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

Western analysis of MGF isolated with or without a 0.5-M sucrose cushion. 10 μg RLG, MGF derived from 10 μg RLG isolated with or without a 0.5-M sucrose cushion, and 10 μg sHeLa mitotic cytosol (1% of input) were fractionated by SDS-PAGE, transferred to nitrocellulose, and probed for GM130, p115, p97, NSF, Mann I, GRASP65, α-SNAP, and rab6 using specific antibodies. Molecular weights in kD are shown on the left. Note the shift in molecular weight of GM130 and GRASP65 in the MGF owing to mitotic phosphorylation of these two proteins.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199741&req=5

Figure 1: Western analysis of MGF isolated with or without a 0.5-M sucrose cushion. 10 μg RLG, MGF derived from 10 μg RLG isolated with or without a 0.5-M sucrose cushion, and 10 μg sHeLa mitotic cytosol (1% of input) were fractionated by SDS-PAGE, transferred to nitrocellulose, and probed for GM130, p115, p97, NSF, Mann I, GRASP65, α-SNAP, and rab6 using specific antibodies. Molecular weights in kD are shown on the left. Note the shift in molecular weight of GM130 and GRASP65 in the MGF owing to mitotic phosphorylation of these two proteins.

Mentions: Analysis of the polypeptide composition of the two sets of fragments revealed that the MGF isolated through the 0.5-M cushion were significantly less contaminated with cytosolic factors (data not shown). In fact, the MGF isolated through the 0.5-M cushion contained 65% less protein (data not shown). Western analysis (Fig. 1) revealed MGF isolated with or without the 0.5-M sucrose cushion contained similar amounts of Mann I, GM130, and GRASP65 (Fig. 1). Therefore, the 0.5-M cushion was not affecting the amount of membranes that were recovered. However, when the MGF are compared with starting RLG, virtually all the Mann I was recovered, but only 40–50% of the GM130 and GRASP65 appeared to be recovered. This may be due to a decrease in the reactivity of the antibodies against mitotically phosphorylated GM130 and GRASP65. MGF isolated with or without the 0.5-M sucrose cushion had similar levels of rab6 and α-SNAP.


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)

Western analysis of MGF isolated with or without a 0.5-M sucrose cushion. 10 μg RLG, MGF derived from 10 μg RLG isolated with or without a 0.5-M sucrose cushion, and 10 μg sHeLa mitotic cytosol (1% of input) were fractionated by SDS-PAGE, transferred to nitrocellulose, and probed for GM130, p115, p97, NSF, Mann I, GRASP65, α-SNAP, and rab6 using specific antibodies. Molecular weights in kD are shown on the left. Note the shift in molecular weight of GM130 and GRASP65 in the MGF owing to mitotic phosphorylation of these two proteins.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Western analysis of MGF isolated with or without a 0.5-M sucrose cushion. 10 μg RLG, MGF derived from 10 μg RLG isolated with or without a 0.5-M sucrose cushion, and 10 μg sHeLa mitotic cytosol (1% of input) were fractionated by SDS-PAGE, transferred to nitrocellulose, and probed for GM130, p115, p97, NSF, Mann I, GRASP65, α-SNAP, and rab6 using specific antibodies. Molecular weights in kD are shown on the left. Note the shift in molecular weight of GM130 and GRASP65 in the MGF owing to mitotic phosphorylation of these two proteins.
Mentions: Analysis of the polypeptide composition of the two sets of fragments revealed that the MGF isolated through the 0.5-M cushion were significantly less contaminated with cytosolic factors (data not shown). In fact, the MGF isolated through the 0.5-M cushion contained 65% less protein (data not shown). Western analysis (Fig. 1) revealed MGF isolated with or without the 0.5-M sucrose cushion contained similar amounts of Mann I, GM130, and GRASP65 (Fig. 1). Therefore, the 0.5-M cushion was not affecting the amount of membranes that were recovered. However, when the MGF are compared with starting RLG, virtually all the Mann I was recovered, but only 40–50% of the GM130 and GRASP65 appeared to be recovered. This may be due to a decrease in the reactivity of the antibodies against mitotically phosphorylated GM130 and GRASP65. MGF isolated with or without the 0.5-M sucrose cushion had similar levels of rab6 and α-SNAP.

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