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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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Golgin-84 is localized to the cis-Golgi network. (a–e) A431 cells were processed for cryoelectron microscopy and labeled with polyclonal antibodies to golgin-84 (a–d) or GM130 (e) followed by secondary antibodies coupled to 10-nm gold. Arrows indicate ER budding profiles, and arrowheads indicate golgin-84 (a–d) or GM130 (e) labeling. (f–g) Quantitation of golgin-84 and GM130 labeling of cryosections. The relative number of gold particles over Golgi cisternae, tubulo-vesicular profiles lateral to the Golgi stack, tubulo-vesicular profiles adjacent to the cis face of the stack (central), or over other structures was quantitated in gluteraldehyde-fixed A431 cells (f) and paraformaldehyde-fixed HeLa cells (g) as described in the Materials and methods section. Note that golgin-84 labeling is present on the cis-most cisternae and more predominantly on tubulo-vesicular profiles lateral to the stack, whereas GM130 labeling is mainly on the cis-most cisternae of the stack (a–c). Bars, 200 nm.
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fig2: Golgin-84 is localized to the cis-Golgi network. (a–e) A431 cells were processed for cryoelectron microscopy and labeled with polyclonal antibodies to golgin-84 (a–d) or GM130 (e) followed by secondary antibodies coupled to 10-nm gold. Arrows indicate ER budding profiles, and arrowheads indicate golgin-84 (a–d) or GM130 (e) labeling. (f–g) Quantitation of golgin-84 and GM130 labeling of cryosections. The relative number of gold particles over Golgi cisternae, tubulo-vesicular profiles lateral to the Golgi stack, tubulo-vesicular profiles adjacent to the cis face of the stack (central), or over other structures was quantitated in gluteraldehyde-fixed A431 cells (f) and paraformaldehyde-fixed HeLa cells (g) as described in the Materials and methods section. Note that golgin-84 labeling is present on the cis-most cisternae and more predominantly on tubulo-vesicular profiles lateral to the stack, whereas GM130 labeling is mainly on the cis-most cisternae of the stack (a–c). Bars, 200 nm.

Mentions: Previous work has shown that golgin-84 is present on the Golgi apparatus (Bascom et al., 1999), but the localization of this protein at the ultrastructural level has not been addressed. To localize golgin-84 within the Golgi apparatus, cryosections of A431 cells were labeled with polyclonal antibodies to golgin-84 and examined under the electron microscope. Golgin-84 was found predominantly on membranes at the cis side of the Golgi stack (Fig. 2). Quantitation revealed that 34% of golgin-84 labeling was on cisternae whereas 66% of labeling was on tubulo-vesicular profiles (often referred to as the cis-Golgi network [CGN]) (Fig. 2 f). Of the tubulo-vesicular profile labeling, the vast majority was on membranes at the lateral edges of the stack rather then on membranes underlying the stacked cisternae (Fig. 2 f). Interestingly, golgin-84 labeling could frequently be detected on tubulo-reticular elements apparently connecting adjacent Golgi stacks (Fig. 2 d). A similar golgin-84 distribution to that observed in A431 cells was also detected in HeLa cells, suggesting that the localization of this protein is not cell type dependent (Fig. 2 g).


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 localized to the cis-Golgi network. (a–e) A431 cells were processed for cryoelectron microscopy and labeled with polyclonal antibodies to golgin-84 (a–d) or GM130 (e) followed by secondary antibodies coupled to 10-nm gold. Arrows indicate ER budding profiles, and arrowheads indicate golgin-84 (a–d) or GM130 (e) labeling. (f–g) Quantitation of golgin-84 and GM130 labeling of cryosections. The relative number of gold particles over Golgi cisternae, tubulo-vesicular profiles lateral to the Golgi stack, tubulo-vesicular profiles adjacent to the cis face of the stack (central), or over other structures was quantitated in gluteraldehyde-fixed A431 cells (f) and paraformaldehyde-fixed HeLa cells (g) as described in the Materials and methods section. Note that golgin-84 labeling is present on the cis-most cisternae and more predominantly on tubulo-vesicular profiles lateral to the stack, whereas GM130 labeling is mainly on the cis-most cisternae of the stack (a–c). Bars, 200 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172652&req=5

fig2: Golgin-84 is localized to the cis-Golgi network. (a–e) A431 cells were processed for cryoelectron microscopy and labeled with polyclonal antibodies to golgin-84 (a–d) or GM130 (e) followed by secondary antibodies coupled to 10-nm gold. Arrows indicate ER budding profiles, and arrowheads indicate golgin-84 (a–d) or GM130 (e) labeling. (f–g) Quantitation of golgin-84 and GM130 labeling of cryosections. The relative number of gold particles over Golgi cisternae, tubulo-vesicular profiles lateral to the Golgi stack, tubulo-vesicular profiles adjacent to the cis face of the stack (central), or over other structures was quantitated in gluteraldehyde-fixed A431 cells (f) and paraformaldehyde-fixed HeLa cells (g) as described in the Materials and methods section. Note that golgin-84 labeling is present on the cis-most cisternae and more predominantly on tubulo-vesicular profiles lateral to the stack, whereas GM130 labeling is mainly on the cis-most cisternae of the stack (a–c). Bars, 200 nm.
Mentions: Previous work has shown that golgin-84 is present on the Golgi apparatus (Bascom et al., 1999), but the localization of this protein at the ultrastructural level has not been addressed. To localize golgin-84 within the Golgi apparatus, cryosections of A431 cells were labeled with polyclonal antibodies to golgin-84 and examined under the electron microscope. Golgin-84 was found predominantly on membranes at the cis side of the Golgi stack (Fig. 2). Quantitation revealed that 34% of golgin-84 labeling was on cisternae whereas 66% of labeling was on tubulo-vesicular profiles (often referred to as the cis-Golgi network [CGN]) (Fig. 2 f). Of the tubulo-vesicular profile labeling, the vast majority was on membranes at the lateral edges of the stack rather then on membranes underlying the stacked cisternae (Fig. 2 f). Interestingly, golgin-84 labeling could frequently be detected on tubulo-reticular elements apparently connecting adjacent Golgi stacks (Fig. 2 d). A similar golgin-84 distribution to that observed in A431 cells was also detected in HeLa cells, suggesting that the localization of this protein is not cell type dependent (Fig. 2 g).

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