Limits...
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.

Show MeSH

Related in: MedlinePlus

Golgin-84 is not part of the Golgi matrix. (a) Rat liver Golgi extracts were immunoprecipitated under native conditions with antibodies to either golgin-84, GM130, or p115, or with a control IgG. The immunoprecipitated (IP) and unbound fractions were analyzed by Western blotting with antibodies to golgin-84, GM130, p115, GRASP65, and mannosidase I as indicated. (b) NRK cells were incubated with 5 μg/ml BFA for 1 h and then fixed and double labeled with antibodies to golgin-84 and GM130 or the ER marker calnexin. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172652&req=5

fig3: Golgin-84 is not part of the Golgi matrix. (a) Rat liver Golgi extracts were immunoprecipitated under native conditions with antibodies to either golgin-84, GM130, or p115, or with a control IgG. The immunoprecipitated (IP) and unbound fractions were analyzed by Western blotting with antibodies to golgin-84, GM130, p115, GRASP65, and mannosidase I as indicated. (b) NRK cells were incubated with 5 μg/ml BFA for 1 h and then fixed and double labeled with antibodies to golgin-84 and GM130 or the ER marker calnexin. Bar, 10 μm.

Mentions: The different distributions of golgin-84 and GM130 in the cis-Golgi suggested that golgin-84 may not be part of the putative Golgi matrix described by Seemann et al. (2000a)(2002). We first tested whether golgin-84 can interact physically with cis-Golgi matrix proteins. Golgin-84 and the cis-Golgi matrix proteins GM130 and p115 were immunoprecipitated from Golgi extracts under mild conditions and the immunoprecipitates tested for the presence of golgin-84, the matrix proteins GM130, p115, and GRASP65, and the Golgi enzyme mannosidase I by immunoblotting. Even though golgin-84 was efficiently precipitated by its antibody (it was depleted from the unbound fraction), no matrix proteins could be detected in the immunoprecipitate. Similarly, no golgin-84 could be detected in the GM130 or p115 immunoprecipitates, which contained significant levels of GM130, p115, and GRASP65 (Fig. 3 a). Thus, golgin-84 does not appear to physically interact with cis-Golgi matrix proteins. To test more directly whether golgin-84 might exist as part of the putative Golgi matrix, we studied its behavior upon treatment of cells with BFA. As shown in Fig. 3 b, golgin-84 redistributed to the ER in BFA-treated cells, as suggested by previous work (Bascom et al., 1999). No golgin-84 was detected in the cytoplasmic GM130-containing punctate structures, indicating that golgin-84 is not part of the putative matrix described by Seemann et al. (2000a)(2002).


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)

Golgin-84 is not part of the Golgi matrix. (a) Rat liver Golgi extracts were immunoprecipitated under native conditions with antibodies to either golgin-84, GM130, or p115, or with a control IgG. The immunoprecipitated (IP) and unbound fractions were analyzed by Western blotting with antibodies to golgin-84, GM130, p115, GRASP65, and mannosidase I as indicated. (b) NRK cells were incubated with 5 μg/ml BFA for 1 h and then fixed and double labeled with antibodies to golgin-84 and GM130 or the ER marker calnexin. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172652&req=5

fig3: Golgin-84 is not part of the Golgi matrix. (a) Rat liver Golgi extracts were immunoprecipitated under native conditions with antibodies to either golgin-84, GM130, or p115, or with a control IgG. The immunoprecipitated (IP) and unbound fractions were analyzed by Western blotting with antibodies to golgin-84, GM130, p115, GRASP65, and mannosidase I as indicated. (b) NRK cells were incubated with 5 μg/ml BFA for 1 h and then fixed and double labeled with antibodies to golgin-84 and GM130 or the ER marker calnexin. Bar, 10 μm.
Mentions: The different distributions of golgin-84 and GM130 in the cis-Golgi suggested that golgin-84 may not be part of the putative Golgi matrix described by Seemann et al. (2000a)(2002). We first tested whether golgin-84 can interact physically with cis-Golgi matrix proteins. Golgin-84 and the cis-Golgi matrix proteins GM130 and p115 were immunoprecipitated from Golgi extracts under mild conditions and the immunoprecipitates tested for the presence of golgin-84, the matrix proteins GM130, p115, and GRASP65, and the Golgi enzyme mannosidase I by immunoblotting. Even though golgin-84 was efficiently precipitated by its antibody (it was depleted from the unbound fraction), no matrix proteins could be detected in the immunoprecipitate. Similarly, no golgin-84 could be detected in the GM130 or p115 immunoprecipitates, which contained significant levels of GM130, p115, and GRASP65 (Fig. 3 a). Thus, golgin-84 does not appear to physically interact with cis-Golgi matrix proteins. To test more directly whether golgin-84 might exist as part of the putative Golgi matrix, we studied its behavior upon treatment of cells with BFA. As shown in Fig. 3 b, golgin-84 redistributed to the ER in BFA-treated cells, as suggested by previous work (Bascom et al., 1999). No golgin-84 was detected in the cytoplasmic GM130-containing punctate structures, indicating that golgin-84 is not part of the putative matrix described by Seemann et al. (2000a)(2002).

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