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

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.

Figure 1.

Golgin-45 is a novel GRASP55 interacting protein. (A) Amino acids 12–404 of golgin-45 were identified using the yeast two- hybrid system by screening GRASP55 against a human testis cDNA library. Golgin-45 and a COOH-terminal deletion mutant were tested for interaction with GRASP55 and GRASP65 in the yeast two-hybrid system compared with GM130 and empty vector as positive and negative controls, respectively. (B) Coimmunoprecipitations of in vitro–translated GRASP55 and GRASP65 with golgin-45 and GM130. (C) GRASP55 complexes were affinity purified from detergent extracts of Golgi membranes. Specifically, interacting proteins were excised (arrowheads) and tryptic digests of the proteins contained therein analyzed by mass spectrometry. (D) Golgi membranes (10 μg) were Western blotted with antibodies to golgin-45 in the presence (+) or absence (−) of 10 μg/ml of recombinant golgin-45. Control blots are also shown for the antibodies to GRASP65, GRASP55, and GM130. (E) Immunoprecipitations (IPs) were performed from 20 μg Golgi membranes with the following antibodies: sheep anti-GRASP55, mouse anti-GRASP65, sheep anti-GM130, and rabbit anti–golgin-45. Immunoprecipitates were blotted with the following antibodies: GRASP55 and GRASP65 IPs with rabbit anti-GM130 and rabbit anti–golgin-45, GM130 IPs with rabbit anti-GRASP55 and mouse anti-GRASP65, and golgin-45 IPs with sheep anti-GRASP55 and mouse anti-GRASP65. Asterisks indicate a nonspecific cross-reactivity of the rabbit anti-GRASP55 antibody to the sheep antibody used for the IP.

Figure 2.

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 (+++).

Figure 3.

Golgin-45 behaves like a Golgi matrix protein upon BFA treatment. (A) Normal rat kidney (NRK) cells were fixed and then stained with antibodies to golgin-45 and GRASP55. In the merged images, DNA is blue, golgin-45 is indicated in red, GRASP55 in green, with yellow indicating areas of golgin-45 and GRASP55 overlap. (B) NRK cells were treated with 5 μg/ml BFA or 200 ng/ml nocodazole for 30 and 60 min, respectively, before fixation and antibody staining. (C) NRK cells were transfected with GFP–golgin-45 for 12 h and then fixed and stained with antibodies to GRASP55. Bar, 10 μm.

Figure 4.

Depletion of golgin-45 disrupts the Golgi apparatus. (A) HeLa cells were treated with duplex RNA to target golgin-45 for 36 h and then transfected for a further 18 h with a plasmid encoding GFP-tagged n-acetylglucosaminyltransferase-I before fixation and staining with antibodies to golgin-45. (B and C) HeLa cells were treated with RNA duplexes to target golgin-45 for 48 h before fixation and staining with antibodies to golgin-45, GRASP55, and GM130. Bars, 10 μm.

Figure 5.

Depletion of golgin-45 disrupts transport of VSV-G protein from the ER to the cell surface. Control (A) or golgin-45 (B) RNAi cells were transfected with a plasmid encoding GFP-tagged VSV-G ts045 protein. VSV-G ts045was arrested in the ER at 39.5°C and then chased out at 31.5°C for 30 and 60 min. Images are shown of cells fixed after 0, 30, and 60 min of chase for both total and cell surface–associated VSV-G. (C) The extent of VSV-G transport after 0, 30, and 60 min chase at 31.5°C was measured in control and golgin-45–depleted cells. This ratio does not approach unity due to the different dyes used to measure surface and total fluorescence. The data shown is representative of three experiments with n = 20 for all data points in each experiment. Live cell videos showing VSV-G ts045 transport in control and golgin-45 depleted cells are available at http://www.jcb.org/cgi/content/full/jcb.200108079/DC1. Bars, 20 μm.