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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 a specific binding partner for rab1. (a) GST-tagged rab1, rab2, and rab6 were loaded with GDP or GTPγS and incubated with Golgi extract, and specifically eluted proteins were analyzed by Western blotting with antibodies to GM130, golgin-45, and golgin-84. (b) Full-length GM130 and golgin-84 lacking the transmembrane domain were tested for interaction in the yeast two-hybrid system with the following rab proteins carrying activating point mutations: rab1Q70L, rab2Q65L, rab5Q79L, rab6Q72L, and rab33bQ92L. Interactions between the indicated proteins results in growth on high selection medium. (c) Full-length and truncation mutants of golgin-84 were tested for interaction with rab1Q70L in the yeast two-hybrid system. The CT mutant corresponds to the ΔHead + CC construct described in Fig. 5 without the membrane anchor.
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fig4: Golgin-84 is a specific binding partner for rab1. (a) GST-tagged rab1, rab2, and rab6 were loaded with GDP or GTPγS and incubated with Golgi extract, and specifically eluted proteins were analyzed by Western blotting with antibodies to GM130, golgin-45, and golgin-84. (b) Full-length GM130 and golgin-84 lacking the transmembrane domain were tested for interaction in the yeast two-hybrid system with the following rab proteins carrying activating point mutations: rab1Q70L, rab2Q65L, rab5Q79L, rab6Q72L, and rab33bQ92L. Interactions between the indicated proteins results in growth on high selection medium. (c) Full-length and truncation mutants of golgin-84 were tested for interaction with rab1Q70L in the yeast two-hybrid system. The CT mutant corresponds to the ΔHead + CC construct described in Fig. 5 without the membrane anchor.

Mentions: Several Golgi-associated coiled-coil proteins interact with the active or GTP-bound forms of the rab family of small GTPases involved in membrane traffic. Specifically, p115, a cis-Golgi vesicle tethering protein, binds to active rab1, and GM130 binds to active rab1 and less efficiently to active rab2, whereas golgin-45, a medial Golgi protein, binds only to active rab2 (Allan et al., 2000; Moyer et al., 2001; Short et al., 2001; Weide et al., 2001). Because golgin-84 shares similarities with these coiled-coil rab-binding proteins, we analyzed whether this protein may itself be a novel rab effector. Golgi extract was incubated with immobilized rab proteins in the inactive (GDP) or active (GTPγS) conformation and bound proteins were analyzed by immunoblotting. As expected, GM130 bound specifically to active rab1, but we could not detect any binding to rab2 (Fig. 4 a). Rab2 did however bind to golgin-45, as previously reported (Short et al., 2001), demonstrating the functionality of this protein in the binding assay (Fig. 4 a). Interestingly, golgin-84 bound specifically to active rab1, with no detectable binding to either rab2 or rab6. To confirm the interaction with rab1, the yeast two-hybrid system was employed. As previously shown, GM130 interacted directly with activated mutants of rab1, rab2, and rab33b (Short et al., 2001; Valsdottir et al., 2001; Weide et al., 2001) (Fig. 4 b). In contrast, golgin-84 interacted only with activated rab1, and not with any of the other rab proteins tested. Therefore, golgin-84 is a specific binding partner for active rab1. The rab1 binding site was mapped using the two-hybrid system to the coiled-coil region of golgin-84 (Fig. 4 c).


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 a specific binding partner for rab1. (a) GST-tagged rab1, rab2, and rab6 were loaded with GDP or GTPγS and incubated with Golgi extract, and specifically eluted proteins were analyzed by Western blotting with antibodies to GM130, golgin-45, and golgin-84. (b) Full-length GM130 and golgin-84 lacking the transmembrane domain were tested for interaction in the yeast two-hybrid system with the following rab proteins carrying activating point mutations: rab1Q70L, rab2Q65L, rab5Q79L, rab6Q72L, and rab33bQ92L. Interactions between the indicated proteins results in growth on high selection medium. (c) Full-length and truncation mutants of golgin-84 were tested for interaction with rab1Q70L in the yeast two-hybrid system. The CT mutant corresponds to the ΔHead + CC construct described in Fig. 5 without the membrane anchor.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172652&req=5

fig4: Golgin-84 is a specific binding partner for rab1. (a) GST-tagged rab1, rab2, and rab6 were loaded with GDP or GTPγS and incubated with Golgi extract, and specifically eluted proteins were analyzed by Western blotting with antibodies to GM130, golgin-45, and golgin-84. (b) Full-length GM130 and golgin-84 lacking the transmembrane domain were tested for interaction in the yeast two-hybrid system with the following rab proteins carrying activating point mutations: rab1Q70L, rab2Q65L, rab5Q79L, rab6Q72L, and rab33bQ92L. Interactions between the indicated proteins results in growth on high selection medium. (c) Full-length and truncation mutants of golgin-84 were tested for interaction with rab1Q70L in the yeast two-hybrid system. The CT mutant corresponds to the ΔHead + CC construct described in Fig. 5 without the membrane anchor.
Mentions: Several Golgi-associated coiled-coil proteins interact with the active or GTP-bound forms of the rab family of small GTPases involved in membrane traffic. Specifically, p115, a cis-Golgi vesicle tethering protein, binds to active rab1, and GM130 binds to active rab1 and less efficiently to active rab2, whereas golgin-45, a medial Golgi protein, binds only to active rab2 (Allan et al., 2000; Moyer et al., 2001; Short et al., 2001; Weide et al., 2001). Because golgin-84 shares similarities with these coiled-coil rab-binding proteins, we analyzed whether this protein may itself be a novel rab effector. Golgi extract was incubated with immobilized rab proteins in the inactive (GDP) or active (GTPγS) conformation and bound proteins were analyzed by immunoblotting. As expected, GM130 bound specifically to active rab1, but we could not detect any binding to rab2 (Fig. 4 a). Rab2 did however bind to golgin-45, as previously reported (Short et al., 2001), demonstrating the functionality of this protein in the binding assay (Fig. 4 a). Interestingly, golgin-84 bound specifically to active rab1, with no detectable binding to either rab2 or rab6. To confirm the interaction with rab1, the yeast two-hybrid system was employed. As previously shown, GM130 interacted directly with activated mutants of rab1, rab2, and rab33b (Short et al., 2001; Valsdottir et al., 2001; Weide et al., 2001) (Fig. 4 b). In contrast, golgin-84 interacted only with activated rab1, and not with any of the other rab proteins tested. Therefore, golgin-84 is a specific binding partner for active rab1. The rab1 binding site was mapped using the two-hybrid system to the coiled-coil region of golgin-84 (Fig. 4 c).

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