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Golgi cisternal unstacking stimulates COPI vesicle budding and protein transport.

Wang Y, Wei JH, Bisel B, Tang D, Seemann J - PLoS ONE (2008)

Bottom Line: We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae.Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes.The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT
The Golgi apparatus in mammalian cells is composed of flattened cisternae that are densely packed to form stacks. We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae. We established an assay to measure protein transport to the cell surface in post-mitotic cells in which the Golgi was unstacked. Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes. Vesicle budding from unstacked cisternae in vitro was significantly increased compared to stacked membranes. These results suggest that Golgi cisternal stacking can directly regulate vesicle formation and thus the rate of protein transport through the Golgi. The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning.

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Inhibition of stacking by antibodies against GRASP65.(A) Rat liver Golgi membranes (RLG) were incubated with cdk1/cyclin B1 and plk1 and fixed either directly or after further incubation with interphase HeLa cell cytosol in the presence of GRASP65 antibodies. Representative EM micrographs of the membranes are shown in (A). Incubation with mitotic kinases led to unstacking of Golgi cisternae (cdk1/cyclin B1+plk1), and the subsequent reformation of stacks upon treatment with interphase cytosol was inhibited by antibodies against GRASP65. Bar, 0.5 µm. (B, C) Quantitation of (A) by the intersection method (mean±SD). Upon treatment with mitotic kinases, membranes in stacks were reduced to 11%, compared to 71% for untreated Golgi membranes. When interphase single cisternae were generated in the presence of GRASP65 antibodies and interphase cytosol, the percentage of membranes in stacks remained unchanged (B). During this process, the single cisternal membranes remained intact and did not vesiculate, as the percentage of membranes in vesicles was unchanged between all groups (C). Statistical significance was assessed by a two-tailed Student's t test.
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pone-0001647-g004: Inhibition of stacking by antibodies against GRASP65.(A) Rat liver Golgi membranes (RLG) were incubated with cdk1/cyclin B1 and plk1 and fixed either directly or after further incubation with interphase HeLa cell cytosol in the presence of GRASP65 antibodies. Representative EM micrographs of the membranes are shown in (A). Incubation with mitotic kinases led to unstacking of Golgi cisternae (cdk1/cyclin B1+plk1), and the subsequent reformation of stacks upon treatment with interphase cytosol was inhibited by antibodies against GRASP65. Bar, 0.5 µm. (B, C) Quantitation of (A) by the intersection method (mean±SD). Upon treatment with mitotic kinases, membranes in stacks were reduced to 11%, compared to 71% for untreated Golgi membranes. When interphase single cisternae were generated in the presence of GRASP65 antibodies and interphase cytosol, the percentage of membranes in stacks remained unchanged (B). During this process, the single cisternal membranes remained intact and did not vesiculate, as the percentage of membranes in vesicles was unchanged between all groups (C). Statistical significance was assessed by a two-tailed Student's t test.

Mentions: We then used an in vitro vesicle budding assay to determine whether unstacked single cisternae can form COPI vesicles more efficiently than stacks. We treated Golgi membranes with the mitotic kinases cdk1/cyclinB1 and plk1 to phosphorylate GRASP65 and thus unstack the Golgi. Dephosphorylation with interphase cytosol in the presence of GRASP65 antibodies inhibited GRASP65 oligomerization [12] and thus restacking, resulting in single interphase cisternae (Fig. 4 A). EM analysis showed that 71% of the cisternae were in stacks (Fig. 4 B). After incubation with cdk1/cyclinB1 and plk1, the majority of the cisternae were separated from each other and the percentage of stacked cisternae dropped to 11%. When further treated with interphase cytosol and GRASP65 antibodies, the percentage of cisternae in stacks remained unchanged at 11% (Fig. 4 B). The percentage of membranes in vesicles remained unchanged at 5% throughout each treatment, showing that the decrease in stacked cisternae was caused by unstacking, rather than by a conversion of the membranes into vesicles (Fig. 4 C).


Golgi cisternal unstacking stimulates COPI vesicle budding and protein transport.

Wang Y, Wei JH, Bisel B, Tang D, Seemann J - PLoS ONE (2008)

Inhibition of stacking by antibodies against GRASP65.(A) Rat liver Golgi membranes (RLG) were incubated with cdk1/cyclin B1 and plk1 and fixed either directly or after further incubation with interphase HeLa cell cytosol in the presence of GRASP65 antibodies. Representative EM micrographs of the membranes are shown in (A). Incubation with mitotic kinases led to unstacking of Golgi cisternae (cdk1/cyclin B1+plk1), and the subsequent reformation of stacks upon treatment with interphase cytosol was inhibited by antibodies against GRASP65. Bar, 0.5 µm. (B, C) Quantitation of (A) by the intersection method (mean±SD). Upon treatment with mitotic kinases, membranes in stacks were reduced to 11%, compared to 71% for untreated Golgi membranes. When interphase single cisternae were generated in the presence of GRASP65 antibodies and interphase cytosol, the percentage of membranes in stacks remained unchanged (B). During this process, the single cisternal membranes remained intact and did not vesiculate, as the percentage of membranes in vesicles was unchanged between all groups (C). Statistical significance was assessed by a two-tailed Student's t test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2249924&req=5

pone-0001647-g004: Inhibition of stacking by antibodies against GRASP65.(A) Rat liver Golgi membranes (RLG) were incubated with cdk1/cyclin B1 and plk1 and fixed either directly or after further incubation with interphase HeLa cell cytosol in the presence of GRASP65 antibodies. Representative EM micrographs of the membranes are shown in (A). Incubation with mitotic kinases led to unstacking of Golgi cisternae (cdk1/cyclin B1+plk1), and the subsequent reformation of stacks upon treatment with interphase cytosol was inhibited by antibodies against GRASP65. Bar, 0.5 µm. (B, C) Quantitation of (A) by the intersection method (mean±SD). Upon treatment with mitotic kinases, membranes in stacks were reduced to 11%, compared to 71% for untreated Golgi membranes. When interphase single cisternae were generated in the presence of GRASP65 antibodies and interphase cytosol, the percentage of membranes in stacks remained unchanged (B). During this process, the single cisternal membranes remained intact and did not vesiculate, as the percentage of membranes in vesicles was unchanged between all groups (C). Statistical significance was assessed by a two-tailed Student's t test.
Mentions: We then used an in vitro vesicle budding assay to determine whether unstacked single cisternae can form COPI vesicles more efficiently than stacks. We treated Golgi membranes with the mitotic kinases cdk1/cyclinB1 and plk1 to phosphorylate GRASP65 and thus unstack the Golgi. Dephosphorylation with interphase cytosol in the presence of GRASP65 antibodies inhibited GRASP65 oligomerization [12] and thus restacking, resulting in single interphase cisternae (Fig. 4 A). EM analysis showed that 71% of the cisternae were in stacks (Fig. 4 B). After incubation with cdk1/cyclinB1 and plk1, the majority of the cisternae were separated from each other and the percentage of stacked cisternae dropped to 11%. When further treated with interphase cytosol and GRASP65 antibodies, the percentage of cisternae in stacks remained unchanged at 11% (Fig. 4 B). The percentage of membranes in vesicles remained unchanged at 5% throughout each treatment, showing that the decrease in stacked cisternae was caused by unstacking, rather than by a conversion of the membranes into vesicles (Fig. 4 C).

Bottom Line: We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae.Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes.The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT
The Golgi apparatus in mammalian cells is composed of flattened cisternae that are densely packed to form stacks. We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae. We established an assay to measure protein transport to the cell surface in post-mitotic cells in which the Golgi was unstacked. Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes. Vesicle budding from unstacked cisternae in vitro was significantly increased compared to stacked membranes. These results suggest that Golgi cisternal stacking can directly regulate vesicle formation and thus the rate of protein transport through the Golgi. The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning.

Show MeSH
Related in: MedlinePlus