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The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation.

Diao A, Rahman D, Pappin DJ, Lucocq J, Lowe M - J. Cell Biol. (2003)

Bottom Line: Using cryoelectron microscopy we could localize golgin-84 to the cis-Golgi network and found that it is enriched on tubules emanating from the lateral edges of, and often connecting, Golgi stacks.These mini-stacks are able to carry out protein transport, though with reduced efficiency compared with a normal Golgi apparatus.Our results suggest that golgin-84 plays a key role in the assembly and maintenance of the Golgi ribbon in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.

ABSTRACT
Fragmentation of the mammalian Golgi apparatus during mitosis requires the phosphorylation of a specific subset of Golgi-associated proteins. We have used a biochemical approach to characterize these proteins and report here the identification of golgin-84 as a novel mitotic target. Using cryoelectron microscopy we could localize golgin-84 to the cis-Golgi network and found that it is enriched on tubules emanating from the lateral edges of, and often connecting, Golgi stacks. Golgin-84 binds to active rab1 but not cis-Golgi matrix proteins. Overexpression or depletion of golgin-84 results in fragmentation of the Golgi ribbon. Strikingly, the Golgi ribbon is converted into mini-stacks constituting only approximately 25% of the volume of a normal Golgi apparatus upon golgin-84 depletion. These mini-stacks are able to carry out protein transport, though with reduced efficiency compared with a normal Golgi apparatus. Our results suggest that golgin-84 plays a key role in the assembly and maintenance of the Golgi ribbon in mammalian cells.

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Depletion of golgin-84 partially inhibits transport of VSV-G from the ER to the cell surface. Control or golgin-84 RNAi–treated HeLa cells were transfected with a plasmid encoding GFP-tagged ts045G VSV-G protein. Cells were incubated at 39.5°C to arrest ts045G VSV-G in the ER, and then chased at 31°C for various times to allow transport before fixation and labeling for cell surface VSV-G. (a) An example of cells shifted to 31°C for 60 min and labeled for cell surface VSV-G. (b) The extent of VSV-G transport was measured as indicated in the Materials and methods and is expressed as the ratio of cell surface to total VSV-G fluorescence. The data shown are representative of two experiments with n = 15 for all data points in each experiment.
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fig8: Depletion of golgin-84 partially inhibits transport of VSV-G from the ER to the cell surface. Control or golgin-84 RNAi–treated HeLa cells were transfected with a plasmid encoding GFP-tagged ts045G VSV-G protein. Cells were incubated at 39.5°C to arrest ts045G VSV-G in the ER, and then chased at 31°C for various times to allow transport before fixation and labeling for cell surface VSV-G. (a) An example of cells shifted to 31°C for 60 min and labeled for cell surface VSV-G. (b) The extent of VSV-G transport was measured as indicated in the Materials and methods and is expressed as the ratio of cell surface to total VSV-G fluorescence. The data shown are representative of two experiments with n = 15 for all data points in each experiment.

Mentions: To study whether golgin-84 plays any role in protein trafficking through the Golgi apparatus, we used a GFP-tagged temperature-sensitive allele of the vesicular stomatitis virus G protein (ts045 VSV-G). This well-characterized secretory cargo marker accumulates in the ER at the nonpermissive temperature of 39.5°C due to a reversible folding defect. When shifted to the permissive temperature (31°C), correctly folded ts045G VSV-G is rapidly transported from the ER, through the Golgi apparatus to the cell surface, where its appearance can be monitored using an antibody against the lumenal domain of the glycoprotein (Pepperkok et al., 1993; Seemann et al., 2000b). As expected, VSV-G was efficiently transported to the cell surface when control cells were shifted to 31°C (Fig. 8 a). In cells depleted of golgin-84, VSV-G could also be detected on the cell surface at 31°C (Fig. 8 a). Golgin-84 depletion therefore does not block VSV-G transport to the cell surface. To determine whether there is a more subtle effect, transport of VSV-G to the cell surface was quantitated in golgin-84–depleted cells relative to control cells. This revealed that VSV-G transport to the cell surface was inhibited by 15%, 39%, and 42% in golgin-84–depleted cells after 30, 60, and 90 min at 31°C, respectively (Fig. 8 b). These results suggest that golgin-84 is required for efficient protein trafficking through the Golgi apparatus.


The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation.

Diao A, Rahman D, Pappin DJ, Lucocq J, Lowe M - J. Cell Biol. (2003)

Depletion of golgin-84 partially inhibits transport of VSV-G from the ER to the cell surface. Control or golgin-84 RNAi–treated HeLa cells were transfected with a plasmid encoding GFP-tagged ts045G VSV-G protein. Cells were incubated at 39.5°C to arrest ts045G VSV-G in the ER, and then chased at 31°C for various times to allow transport before fixation and labeling for cell surface VSV-G. (a) An example of cells shifted to 31°C for 60 min and labeled for cell surface VSV-G. (b) The extent of VSV-G transport was measured as indicated in the Materials and methods and is expressed as the ratio of cell surface to total VSV-G fluorescence. The data shown are representative of two experiments with n = 15 for all data points in each experiment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172652&req=5

fig8: Depletion of golgin-84 partially inhibits transport of VSV-G from the ER to the cell surface. Control or golgin-84 RNAi–treated HeLa cells were transfected with a plasmid encoding GFP-tagged ts045G VSV-G protein. Cells were incubated at 39.5°C to arrest ts045G VSV-G in the ER, and then chased at 31°C for various times to allow transport before fixation and labeling for cell surface VSV-G. (a) An example of cells shifted to 31°C for 60 min and labeled for cell surface VSV-G. (b) The extent of VSV-G transport was measured as indicated in the Materials and methods and is expressed as the ratio of cell surface to total VSV-G fluorescence. The data shown are representative of two experiments with n = 15 for all data points in each experiment.
Mentions: To study whether golgin-84 plays any role in protein trafficking through the Golgi apparatus, we used a GFP-tagged temperature-sensitive allele of the vesicular stomatitis virus G protein (ts045 VSV-G). This well-characterized secretory cargo marker accumulates in the ER at the nonpermissive temperature of 39.5°C due to a reversible folding defect. When shifted to the permissive temperature (31°C), correctly folded ts045G VSV-G is rapidly transported from the ER, through the Golgi apparatus to the cell surface, where its appearance can be monitored using an antibody against the lumenal domain of the glycoprotein (Pepperkok et al., 1993; Seemann et al., 2000b). As expected, VSV-G was efficiently transported to the cell surface when control cells were shifted to 31°C (Fig. 8 a). In cells depleted of golgin-84, VSV-G could also be detected on the cell surface at 31°C (Fig. 8 a). Golgin-84 depletion therefore does not block VSV-G transport to the cell surface. To determine whether there is a more subtle effect, transport of VSV-G to the cell surface was quantitated in golgin-84–depleted cells relative to control cells. This revealed that VSV-G transport to the cell surface was inhibited by 15%, 39%, and 42% in golgin-84–depleted cells after 30, 60, and 90 min at 31°C, respectively (Fig. 8 b). These results suggest that golgin-84 is required for efficient protein trafficking through the Golgi apparatus.

Bottom Line: Using cryoelectron microscopy we could localize golgin-84 to the cis-Golgi network and found that it is enriched on tubules emanating from the lateral edges of, and often connecting, Golgi stacks.These mini-stacks are able to carry out protein transport, though with reduced efficiency compared with a normal Golgi apparatus.Our results suggest that golgin-84 plays a key role in the assembly and maintenance of the Golgi ribbon in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.

ABSTRACT
Fragmentation of the mammalian Golgi apparatus during mitosis requires the phosphorylation of a specific subset of Golgi-associated proteins. We have used a biochemical approach to characterize these proteins and report here the identification of golgin-84 as a novel mitotic target. Using cryoelectron microscopy we could localize golgin-84 to the cis-Golgi network and found that it is enriched on tubules emanating from the lateral edges of, and often connecting, Golgi stacks. Golgin-84 binds to active rab1 but not cis-Golgi matrix proteins. Overexpression or depletion of golgin-84 results in fragmentation of the Golgi ribbon. Strikingly, the Golgi ribbon is converted into mini-stacks constituting only approximately 25% of the volume of a normal Golgi apparatus upon golgin-84 depletion. These mini-stacks are able to carry out protein transport, though with reduced efficiency compared with a normal Golgi apparatus. Our results suggest that golgin-84 plays a key role in the assembly and maintenance of the Golgi ribbon in mammalian cells.

Show MeSH
Related in: MedlinePlus