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First published online 16 September 2003
doi: 10.1242/jcs.00746

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A role for GRIP domain proteins and/or their ligands in structure and function of the trans Golgi network

¹ Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
² Integrated Imaging Center, Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA

View larger version (61K): [in a new window]   Fig. 1. Saturation of tGolgin-1 C312 localization in HeLa cells. HeLa cells transiently transfected with plasmid expressing HA-tagged C312 were analyzed 2 days later by IFM with anti-HA and RRX-conjugated secondary antibodies. (a-c) Examples of cells with different expression levels of C312 classified as levels I, II and III as indicated. (d) Comparison of semiquantitative total cell fluorescence levels with phenotypic classification. Total fluorescence from individual cells (in arbitrary units) was measured using OpenLab software as indicated in Materials and methods, and plotted on the y axis. Each dot represents the measurement from a single cell; the bar represents the median expression level from all cells.

View larger version (81K): [in a new window]   Fig. 3. Parallel displacement of TGN46 and endogenous GRIP domain proteins at similar expression levels of C312. (a-j) HeLa cells that were transiently transfected with C312 were analyzed by three-color IFM using rat anti-HA, mouse anti-tGolgin-1 and sheep anti-TGN46 with AMCA-, RRX- and FITC-conjugated species-specific antibodies, respectively. (a-i) Representative images of staining patterns for HA-C312 (a,d,g), TGN46 (b,e,h) and tGolgin-1 (c,f,i) obtained with C312 expressed at level I (a-c), II (d-f) and III (g-i). (j) Semiquantitative analyses of total cell expression level of AMCA fluorescence (representing C312) in cells characterized as having a tight pericentriolar Golgi staining pattern (intact), diffuse paranuclear staining (diffuse), or diffuse cytoplasmic distribution (cytoplasmic) for TGN46 and endogenous tGolgin-1. C312 expression is plotted in arbitrary units on a log scale on the y axis; circles represent values for individual cells, and bars represent the median of all analyzed cells. (k,l) Cells transiently transfected with C312 were analyzed by IFM using antibodies to HA (k) and golgin-97 (l). (m,n) Cells transiently transfected with g97-C179 were analyzed by IFM using antibodies to HA (m) and to TGN46 (n).

View larger version (112K): [in a new window]   Fig. 2. Overexpression of C312 disrupts the localization of TGN resident proteins. HeLa cells (a-f,m-o) or stable transfectants of TRVb-1 cells expressing TGG (g-i) or TTF (j-l) were transiently transfected with C312 or C312(Y2187A), as indicated, and analyzed by IFM using antibodies to the indicated proteins and to the HA tag (' columns) and appropriate secondary RRX- and FITC-conjugated secondary antibodies. The cells in d-f were cotransfected with furin-HA (50-100 ng/six-well dish) and T7-epitope-tagged C312 (5-7 µg/six-well dish); anti-HA was used to detect furin and anti-T7 to detect C312. (a,d,g,j,m) Cells with low C312 expression (level I); (b,e,h,k,n) cells with high expression (level III);(c,f,i,l,o) selected cells in which semiquantitative analyses showed levels of HA staining comparable to those in b, e, h, k, and n.

View larger version (24K): [in a new window]   Fig. 4. Reversibility of TGN46 displacement induced by GRIP domain overexpression. (a) HeLa cells transiently transfected with C312 or C312(Y2187A) or control untransfected cells were metabolically labeled with [35S]methionine/cysteine for 30 minutes and then chased for 0, 1, 2, or 4 hours. C312 or C312(Y2187A) was immunoprecipitated from cell lysates at each time point using anti-HA antibodies, fractionated by SDS-PAGE, and total C312 levels were determined by phosphorimaging analysis of the 40 kDa band that was absent in the controls (see Fig. 7). The amount at time 0 was set to 100%, and the percentage remaining at each time point is plotted. A representative of 3 experiments is shown. (b,c) HeLa cells from the same well transiently transfected with C312 were treated with 10 µg/ml CHX for 0, 2 or 4 hours as indicated, fixed, and then analyzed by IFM using antibodies to the HA-epitope (b) and to TGN46 (c). (b) The percentage of cells in a representative experiment that were positively stained with anti-HA (n=238 to 269 per time point in this experiment) were characterized for phenotypic expression level of C312 expression. (c) The percentage of cells in the same experiment that were positively stained with anti-HA were characterized for phenotypic appearance of TGN46 staining; `intact' TGN46 indicates a tight Golgi ribbon as in Fig. 2a.

View larger version (46K): [in a new window]   Fig. 7. GRIP domain overexpression affects proprotein convertase activity on a substrate protein, Pmel17. (a) Schematic diagram of Pmel17 primary structure and processed forms, as shown by Berson et al. (Berson et al., 2001). (b) HeLa cells were transiently transfected with expression vectors for Pmel17 (10 µg/10 cm dish) and either vector alone (lanes 1-4), C312 (lanes 5-8) or C312(Y2187A) (lanes 9-12) at 39 µg/10 cm dish. Two days post-transfection cells were harvested and pulse labeled with [35S]methionine/cysteine for 30 minutes, and then chased for the indicated times. Pmel17 was immunoprecipitated from cell lysates at each time point, fractionated by SDS-PAGE, and visualized by phosphorimaging analysis. (c) Anti-HA immunoprecipitates from the same samples analyzed in the same way. Only the relevant portion of the gel encompassing C312 or C312(Y2187A) transgene products is shown.

View larger version (87K): [in a new window]   Fig. 5. Localization of Golgi stack residents in cells overexpressing C312. HeLa cells transiently transfected with C312 were analyzed by IFM using antibodies to the HA-epitope (to detect C312) and to endogenous Golgi markers as indicated. (Aa-h) Cells overexpressing C312 and co-stained for giantin, mannosidase II (ManII), GM130 or p115. Examples are representative of all cells examined. (B) Cells co-stained for galactosyltransferase (GalT; a-f) or Rab6 (g-l). In approximately 46% of analyzed C312-overexpressing cells (n=369 over 3 experiments), the GalT staining pattern was similar to untransfected cells in the same field (a,b) and to cells transfected with C312(Y2187A) (e,f), whereas in the other 54%, GalT staining was diffuse (c,d). A similar disparity was seen for Rab6; in approximately 74% of analyzed C312-overexpressing cells (n=175 over 2 representative experiments), Rab6 staining was similar to controls (g,h compared with k,l) and in 26%, Rab6 staining was diffuse (i,j).

View larger version (118K): [in a new window]   Fig. 6. GRIP domain overexpression affects Golgi to plasma membrane transport. HeLa cells were transiently transfected with expression vectors for VSV-G-EGFP (2.5 µg/six-well dish) and either vector alone (a-c'), C312(Y2187A) (d-f') or C312 (g-m') at 5µg/six-well dish. Cells were grown at 39°C for 1 day and then fixed either before (a,d,g) or after shifting to 32°C for the indicated times. Cells were analyzed by fluorescence microscopy after immunostaining with anti-HA and RRX-conjugated secondary antibodies (' panels); EGFP was visualized directly (non-' panels). (g-k') Examples of cells with similar anti-HA staining intensities to those shown in d-f'; (l,l') an example of a cell with low C312 expression, and (m,m') an example of a cell with very high C312 expression. All ' panels were taken at the same exposure time on samples prepared and analyzed on the same day. The `spotty' appearance of VSV-G-EGFP localization in a, d and g may result from a fixation artifact.

View larger version (87K): [in a new window]   Fig. 8. GRIP domain overexpression disrupts Golgi morphology. HeLa cells transfected with expression vectors for Tac and for (A) C312(Y2187A) or (B,C) C312 were sorted for Tac cell surface staining, exposed to HRP for 15-30 minutes at 37°C, and then fixed and embedded in epon for conventional EM analyses. Note the flattened stacks of Golgi complex (Gc) cisternae in A and their absence in B and C. m, mitochondrion; n, nucleus. In C, stars are placed next to multivesicular bodies, which were abnormally abundant in most profiles from C312-transfected cells. Bars, 0.1 µm.

View larger version (141K): [in a new window]   Fig. 9. TGN46 localizes to vacuoles and to multivesicular bodies in C312-overexpressing cells. HeLa cells transfected with expression vectors for Tac and for (A) C312(Y2187A) or (B-F) C312 were sorted for Tac cell surface staining. Positive cells were exposed to HRP for 15-30 minutes at 37°C, then fixed, and ultrathin cryosections were labeled with antibodies to HA, TGN46 and or HRP and gold-conjugated secondary antibodies for immuno-EM analyses. (A-C) Sections were labeled with anti-HA and 10 nm gold-conjugated anti-mouse immunoglobulin, and anti-TGN46 and 5 nm gold-conjugated anti-sheep immunoglobulin. Arrowheads in B and C point to 5 nm gold particles. Note the labeling of the trans face of the Golgi complex by both anti-HA and anti-TGN46 in A, and the dense labeling of vacuoles with anti-HA in B and C. These examples show TGN46 in the vacuolated structures. (D-F) Sections were labeled with anti-HRP and 10 nm gold-conjugated anti-rabbit immunoglobulin, and anti-TGN46 and 5 nm gold-conjugated anti-sheep immunoglobulin. Arrowheads point to 5 nm gold particles (TGN46) in multivesicular endosomes (mve) that are also labeled by anti-HRP (10 nm gold). Bars, 0.1 µm.

View larger version (38K): [in a new window]   Fig. 10. Model for functional effects of GRIP domain overexpression. (a) Simplified model of vesicular traffic in and out of the TGN in HeLa cells, showing relevant endosomal compartments, the TGN, and the Golgi stack. Early and recycling endosomes are grouped together for simplicity. Cargo following the indicated pathways between organelles are boxed. GRIP proteins or GRIP-interacting molecules are shown to facilitate recycling of proteins from early/recycling endosomes to the TGN. (b) Perturbation of these pathways by overexpression of GRIP domain proteins.

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