and then quantitated for percentage total membrane as cisternae ± SEM and percentage total membrane present as stacked regions of cisternae ± SEM . Results are displayed in graphic form at the left and representative fields of the two extreme p115 concentrations are positioned adjacent. Note the long single, wrinkled cisternae formed by the p97 pathway in the absence of p115 (arrow in A) and the long stacks of two cisternae with maximum p115 (arrowheads in A). Note the virtual absence of cisternae for the NSF pathway in the absence of p115 (B) and the short stacks of three or more cisternae with maximum p115 (arrowheads in B). Note the long, wrinkled single cisternae formed by the NSF/p97 pathway in the absence of p115 (arrow in C) and the stacks of two or three cisternae with maximum p115 (arrowheads in C). Bar, 0.5 μm.

Mentions: Titration of p115 into the p97 reaction revealed that this fusion pathway was insensitive to added p115 (Fig. 5 A). However, only single cisternae with a mean cisternal cross-sectional length of 1.4 μm formed in the absence of p115 (Table ). These single cisternae had a wrinkled appearance reminiscent of those formed in p115-depleted cytosol (compare Fig. 2 G and 5 A). Upon addition of p115, these cisternae had a smoother appearance and formed stacks (Fig. 5 A). However, the stacks formed rarely had more than two cisternae per stack.


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)

Titration of p115 into the p97, NSF, and NSF/p97 catalyzed reassembly reactions. MGF isolated through a 0.5-M sucrose cushion were incubated for 60 min at 37°C with: A, p97 (70 ng/μl) and p47 (37.5 ng/μl); B, NSF (100 ng/μl), α-SNAP (25 ng/μl) and γ-SNAP (25 ng/μl); or C, all these components combined with from 0–30 ng/μl p115. Samples were fixed and processed for EM, and then quantitated for percentage total membrane as cisternae ± SEM  and percentage total membrane present as stacked regions of cisternae ± SEM . Results are displayed in graphic form at the left and representative fields of the two extreme p115 concentrations are positioned adjacent. Note the long single, wrinkled cisternae formed by the p97 pathway in the absence of p115 (arrow in A) and the long stacks of two cisternae with maximum p115 (arrowheads in A). Note the virtual absence of cisternae for the NSF pathway in the absence of p115 (B) and the short stacks of three or more cisternae with maximum p115 (arrowheads in B). Note the long, wrinkled single cisternae formed by the NSF/p97 pathway in the absence of p115 (arrow in C) and the stacks of two or three cisternae with maximum p115 (arrowheads in C). Bar, 0.5 μm.
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Related In: Results  -  Collection

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Figure 5: Titration of p115 into the p97, NSF, and NSF/p97 catalyzed reassembly reactions. MGF isolated through a 0.5-M sucrose cushion were incubated for 60 min at 37°C with: A, p97 (70 ng/μl) and p47 (37.5 ng/μl); B, NSF (100 ng/μl), α-SNAP (25 ng/μl) and γ-SNAP (25 ng/μl); or C, all these components combined with from 0–30 ng/μl p115. Samples were fixed and processed for EM, and then quantitated for percentage total membrane as cisternae ± SEM and percentage total membrane present as stacked regions of cisternae ± SEM . Results are displayed in graphic form at the left and representative fields of the two extreme p115 concentrations are positioned adjacent. Note the long single, wrinkled cisternae formed by the p97 pathway in the absence of p115 (arrow in A) and the long stacks of two cisternae with maximum p115 (arrowheads in A). Note the virtual absence of cisternae for the NSF pathway in the absence of p115 (B) and the short stacks of three or more cisternae with maximum p115 (arrowheads in B). Note the long, wrinkled single cisternae formed by the NSF/p97 pathway in the absence of p115 (arrow in C) and the stacks of two or three cisternae with maximum p115 (arrowheads in C). Bar, 0.5 μm.
Mentions: Titration of p115 into the p97 reaction revealed that this fusion pathway was insensitive to added p115 (Fig. 5 A). However, only single cisternae with a mean cisternal cross-sectional length of 1.4 μm formed in the absence of p115 (Table ). These single cisternae had a wrinkled appearance reminiscent of those formed in p115-depleted cytosol (compare Fig. 2 G and 5 A). Upon addition of p115, these cisternae had a smoother appearance and formed stacks (Fig. 5 A). However, the stacks formed rarely had more than two cisternae per stack.

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