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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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Overexpression of golgin-84 fragments the Golgi ribbon. (a) Schematic representation of the structure of golgin-84 showing the constructs that were expressed in HeLa cells. Each construct was tagged at the NH2 terminus with GFP. The predicted coiled-coil region is shaded gray and the predicted transmembrane region is black. A summary of the localization and effects upon Golgi structure of each construct is shown on the right. (b) HeLa cells transfected with GFP-tagged WT (top), ΔHead (middle), and ΔHead + CC (bottom) golgin-84 constructs were fixed and stained with antibodies to GM130. In the merged images on the right, DNA is blue, GFP–golgin-84 is indicated in green, GM130 is red, and yellow indicates regions of overlap between GFP–golgin-84 and GM130. Bar, 10 μm. (c) HeLa cells expressing GFP-tagged WT golgin-84 were processed for cryoelectron microscopy and labeled with polyclonal antibodies to GFP followed by rabbit anti–sheep antibodies and protein A coupled to 8-nm gold. Bars, 100 nm.
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fig5: Overexpression of golgin-84 fragments the Golgi ribbon. (a) Schematic representation of the structure of golgin-84 showing the constructs that were expressed in HeLa cells. Each construct was tagged at the NH2 terminus with GFP. The predicted coiled-coil region is shaded gray and the predicted transmembrane region is black. A summary of the localization and effects upon Golgi structure of each construct is shown on the right. (b) HeLa cells transfected with GFP-tagged WT (top), ΔHead (middle), and ΔHead + CC (bottom) golgin-84 constructs were fixed and stained with antibodies to GM130. In the merged images on the right, DNA is blue, GFP–golgin-84 is indicated in green, GM130 is red, and yellow indicates regions of overlap between GFP–golgin-84 and GM130. Bar, 10 μm. (c) HeLa cells expressing GFP-tagged WT golgin-84 were processed for cryoelectron microscopy and labeled with polyclonal antibodies to GFP followed by rabbit anti–sheep antibodies and protein A coupled to 8-nm gold. Bars, 100 nm.

Mentions: The stiochiometric phosphorylation of golgin-84 in mitosis together with its binding to active rab1 suggested that it may play a role in Golgi structure and/or membrane trafficking through the Golgi apparatus. To address a possible structural role for golgin-84, GFP-tagged full-length and truncated versions of the protein were expressed in HeLa cells, and effects upon Golgi structure were analyzed by immunofluorescence microscopy (Fig. 5 a). At moderate expression levels, none of the golgin-84 constructs elicited any significant change in Golgi structure (unpublished data). Furthermore, constructs that failed to target to the Golgi apparatus had no effects upon Golgi structure even at very high levels of expression (Fig. 5 a; unpublished data). In contrast, expression at high levels of both full-length and golgin-84 lacking the head region had dramatic effects upon Golgi structure, converting the ribbon into punctate structures dispersed throughout the cytoplasm (Fig. 5 b). EM analysis of the Golgi fragments in cells overexpressing full-length golgin-84 revealed that they are similar in overall organization to the Golgi apparatus in control cells, comprising three to four stacked cisternae and vesiculo-tubular profiles that label heavily for the overexpressed protein (Fig. 5 c). Thus, overexpression of golgin-84 does not lead to vesiculation of Golgi stacks, just break-up of the ribbon. Fragmentation was not due to the presence of GFP at the NH2 terminus because identical results were obtained with golgin-84 constructs containing an NH2-terminal myc tag instead of GFP (unpublished data). Fragmentation appeared more extensive with the construct lacking the head region, suggesting that the head may play some role in regulating Golgi structure. Interestingly, golgin-84 lacking most of the coiled-coil region in addition to the head domain had only a minor effect upon Golgi structure, even at extremely high expression levels. Together, the results suggest that the coiled-coil domain is important for the fragmentation observed, and that it is only able to induce fragmentation when properly targeted to the Golgi membranes.


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)

Overexpression of golgin-84 fragments the Golgi ribbon. (a) Schematic representation of the structure of golgin-84 showing the constructs that were expressed in HeLa cells. Each construct was tagged at the NH2 terminus with GFP. The predicted coiled-coil region is shaded gray and the predicted transmembrane region is black. A summary of the localization and effects upon Golgi structure of each construct is shown on the right. (b) HeLa cells transfected with GFP-tagged WT (top), ΔHead (middle), and ΔHead + CC (bottom) golgin-84 constructs were fixed and stained with antibodies to GM130. In the merged images on the right, DNA is blue, GFP–golgin-84 is indicated in green, GM130 is red, and yellow indicates regions of overlap between GFP–golgin-84 and GM130. Bar, 10 μm. (c) HeLa cells expressing GFP-tagged WT golgin-84 were processed for cryoelectron microscopy and labeled with polyclonal antibodies to GFP followed by rabbit anti–sheep antibodies and protein A coupled to 8-nm gold. Bars, 100 nm.
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Related In: Results  -  Collection

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fig5: Overexpression of golgin-84 fragments the Golgi ribbon. (a) Schematic representation of the structure of golgin-84 showing the constructs that were expressed in HeLa cells. Each construct was tagged at the NH2 terminus with GFP. The predicted coiled-coil region is shaded gray and the predicted transmembrane region is black. A summary of the localization and effects upon Golgi structure of each construct is shown on the right. (b) HeLa cells transfected with GFP-tagged WT (top), ΔHead (middle), and ΔHead + CC (bottom) golgin-84 constructs were fixed and stained with antibodies to GM130. In the merged images on the right, DNA is blue, GFP–golgin-84 is indicated in green, GM130 is red, and yellow indicates regions of overlap between GFP–golgin-84 and GM130. Bar, 10 μm. (c) HeLa cells expressing GFP-tagged WT golgin-84 were processed for cryoelectron microscopy and labeled with polyclonal antibodies to GFP followed by rabbit anti–sheep antibodies and protein A coupled to 8-nm gold. Bars, 100 nm.
Mentions: The stiochiometric phosphorylation of golgin-84 in mitosis together with its binding to active rab1 suggested that it may play a role in Golgi structure and/or membrane trafficking through the Golgi apparatus. To address a possible structural role for golgin-84, GFP-tagged full-length and truncated versions of the protein were expressed in HeLa cells, and effects upon Golgi structure were analyzed by immunofluorescence microscopy (Fig. 5 a). At moderate expression levels, none of the golgin-84 constructs elicited any significant change in Golgi structure (unpublished data). Furthermore, constructs that failed to target to the Golgi apparatus had no effects upon Golgi structure even at very high levels of expression (Fig. 5 a; unpublished data). In contrast, expression at high levels of both full-length and golgin-84 lacking the head region had dramatic effects upon Golgi structure, converting the ribbon into punctate structures dispersed throughout the cytoplasm (Fig. 5 b). EM analysis of the Golgi fragments in cells overexpressing full-length golgin-84 revealed that they are similar in overall organization to the Golgi apparatus in control cells, comprising three to four stacked cisternae and vesiculo-tubular profiles that label heavily for the overexpressed protein (Fig. 5 c). Thus, overexpression of golgin-84 does not lead to vesiculation of Golgi stacks, just break-up of the ribbon. Fragmentation was not due to the presence of GFP at the NH2 terminus because identical results were obtained with golgin-84 constructs containing an NH2-terminal myc tag instead of GFP (unpublished data). Fragmentation appeared more extensive with the construct lacking the head region, suggesting that the head may play some role in regulating Golgi structure. Interestingly, golgin-84 lacking most of the coiled-coil region in addition to the head domain had only a minor effect upon Golgi structure, even at extremely high expression levels. Together, the results suggest that the coiled-coil domain is important for the fragmentation observed, and that it is only able to induce fragmentation when properly targeted to the Golgi membranes.

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