The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation

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.

Figure 1.

Identification of golgin-84 as a mitotic phosphoprotein. (a–c) Rat liver Golgi membranes were incubated with interphase or mitotic HeLa cytosol in the presence of [γ-³²P]ATP for 30 min at 30°C and reisolated by centrifugation. (a) Samples were subjected to 16-BAC/SDS-PAGE 2D electrophoresis and radiolabeled proteins were detected by autoradiography. Positions of the known mitotic phosphoproteins GM130, GRASP65, GRASP55, and rab1 are indicated. (b) The radiolabeled membranes were washed with sodium carbonate and the carbonate pellet was extracted with Triton X-114. Proteins partitioning into the aqueous phase were analyzed by 2D 16-BAC/SDS-PAGE and silver staining, followed by autoradiography. Arrows indicate the doublet of spots corresponding to golgin-84. The lower spot is a proteolytic cleavage product. (c) The radiolabeled membranes were solubilized in SDS and subjected to immunoprecipitation with antibodies to golgin-84. The membrane extracts and immunoprecipitates were analyzed by 1D SDS-PAGE followed by autoradiography. (d) Golgi membranes incubated with interphase or mitotic cytosol (in vitro) or total membrane fractions prepared from interphase and mitotic HeLa cells (in vivo) were analyzed by 1D SDS-PAGE and immunoblotting with antibodies to golgin-84.

Figure 2.

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.

Figure 3.

Golgin-84 is not part of the Golgi matrix. (a) Rat liver Golgi extracts were immunoprecipitated under native conditions with antibodies to either golgin-84, GM130, or p115, or with a control IgG. The immunoprecipitated (IP) and unbound fractions were analyzed by Western blotting with antibodies to golgin-84, GM130, p115, GRASP65, and mannosidase I as indicated. (b) NRK cells were incubated with 5 μg/ml BFA for 1 h and then fixed and double labeled with antibodies to golgin-84 and GM130 or the ER marker calnexin. Bar, 10 μm.

Figure 3.

Golgin-84 is not part of the Golgi matrix. (a) Rat liver Golgi extracts were immunoprecipitated under native conditions with antibodies to either golgin-84, GM130, or p115, or with a control IgG. The immunoprecipitated (IP) and unbound fractions were analyzed by Western blotting with antibodies to golgin-84, GM130, p115, GRASP65, and mannosidase I as indicated. (b) NRK cells were incubated with 5 μg/ml BFA for 1 h and then fixed and double labeled with antibodies to golgin-84 and GM130 or the ER marker calnexin. Bar, 10 μm.

Figure 4.

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.

Figure 5.

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.

Figure 6.

Depletion of golgin-84 using RNAi fragments the Golgi ribbon. (a) HeLa cells were either mock transfected (no RNAi) or transfected with duplex RNA to target lamin A or golgin-84 and, after 1–4 d, were subjected to Western blotting with antibodies to GM130 or golgin-84. (b) Mock transfected or RNAi-treated HeLa cells were fixed and double labeled with antibodies to golgin-84, lamin A, GalNacT2, mSec23p, and GM130. In the merged images on the right, DNA is blue, golgin-84, lamin A, GalNacT2, mSec23p are in green, and GM130 is red, with regions of overlap between these proteins indicated in yellow. Bar, 10 μm. (c) Quantitation of golgin-84 levels and Golgi fragmentation in RNAi-treated cells. Golgin-84 levels were measured by quantitating Western blots of RNAi-treated HeLa cells as described in the Materials and methods section. Golgi fragmentation was measured by immunofluorescence microscopy using antibodies to GM130 to assess Golgi morphology. For each time point, 200 cells were counted. The data are an average of two independent experiments.

Figure 7.

Golgin-84 depletion results in a significant loss of membrane from the Golgi apparatus and an exaggerated ER. Control (a) or golgin-84 RNAi–treated HeLa cells (b and c) were fixed and embedded in Epon for electron microscopy. Large arrows indicate Golgi membranes whereas small arrows indicate the ER. Note the decrease in size of the Golgi apparatus and the exaggerated and swollen ER in golgin-84–depleted cells. (d) Quantitation of the amount of membrane in Golgi cisternae, Golgi vesicles, ER, and nuclear envelope (NE) in golgin-84–depleted cells relative to control cells expressed as density of membrane surface in cell volume. For controls and RNAi samples, n = 23 and 22 micrographs, respectively. Bars, 200 nm.

Figure 8.

Depletion of golgin-84 partially inhibits transport of VSV-G from the ER to the cell surface. Control or golgin-84 RNAi–treated HeLa cells were transfected with a plasmid encoding GFP-tagged ts045G VSV-G protein. Cells were incubated at 39.5°C to arrest ts045G VSV-G in the ER, and then chased at 31°C for various times to allow transport before fixation and labeling for cell surface VSV-G. (a) An example of cells shifted to 31°C for 60 min and labeled for cell surface VSV-G. (b) The extent of VSV-G transport was measured as indicated in the Materials and methods and is expressed as the ratio of cell surface to total VSV-G fluorescence. The data shown are representative of two experiments with n = 15 for all data points in each experiment.