Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of interphase cytosol depleted of p115 on the reassembly process. (A) Rat liver cytosol was depleted of p115, as described in Materials and Methods, using either the anti-p115 mAb antibody 4H1 or the N73pep. 20 μg of cytosol was fractionated by SDS-PAGE using a 7.5% gel, transferred to nitrocellulose, and probed with the anti-p115 mAb 8A6. (B–J) 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 (B–D), p115-depleted cytosol (E–G), or p115-depleted cytosol supplemented with purified rat liver p115 (H–J), fixed, and processed for EM. Representative fields are shown. Note the presence of stacks (arrowheads) in B–D and H–J, but only single cisternae (arrows) in E–G. The reduced number of cisternae in E suggests poor reassembly at this concentration of p115-depleted cytosol. Note that the cisternae formed often have a wrinkled appearance (asterisks in G) in p115-depleted cytosol and are often blunt-ended with few associated vesicles (compare asterisks in F and I), in contrast to when p115 is present. Bar, 0.5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199741&req=5

Figure 2: Effect of interphase cytosol depleted of p115 on the reassembly process. (A) Rat liver cytosol was depleted of p115, as described in Materials and Methods, using either the anti-p115 mAb antibody 4H1 or the N73pep. 20 μg of cytosol was fractionated by SDS-PAGE using a 7.5% gel, transferred to nitrocellulose, and probed with the anti-p115 mAb 8A6. (B–J) 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 (B–D), p115-depleted cytosol (E–G), or p115-depleted cytosol supplemented with purified rat liver p115 (H–J), fixed, and processed for EM. Representative fields are shown. Note the presence of stacks (arrowheads) in B–D and H–J, but only single cisternae (arrows) in E–G. The reduced number of cisternae in E suggests poor reassembly at this concentration of p115-depleted cytosol. Note that the cisternae formed often have a wrinkled appearance (asterisks in G) in p115-depleted cytosol and are often blunt-ended with few associated vesicles (compare asterisks in F and I), in contrast to when p115 is present. Bar, 0.5 μm.

Mentions: To assess p115 function in the reassembly process, rat liver cytosol, p115-depleted cytosol, and p115-depleted cytosol with purified p115 added back were titrated into the reassembly assay. p115 was depleted >95% from rat liver cytosol using either the mAb 4H1 or N73pep (Fig. 2 A). p115 was purified to near homogeneity from rat liver cytosol to add back to this depleted cytosol. MGF were resuspended in cytosol of increasing concentrations and incubated for 60 min at 37°C.


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 interphase cytosol depleted of p115 on the reassembly process. (A) Rat liver cytosol was depleted of p115, as described in Materials and Methods, using either the anti-p115 mAb antibody 4H1 or the N73pep. 20 μg of cytosol was fractionated by SDS-PAGE using a 7.5% gel, transferred to nitrocellulose, and probed with the anti-p115 mAb 8A6. (B–J) 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 (B–D), p115-depleted cytosol (E–G), or p115-depleted cytosol supplemented with purified rat liver p115 (H–J), fixed, and processed for EM. Representative fields are shown. Note the presence of stacks (arrowheads) in B–D and H–J, but only single cisternae (arrows) in E–G. The reduced number of cisternae in E suggests poor reassembly at this concentration of p115-depleted cytosol. Note that the cisternae formed often have a wrinkled appearance (asterisks in G) in p115-depleted cytosol and are often blunt-ended with few associated vesicles (compare asterisks in F and I), in contrast to when p115 is present. Bar, 0.5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199741&req=5

Figure 2: Effect of interphase cytosol depleted of p115 on the reassembly process. (A) Rat liver cytosol was depleted of p115, as described in Materials and Methods, using either the anti-p115 mAb antibody 4H1 or the N73pep. 20 μg of cytosol was fractionated by SDS-PAGE using a 7.5% gel, transferred to nitrocellulose, and probed with the anti-p115 mAb 8A6. (B–J) 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 (B–D), p115-depleted cytosol (E–G), or p115-depleted cytosol supplemented with purified rat liver p115 (H–J), fixed, and processed for EM. Representative fields are shown. Note the presence of stacks (arrowheads) in B–D and H–J, but only single cisternae (arrows) in E–G. The reduced number of cisternae in E suggests poor reassembly at this concentration of p115-depleted cytosol. Note that the cisternae formed often have a wrinkled appearance (asterisks in G) in p115-depleted cytosol and are often blunt-ended with few associated vesicles (compare asterisks in F and I), in contrast to when p115 is present. Bar, 0.5 μm.
Mentions: To assess p115 function in the reassembly process, rat liver cytosol, p115-depleted cytosol, and p115-depleted cytosol with purified p115 added back were titrated into the reassembly assay. p115 was depleted >95% from rat liver cytosol using either the mAb 4H1 or N73pep (Fig. 2 A). p115 was purified to near homogeneity from rat liver cytosol to add back to this depleted cytosol. MGF were resuspended in cytosol of increasing concentrations and incubated for 60 min at 37°C.

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