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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 using RNAi fragments the Golgi ribbon. (a) HeLa cells were either mock transfected (no RNAi) or transfected with duplex RNA to target lamin A or golgin-84 and, after 1–4 d, were subjected to Western blotting with antibodies to GM130 or golgin-84. (b) Mock transfected or RNAi-treated HeLa cells were fixed and double labeled with antibodies to golgin-84, lamin A, GalNacT2, mSec23p, and GM130. In the merged images on the right, DNA is blue, golgin-84, lamin A, GalNacT2, mSec23p are in green, and GM130 is red, with regions of overlap between these proteins indicated in yellow. Bar, 10 μm. (c) Quantitation of golgin-84 levels and Golgi fragmentation in RNAi-treated cells. Golgin-84 levels were measured by quantitating Western blots of RNAi-treated HeLa cells as described in the Materials and methods section. Golgi fragmentation was measured by immunofluorescence microscopy using antibodies to GM130 to assess Golgi morphology. For each time point, 200 cells were counted. The data are an average of two independent experiments.
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fig6: Depletion of golgin-84 using RNAi fragments the Golgi ribbon. (a) HeLa cells were either mock transfected (no RNAi) or transfected with duplex RNA to target lamin A or golgin-84 and, after 1–4 d, were subjected to Western blotting with antibodies to GM130 or golgin-84. (b) Mock transfected or RNAi-treated HeLa cells were fixed and double labeled with antibodies to golgin-84, lamin A, GalNacT2, mSec23p, and GM130. In the merged images on the right, DNA is blue, golgin-84, lamin A, GalNacT2, mSec23p are in green, and GM130 is red, with regions of overlap between these proteins indicated in yellow. Bar, 10 μm. (c) Quantitation of golgin-84 levels and Golgi fragmentation in RNAi-treated cells. Golgin-84 levels were measured by quantitating Western blots of RNAi-treated HeLa cells as described in the Materials and methods section. Golgi fragmentation was measured by immunofluorescence microscopy using antibodies to GM130 to assess Golgi morphology. For each time point, 200 cells were counted. The data are an average of two independent experiments.

Mentions: To further examine the role of golgin-84 in maintaining normal Golgi structure, we used small interfering RNA (siRNA) to deplete cellular golgin-84 (Elbashir et al., 2001). Transfection of HeLa cells with an siRNA duplex matching the nucleotide sequence of golgin-84 resulted in a reduction in golgin-84 levels of ∼90% after 3 d in culture (Fig. 6 a). This reduction was specific because levels of GM130 were unaffected, and transfection of cells with an siRNA duplex targeting lamin A had no effect upon golgin-84 (or GM130) levels. Immunofluorescence microscopy with antibodies to GM130 showed that there was little effect upon Golgi morphology in mock transfected cells or cells transfected with an siRNA that effectively depleted cellular lamin A (Fig. 6 b). Depletion of golgin-84, however, had a drastic effect upon Golgi structure, breaking the ribbon into large fragments dispersed in the cytoplasm (Fig. 6 b). The extent of Golgi fragmentation correlated well with loss of golgin-84 over time (Fig. 6 c). This, combined with the lack of fragmentation induced by prolonged exposure to the control lamin A oligo, suggests that fragmentation is a consequence of golgin-84 depletion and not cellular toxicity resulting from the RNA interference (RNAi) treatment itself. Although predominantly punctate in appearance, the Golgi fragments occasionally resembled short tubules. Fragments resulting from golgin-84 depletion contained markers of the cis- (GM130), medial (GalNacT2; Fig. 6 b), and trans-Golgi (TGN46; unpublished data), suggesting that some degree of Golgi organization may be retained in these cells. The Golgi fragments did not extensively colocalize with the ER exit site marker mSec23p, suggesting that they arise directly from the break-up of the Golgi ribbon rather than the cycling through the ER (Fig. 6 b).


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 using RNAi fragments the Golgi ribbon. (a) HeLa cells were either mock transfected (no RNAi) or transfected with duplex RNA to target lamin A or golgin-84 and, after 1–4 d, were subjected to Western blotting with antibodies to GM130 or golgin-84. (b) Mock transfected or RNAi-treated HeLa cells were fixed and double labeled with antibodies to golgin-84, lamin A, GalNacT2, mSec23p, and GM130. In the merged images on the right, DNA is blue, golgin-84, lamin A, GalNacT2, mSec23p are in green, and GM130 is red, with regions of overlap between these proteins indicated in yellow. Bar, 10 μm. (c) Quantitation of golgin-84 levels and Golgi fragmentation in RNAi-treated cells. Golgin-84 levels were measured by quantitating Western blots of RNAi-treated HeLa cells as described in the Materials and methods section. Golgi fragmentation was measured by immunofluorescence microscopy using antibodies to GM130 to assess Golgi morphology. For each time point, 200 cells were counted. The data are an average of two independent experiments.
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Related In: Results  -  Collection

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fig6: Depletion of golgin-84 using RNAi fragments the Golgi ribbon. (a) HeLa cells were either mock transfected (no RNAi) or transfected with duplex RNA to target lamin A or golgin-84 and, after 1–4 d, were subjected to Western blotting with antibodies to GM130 or golgin-84. (b) Mock transfected or RNAi-treated HeLa cells were fixed and double labeled with antibodies to golgin-84, lamin A, GalNacT2, mSec23p, and GM130. In the merged images on the right, DNA is blue, golgin-84, lamin A, GalNacT2, mSec23p are in green, and GM130 is red, with regions of overlap between these proteins indicated in yellow. Bar, 10 μm. (c) Quantitation of golgin-84 levels and Golgi fragmentation in RNAi-treated cells. Golgin-84 levels were measured by quantitating Western blots of RNAi-treated HeLa cells as described in the Materials and methods section. Golgi fragmentation was measured by immunofluorescence microscopy using antibodies to GM130 to assess Golgi morphology. For each time point, 200 cells were counted. The data are an average of two independent experiments.
Mentions: To further examine the role of golgin-84 in maintaining normal Golgi structure, we used small interfering RNA (siRNA) to deplete cellular golgin-84 (Elbashir et al., 2001). Transfection of HeLa cells with an siRNA duplex matching the nucleotide sequence of golgin-84 resulted in a reduction in golgin-84 levels of ∼90% after 3 d in culture (Fig. 6 a). This reduction was specific because levels of GM130 were unaffected, and transfection of cells with an siRNA duplex targeting lamin A had no effect upon golgin-84 (or GM130) levels. Immunofluorescence microscopy with antibodies to GM130 showed that there was little effect upon Golgi morphology in mock transfected cells or cells transfected with an siRNA that effectively depleted cellular lamin A (Fig. 6 b). Depletion of golgin-84, however, had a drastic effect upon Golgi structure, breaking the ribbon into large fragments dispersed in the cytoplasm (Fig. 6 b). The extent of Golgi fragmentation correlated well with loss of golgin-84 over time (Fig. 6 c). This, combined with the lack of fragmentation induced by prolonged exposure to the control lamin A oligo, suggests that fragmentation is a consequence of golgin-84 depletion and not cellular toxicity resulting from the RNA interference (RNAi) treatment itself. Although predominantly punctate in appearance, the Golgi fragments occasionally resembled short tubules. Fragments resulting from golgin-84 depletion contained markers of the cis- (GM130), medial (GalNacT2; Fig. 6 b), and trans-Golgi (TGN46; unpublished data), suggesting that some degree of Golgi organization may be retained in these cells. The Golgi fragments did not extensively colocalize with the ER exit site marker mSec23p, suggesting that they arise directly from the break-up of the Golgi ribbon rather than the cycling through the ER (Fig. 6 b).

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