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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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Kinetic analysis of Golgi membrane reassembly in rat liver cytosol and p115-depleted cytosol. (A–H) MGF isolated through a 0.5-M sucrose cushion were incubated for increasing time at 37°C with either rat liver cytosol (A–D) or p115-depleted cytosol (E–H) with the cytosol concentration set at 10 mg/ml, fixed and processed for EM, and quantitated as described in Materials and Methods. Representative fields are shown. In rat liver cytosol, note that the first intermediate formed is the single cisterna after 5 min (arrows in A). By 15 min, these single cisternae had grown in length, had tubular networks associated with their rims (asterisks in B), and had begun to align and dock to form stacks (arrowheads in B). Many discrete stacks had formed by 45 min (arrowheads in C), which had joined up by 120 min (arrowheads in D). In p115-depleted cytosol, single cisternae were again present after 5 min (arrows in E), and had increased in length by 15 min (arrows in F), but were often blunt-ended (asterisk in F) and were not stacked. At 45 min, these cisternae remained blunt-ended (asterisk in G) and unstacked. By 120 min, some stacks of blunt-ended cisternae had begun to form (arrowhead in H), but many single cisternae remained (arrows in H). Bar, 0.5 μm. (I) Quantitation of the time course. The percentage total membrane present as cisternae ± SEM  and stacked regions of cisternae ± SEM  are presented for each cytosol at every time point tested. (J) MGF were reassembled in p115-depleted cytosol and supplemented with p115 at different times (time of addition of p115). The reaction was allowed to proceed for a total time of 120 min at 37°C. Samples were then fixed and processed for EM, and the percentage total membrane as stacked regions of cisternae ± SEM  are presented for each time point tested.
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Figure 4: Kinetic analysis of Golgi membrane reassembly in rat liver cytosol and p115-depleted cytosol. (A–H) MGF isolated through a 0.5-M sucrose cushion were incubated for increasing time at 37°C with either rat liver cytosol (A–D) or p115-depleted cytosol (E–H) with the cytosol concentration set at 10 mg/ml, fixed and processed for EM, and quantitated as described in Materials and Methods. Representative fields are shown. In rat liver cytosol, note that the first intermediate formed is the single cisterna after 5 min (arrows in A). By 15 min, these single cisternae had grown in length, had tubular networks associated with their rims (asterisks in B), and had begun to align and dock to form stacks (arrowheads in B). Many discrete stacks had formed by 45 min (arrowheads in C), which had joined up by 120 min (arrowheads in D). In p115-depleted cytosol, single cisternae were again present after 5 min (arrows in E), and had increased in length by 15 min (arrows in F), but were often blunt-ended (asterisk in F) and were not stacked. At 45 min, these cisternae remained blunt-ended (asterisk in G) and unstacked. By 120 min, some stacks of blunt-ended cisternae had begun to form (arrowhead in H), but many single cisternae remained (arrows in H). Bar, 0.5 μm. (I) Quantitation of the time course. The percentage total membrane present as cisternae ± SEM and stacked regions of cisternae ± SEM are presented for each cytosol at every time point tested. (J) MGF were reassembled in p115-depleted cytosol and supplemented with p115 at different times (time of addition of p115). The reaction was allowed to proceed for a total time of 120 min at 37°C. Samples were then fixed and processed for EM, and the percentage total membrane as stacked regions of cisternae ± SEM are presented for each time point tested.

Mentions: Kinetic analysis revealed the reassembly reaction was complete for both cisternal regrowth and stacking after 60 min in rat liver cytosol (10 mg/ml; Fig. 4 I). The first intermediates that formed quickly during the first 15 min of the incubation were single cisternae (Fig. 4A and Fig. B), frequently with tubular networks at their rims (asterisks in Fig. 4 B). By 15 min, these intermediates had begun to dock and align to form the beginnings of stacked Golgi structure (arrowheads in Fig. 4 B). The lag in the formation of stacked structures (Fig. 4 I) therefore may be considered due to the need to form single cisternae first. By 45 min, this process was well advanced and Golgi stacks with two or more cisternae per stack were prevalent and these discrete stacks were becoming linked via tubular networks (Fig. 4 C). By 60–120 min, these linkages had been made, the tubular networks were less apparent, and long cisternal stacks were the end product, which often adopted an approximate closed concentric circular morphology (compare Fig. 2 D and 4 D).


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)

Kinetic analysis of Golgi membrane reassembly in rat liver cytosol and p115-depleted cytosol. (A–H) MGF isolated through a 0.5-M sucrose cushion were incubated for increasing time at 37°C with either rat liver cytosol (A–D) or p115-depleted cytosol (E–H) with the cytosol concentration set at 10 mg/ml, fixed and processed for EM, and quantitated as described in Materials and Methods. Representative fields are shown. In rat liver cytosol, note that the first intermediate formed is the single cisterna after 5 min (arrows in A). By 15 min, these single cisternae had grown in length, had tubular networks associated with their rims (asterisks in B), and had begun to align and dock to form stacks (arrowheads in B). Many discrete stacks had formed by 45 min (arrowheads in C), which had joined up by 120 min (arrowheads in D). In p115-depleted cytosol, single cisternae were again present after 5 min (arrows in E), and had increased in length by 15 min (arrows in F), but were often blunt-ended (asterisk in F) and were not stacked. At 45 min, these cisternae remained blunt-ended (asterisk in G) and unstacked. By 120 min, some stacks of blunt-ended cisternae had begun to form (arrowhead in H), but many single cisternae remained (arrows in H). Bar, 0.5 μm. (I) Quantitation of the time course. The percentage total membrane present as cisternae ± SEM  and stacked regions of cisternae ± SEM  are presented for each cytosol at every time point tested. (J) MGF were reassembled in p115-depleted cytosol and supplemented with p115 at different times (time of addition of p115). The reaction was allowed to proceed for a total time of 120 min at 37°C. Samples were then fixed and processed for EM, and the percentage total membrane as stacked regions of cisternae ± SEM  are presented for each time point tested.
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Related In: Results  -  Collection

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Figure 4: Kinetic analysis of Golgi membrane reassembly in rat liver cytosol and p115-depleted cytosol. (A–H) MGF isolated through a 0.5-M sucrose cushion were incubated for increasing time at 37°C with either rat liver cytosol (A–D) or p115-depleted cytosol (E–H) with the cytosol concentration set at 10 mg/ml, fixed and processed for EM, and quantitated as described in Materials and Methods. Representative fields are shown. In rat liver cytosol, note that the first intermediate formed is the single cisterna after 5 min (arrows in A). By 15 min, these single cisternae had grown in length, had tubular networks associated with their rims (asterisks in B), and had begun to align and dock to form stacks (arrowheads in B). Many discrete stacks had formed by 45 min (arrowheads in C), which had joined up by 120 min (arrowheads in D). In p115-depleted cytosol, single cisternae were again present after 5 min (arrows in E), and had increased in length by 15 min (arrows in F), but were often blunt-ended (asterisk in F) and were not stacked. At 45 min, these cisternae remained blunt-ended (asterisk in G) and unstacked. By 120 min, some stacks of blunt-ended cisternae had begun to form (arrowhead in H), but many single cisternae remained (arrows in H). Bar, 0.5 μm. (I) Quantitation of the time course. The percentage total membrane present as cisternae ± SEM and stacked regions of cisternae ± SEM are presented for each cytosol at every time point tested. (J) MGF were reassembled in p115-depleted cytosol and supplemented with p115 at different times (time of addition of p115). The reaction was allowed to proceed for a total time of 120 min at 37°C. Samples were then fixed and processed for EM, and the percentage total membrane as stacked regions of cisternae ± SEM are presented for each time point tested.
Mentions: Kinetic analysis revealed the reassembly reaction was complete for both cisternal regrowth and stacking after 60 min in rat liver cytosol (10 mg/ml; Fig. 4 I). The first intermediates that formed quickly during the first 15 min of the incubation were single cisternae (Fig. 4A and Fig. B), frequently with tubular networks at their rims (asterisks in Fig. 4 B). By 15 min, these intermediates had begun to dock and align to form the beginnings of stacked Golgi structure (arrowheads in Fig. 4 B). The lag in the formation of stacked structures (Fig. 4 I) therefore may be considered due to the need to form single cisternae first. By 45 min, this process was well advanced and Golgi stacks with two or more cisternae per stack were prevalent and these discrete stacks were becoming linked via tubular networks (Fig. 4 C). By 60–120 min, these linkages had been made, the tubular networks were less apparent, and long cisternal stacks were the end product, which often adopted an approximate closed concentric circular morphology (compare Fig. 2 D and 4 D).

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