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A GRASP55-rab2 effector complex linking Golgi structure to membrane traffic.

Short B, Preisinger C, Körner R, Kopajtich R, Byron O, Barr FA - J. Cell Biol. (2001)

Bottom Line: Here, we find that a novel coiled-coil protein golgin-45 interacts with the medial-Golgi matrix protein GRASP55 and the GTP form of rab2 but not other Golgi rab proteins.Depletion of golgin-45 disrupts the Golgi apparatus and causes a block in secretory protein transport.These results demonstrate that GRASP55 and golgin-45 form a rab2 effector complex on medial-Golgi essential for normal protein transport and Golgi structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Max-Planck-Institute of Biochemistry, Martinsried D-82152, Germany.

ABSTRACT
Membrane traffic between the endoplasmic reticulum (ER) and Golgi apparatus and through the Golgi apparatus is a highly regulated process controlled by members of the rab GTPase family. The GTP form of rab1 regulates ER to Golgi transport by interaction with the vesicle tethering factor p115 and the cis-Golgi matrix protein GM130, also part of a complex with GRASP65 important for the organization of cis-Golgi cisternae. Here, we find that a novel coiled-coil protein golgin-45 interacts with the medial-Golgi matrix protein GRASP55 and the GTP form of rab2 but not other Golgi rab proteins. Depletion of golgin-45 disrupts the Golgi apparatus and causes a block in secretory protein transport. These results demonstrate that GRASP55 and golgin-45 form a rab2 effector complex on medial-Golgi essential for normal protein transport and Golgi structure.

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Golgin-45 is a specific binding partner for activated rab2, whereas GM130 binds both rab1 and 2. (A) Rab1, 2, and 6 beads loaded with either GDP or GTPγS were incubated with Golgi extract and the specifically eluted proteins analyzed by Western blotting. Note that golgin-45 is depleted from the supernatant of rab2-GTP beads. (B) Full-length golgin-45 and p115 were tested for interaction with wild-type rab2 and the following rab proteins carrying activating point mutations rab1Q70L, rab2Q65L, rab5Q79L, and rab6Q72L. (C) Full-length and truncation mutants of golgin-45 were tested for interaction with rab2Q65L and GRASP55 in the yeast two-hybrid system. Interaction was scored by assessing growth on QDO plates, from none (−) to strong (+++).
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fig2: Golgin-45 is a specific binding partner for activated rab2, whereas GM130 binds both rab1 and 2. (A) Rab1, 2, and 6 beads loaded with either GDP or GTPγS were incubated with Golgi extract and the specifically eluted proteins analyzed by Western blotting. Note that golgin-45 is depleted from the supernatant of rab2-GTP beads. (B) Full-length golgin-45 and p115 were tested for interaction with wild-type rab2 and the following rab proteins carrying activating point mutations rab1Q70L, rab2Q65L, rab5Q79L, and rab6Q72L. (C) Full-length and truncation mutants of golgin-45 were tested for interaction with rab2Q65L and GRASP55 in the yeast two-hybrid system. Interaction was scored by assessing growth on QDO plates, from none (−) to strong (+++).

Mentions: Several Golgi-associated coiled-coil proteins have been shown to interact with the activated or GTP forms of the rab family of GTPases involved in membrane traffic. In particular, both p115 and GM130 bind to the GTP form of the small GTPase rab1 (Allan et al., 2000; Moyer et al., 2001; Weide et al., 2001). Since golgin-45 shares several properties with GM130, we tested if it interacted with Golgi-localized rab GTPases. Golgin-45 from Golgi membranes bound quantitatively and specifically to the GTP form of rab2 and showed negligible binding to other inactive or activated rabs (Fig. 2 A). GRASP55 was also specifically eluted from rab2-GTP beads consistent with its interaction with golgin-45. Intriguingly both GM130 and p115 bound to both activated rab1 and rab2 but not the inactive forms of these rabs or any form of rab6 (Fig. 2 A). The yeast two-hybrid system was then used to screen for interactions between golgin-45, GM130, or p115 and the Golgi rabs 1, 2, and 6 with endosomal rab5 as a negative control. Golgin-45 interacted strongly with the rab2 activated mutant but only weakly with wild-type rab2 (Fig. 2 B). None of the other rab proteins tested showed an interaction with golgin-45. The ability to detect the known interactions between an activated form of rab1 and p115 or GM130 (Fig. 2 B) demonstrates the validity of this approach for studying specific rab-effector protein interactions. GM130 was also able to interact strongly with the activated mutant of rab2 and much less with the wild-type protein (Fig. 2 B), confirming the results of the rab2 binding assays from Golgi membranes. These findings are consistent with direct interaction between the active form of rab2 and golgin-45, rab1 and p115, rab 1 or rab2, and GM130. The presence of p115 in rab2-GTP binding assays is probably due to its binding to GM130, since it interacts with rab1 but not with rab2 in the two-hybrid system. The rab2-binding site in golgin-45 lies in the predicted coiled-coil domain, demonstrating the functional significance of the weak homology to other golgins in this region (Fig. 2 C).


A GRASP55-rab2 effector complex linking Golgi structure to membrane traffic.

Short B, Preisinger C, Körner R, Kopajtich R, Byron O, Barr FA - J. Cell Biol. (2001)

Golgin-45 is a specific binding partner for activated rab2, whereas GM130 binds both rab1 and 2. (A) Rab1, 2, and 6 beads loaded with either GDP or GTPγS were incubated with Golgi extract and the specifically eluted proteins analyzed by Western blotting. Note that golgin-45 is depleted from the supernatant of rab2-GTP beads. (B) Full-length golgin-45 and p115 were tested for interaction with wild-type rab2 and the following rab proteins carrying activating point mutations rab1Q70L, rab2Q65L, rab5Q79L, and rab6Q72L. (C) Full-length and truncation mutants of golgin-45 were tested for interaction with rab2Q65L and GRASP55 in the yeast two-hybrid system. Interaction was scored by assessing growth on QDO plates, from none (−) to strong (+++).
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Related In: Results  -  Collection

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fig2: Golgin-45 is a specific binding partner for activated rab2, whereas GM130 binds both rab1 and 2. (A) Rab1, 2, and 6 beads loaded with either GDP or GTPγS were incubated with Golgi extract and the specifically eluted proteins analyzed by Western blotting. Note that golgin-45 is depleted from the supernatant of rab2-GTP beads. (B) Full-length golgin-45 and p115 were tested for interaction with wild-type rab2 and the following rab proteins carrying activating point mutations rab1Q70L, rab2Q65L, rab5Q79L, and rab6Q72L. (C) Full-length and truncation mutants of golgin-45 were tested for interaction with rab2Q65L and GRASP55 in the yeast two-hybrid system. Interaction was scored by assessing growth on QDO plates, from none (−) to strong (+++).
Mentions: Several Golgi-associated coiled-coil proteins have been shown to interact with the activated or GTP forms of the rab family of GTPases involved in membrane traffic. In particular, both p115 and GM130 bind to the GTP form of the small GTPase rab1 (Allan et al., 2000; Moyer et al., 2001; Weide et al., 2001). Since golgin-45 shares several properties with GM130, we tested if it interacted with Golgi-localized rab GTPases. Golgin-45 from Golgi membranes bound quantitatively and specifically to the GTP form of rab2 and showed negligible binding to other inactive or activated rabs (Fig. 2 A). GRASP55 was also specifically eluted from rab2-GTP beads consistent with its interaction with golgin-45. Intriguingly both GM130 and p115 bound to both activated rab1 and rab2 but not the inactive forms of these rabs or any form of rab6 (Fig. 2 A). The yeast two-hybrid system was then used to screen for interactions between golgin-45, GM130, or p115 and the Golgi rabs 1, 2, and 6 with endosomal rab5 as a negative control. Golgin-45 interacted strongly with the rab2 activated mutant but only weakly with wild-type rab2 (Fig. 2 B). None of the other rab proteins tested showed an interaction with golgin-45. The ability to detect the known interactions between an activated form of rab1 and p115 or GM130 (Fig. 2 B) demonstrates the validity of this approach for studying specific rab-effector protein interactions. GM130 was also able to interact strongly with the activated mutant of rab2 and much less with the wild-type protein (Fig. 2 B), confirming the results of the rab2 binding assays from Golgi membranes. These findings are consistent with direct interaction between the active form of rab2 and golgin-45, rab1 and p115, rab 1 or rab2, and GM130. The presence of p115 in rab2-GTP binding assays is probably due to its binding to GM130, since it interacts with rab1 but not with rab2 in the two-hybrid system. The rab2-binding site in golgin-45 lies in the predicted coiled-coil domain, demonstrating the functional significance of the weak homology to other golgins in this region (Fig. 2 C).

Bottom Line: Here, we find that a novel coiled-coil protein golgin-45 interacts with the medial-Golgi matrix protein GRASP55 and the GTP form of rab2 but not other Golgi rab proteins.Depletion of golgin-45 disrupts the Golgi apparatus and causes a block in secretory protein transport.These results demonstrate that GRASP55 and golgin-45 form a rab2 effector complex on medial-Golgi essential for normal protein transport and Golgi structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Max-Planck-Institute of Biochemistry, Martinsried D-82152, Germany.

ABSTRACT
Membrane traffic between the endoplasmic reticulum (ER) and Golgi apparatus and through the Golgi apparatus is a highly regulated process controlled by members of the rab GTPase family. The GTP form of rab1 regulates ER to Golgi transport by interaction with the vesicle tethering factor p115 and the cis-Golgi matrix protein GM130, also part of a complex with GRASP65 important for the organization of cis-Golgi cisternae. Here, we find that a novel coiled-coil protein golgin-45 interacts with the medial-Golgi matrix protein GRASP55 and the GTP form of rab2 but not other Golgi rab proteins. Depletion of golgin-45 disrupts the Golgi apparatus and causes a block in secretory protein transport. These results demonstrate that GRASP55 and golgin-45 form a rab2 effector complex on medial-Golgi essential for normal protein transport and Golgi structure.

Show MeSH
Related in: MedlinePlus