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First published online 2 November 2004
doi: 10.1242/jcs.01497

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Mammalian GRIP domain proteins differ in their membrane binding properties and are recruited to distinct domains of the TGN

¹ The Russell Grimwade School of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia
² Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia
³ School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia
⁴ Laboratory of Membrane Biology, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Rep. of Singapore

View larger version (78K): [in a new window]   Fig. 1. Expression of full-length GCC185 results in dispersal of GCC185-labelled structures. (A) COS cells were transfected with myc-GCC185, fixed, permeabilized and stained with anti-myc monoclonal antibodies and FITC-anti-mouse Ig. Shown are three images expressing different levels of GCC185. (B) COS cells were co-transfected with myc-GCC185 and GFP-GCC185GRIP constructs, fixed, permeabilized and stained with anti-myc monoclonal antibody and Alexa 568 anti-mouse IgG. Superimposed images (merge) reveal regions of colocalization (arrows). (C) COS cells were co-transfected with myc-GCC185 and GFP-golgin97GRIP constructs, fixed, permeabilized and stained with anti-myc monoclonal antibody and Alexa 568 anti-mouse IgG. (D) COS cells were co-transfected with constructs encoding GFP-GCC185GRIP and myc-GCC185. Total extracts were prepared after 48 hours transfection as described in Materials and Methods, and lysates were either immunoprecipitated (IP) with anti-GFP antibody (GFP), anti-myc monoclonal antibody (Myc) or with an irrelevant monoclonal antibody (Control). Immune complexes were collected and subjected to SDS-PAGE under reducing conditions. After transfer to PVDF membranes, membranes were immunoblotted with anti-GFP antibody or anti-myc antibody as indicated using a chemiluminescence detection system. Bars: 10 µm.

View larger version (89K): [in a new window]   Fig. 3. Distribution of Golgi markers in HeLa cells expressing high levels of GCC185. HeLa cells were transiently transfected as indicated, with either GFP-GCC185 or myc-GCC185, fixed, permeabilized and co-stained for GM130, TGN46 or ß-COP. GM130 was detected with mouse monoclonal antibody and Alexa 568 goat anti-mouse IgG, TGN46 with sheep anti-TGN46 and FITC-donkey anti-sheep Ig, ß-COP with rabbit anti-ß-COP and Alexa 568 goatanti-rabbit IgG. Images of transfected cells expressing high levels of GCC185 and also superimposed images (merge) are shown. Control incubations demonstrated no cross-reactivity between the anti-Ig conjugates and the irrelevant primary antibody. Bars, 10 µm.

View larger version (175K): [in a new window]   Fig. 2. Overexpression of GCC185 leads to the formation of GCC185 labelled tubular structures throughout the cytoplasm. COS cells were transfected with myc-GCC185, fixed with glutaraldehyde and processed for cryoelectron microscopy. Ultrathin cryosections were labelled with monoclonal antibodies to myc. Antibodies were detected with 10 nm protein A gold particles (c-myc 10 nm). Note the labelling on tubulo-vesicular structures (arrows) scattered throughout the cytoplasm that get close to the plasma membrane (B). G, Golgi; mc, mitochondria; pm, plasma membrane. Bars, 200 nm.

View larger version (59K): [in a new window]   Fig. 4. GCC185 is recruited to structures distinct from the other three TGN golgins. (A) HeLa cells were transiently transfected as indicated, with either GFP-GCC185 or myc-GCC185, fixed, permeabilized and co-stained for endogenous golgin-97, p230 or GCC88. Golgin-97 was detected with a mouse monoclonal antibody and Alexa 568 goat anti-mouse IgG, p230 detected with human anti-p230 antibodies and Alexa 594 goat anti-human IgG and GCC88 detected with rabbit anti-GCC88 antibodies followed by Alexa 594 goat anti-rabbit IgG. (B) COS cells were transfected with myc-GCC185 and untagged GCC88, fixed, permeablized and stained with rabbit anti-GCC88 followed by Alexa 568 goat anti-rabbit IgG, and anti-myc monoclonal antibody followed by FITC goat anti-mouse Ig. Shown are images of transfected cells expressing high levels of both myc-GCC185 and GCC88. Bar, 10 µm.

View larger version (88K): [in a new window]   Fig. 5. The TGN resident membrane protein, sialyltransferase, segregates into GCC185-labelled structures. HeLa cells, stably expressing SialylT fused to the VSV-G epitope, were transiently transfected with (A) GFP-GCC185 or (B) GFP-GCC88, fixed, permeabilized and stained for VSV-G epitope tagged SialylT with mouse monoclonal antibodies and Alexa 568 anti-mouse IgG. Superimposed images (merge) are shown. Arrrows in A indicate structures in the periphery of the cell, labelled with both GFP-GCC185 and SialylT. Bar, 10 µm.

View larger version (51K): [in a new window]   Fig. 6. Differential displacement of endogenous GRIP proteins by expression of GFP-GRIP fusion proteins. (A,B) Displacement of endogenous GRIP proteins by GFP-p230GRIP or GFP-golgin-97GRIP. COS cells were transfected with (A) GFP-p230GRIP or (B) GFP-golgin-97GRIP, fixed and stained for each of the endogenous golgins using mouse and rabbit antibodies and detected with TRITC conjugated secondary antibodies as indicated. (C,D) Localization of endogenous p230 (C) and endogenous GCC185 and GCC88 (D) in cells overexpressing GFP-GCC88GRIP and GPF-GCC185GRIP. COS cells were transfected with constructs encoding either GFP-GCC88GRIP or GFP-GCC185GRIP fusion proteins, as indicated. Cells were fixed and stained with human anti-p230 followed by anti-human-Ig-TRITC in C, and rabbit anti-GCC88 or rabbit GCC185 followed by Alexa 568 goat anti-rabbit IgG in D. Arrows indicate the Golgi region in cells expressing GFP-GRIP fusion proteins in paired images of the same field. Green and red images were collected separately. Bars, 20 µm.

View larger version (85K): [in a new window]   Fig. 7. Targeting of Arl1(Q71L) to endosomes results in recruitment of p230, but not GCC88 or GCC185 to endosomal membranes. HeLa cells were transiently transfected with either (A) GFP-Arl1(WT) or (B) myc-SNX3-Arl1(Q71L), fixed and permeabilized. (A) Transfected cells were stained for p230 with human anti-p230 antibodies followed by Alexa 594 goat anti-human IgG. (B) Transfected cells were co-stained for myc-SNX3-Arl1(Q71L) and p230 and either GCC88 or GCC185, as indicated. myc-SNX3-Arl1(Q71L) was detected with mouse anti-myc monoclonal antibodies and Alexa 568 goat anti-mouse IgG, p230 detected with human anti-p230 antibodies and FITC-sheep anti-human IgG, and GCC88 and GCC185 detected with rabbit anti-GCC88 or anti-GCC185 antibodies followed by Cy5-labelled goat anti-rabbit IgG. Transfected cells expressing high levels of myc-SNX3-Arl1(Q71L) resulted in recruitment of endogenous p230 onto endosomal structures, whereas both GCC88 and GCC185 retained their normal Golgi localization. Bars, 10 µm.

View larger version (63K): [in a new window]   Fig. 8. Invariant tyrosine of the GRIP domain is not required for Golgi targeting of GCC185GRIP. (A) DNA sequence profile of GFP-GCC185GRIP with the TAC coding for the invariant tyrosine highlighted, and an image of COS cells transfected with GFP-GCC185GRIP. (B) DNA sequence profile of GFP-GCC185GRIP (Y4A) showing the GCA substitution (boxed), with an image of COS cells transfected with GFP-GCC185GRIP (Y4A). Bars, 20 µm.

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