Three homologous ArfGAPs participate in coat protein I-mediated transport

Abstract

ArfGAP1 is a prototype of GTPase-activating proteins for ADP-ribosylation factors (ARFs) and has been proposed to be involved in retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER) by regulating the uncoating of coat protein I (COPI)-coated vesicles. Depletion of ArfGAP1 by RNA interference, however, causes neither a discernible phenotypic change in the COPI localization nor a change in the Golgi-to-ER retrograde transport. Therefore, we also examined ArfGAP2 and ArfGAP3, closely related homologues of ArfGAP1. Cells in which ArfGAP1, ArfGAP2, and ArfGAP3 are simultaneously knocked down show an increase in the GTP-bound ARF level. Furthermore, in these cells proteins resident in or cycling through the cis-Golgi, including ERGIC-53, beta-COP, and GM130, accumulate in the ER-Golgi intermediate compartment, and Golgi-to-ER retrograde transport is blocked. The phenotypes observed in the triple ArfGAP knockdown cells are similar to those seen in beta-COP-depleted cells. Both the triple ArfGAP- and beta-COP-depleted cells accumulate characteristic vacuolar structures that are visible under electron microscope. Furthermore, COPI is concentrated at rims of the vacuolar structures in the ArfGAP-depleted cells. On the basis of these observations, we conclude that ArfGAP1, ArfGAP2, and ArfGAP3 have overlapping roles in regulating COPI function in Golgi-to-ER retrograde transport.

FIGURE 1.

Disorganization of the Golgi apparatus in cells simultaneously depleted of ArfGAP1, ArfGAP2, and ArfGAP3. HeLa cells were treated for 120 h with siRNAs for LacZ (A-D) or for ArfGAP1+ArfGAP2 (E-H), ArfGAP1+ArfGAP3 (I-L), ArfGAP2+ArfGAP3 (M-P), or ArfGAP1+ArfGAP2+ArfGAP3 (Q-T) and stained for protein disulfide isomerase (A, E, I, M, and Q), ERGIC-53 (B, F, J, N, and R), β-COP (C, G, K, O, and S), or GM130 (D, H, L, P, and T).

FIGURE 2.

Increase in the GTP-bound ARF level in cells simultaneously knocked down of ArfGAP1, ArfGAP2, and ArfGAP3. Lysates were prepared from HeLa cells treated for 120 h with siRNAs for LacZ (lanes 1, 6, and 11) or for ArfGAP1 (lanes 2, 7, and 12), ArfGAP2 (lanes 3, 8, and 13), ArfGAP3 (lanes 4, 9, and 14), or ArfGAP1+ArfGAP2+ArfGAP3 (lanes 5, 10, and 15) and subjected to pulldown with glutathione S-transferase (GST)-GGA1(GAT) (lanes 6-10) or GST (lanes 11-15). The data are representatives of five independent experiments for the control and triple ArfGAP knockdown cells and two independent experiments for the single ArfGAP knockdown cells.

FIGURE 3.

Two distinct phenotypic stages in cells triple-depleted of ArfGAP1, ArfGAP2, and ArfGAP3. A, HeLa cells treated for 120 h with siRNAs for ArfGAP1+ArfGAP2+ArfGAP3 were double-stained for ERGIC-53 (a-c) and β-COP (a′-c′). Cells with normal distribution of ERGIC-53 and β-COP (a and a′) or with typical stage 1 (b and b′) or stage 2 (c and c′) distributions are shown. B, HeLa cells treated with siRNAs for ArfGAP1+ArfGAP2+ArfGAP3 for 96 or 120 h were classified as having normal, stage 1, and stage 2 distributions of ERGIC-53, and the number of cells with each distribution was counted. The number (n) is the sum of counted cells in three independent experiments. C, HeLa cells treated with siRNAs for ArfGAP1+ArfGAP2+ArfGAP3 for 96 h were transfected with an expression vector for C-terminal-HA-tagged ArfGAP2 (a-a″) or ArfGAP3 (b-b″) and incubated for 24 h, then triply stained for HA (a and b), ERGIC-53 (a′ and b′) and β-COP (a′ and b′). Cells overexpressing ArfGAP2-HA or ArfGAP3-HA are indicated by asterisks. D, the number of cells with normal, stage 1, and stage 2 distribution of ERGIC-53 in mock-transfected cells or cells overexpressing either ArfGAP2-HA or ArfGAP3-HA. The number (n) is the sum of counted cells in three independent experiments.

FIGURE 4.

Redistribution of Golgi proteins in cells triple-depleted of ArfGAPs. HeLa cells were treated for 120 h with siRNAs for LacZ (A) or ArfGAP1+ArfGAP2+ArfGAP3 (B) and processed for double staining for β-COP (a and d), GM130 (b), or golgin-97 (c), and either ERGIC-53 (a′-c′) or Sec31A (d′). Merged images are shown in a″-d″. Boxed regions are enlarged and shown in the corresponding insets. C, colocalization of the indicated proteins in cells treated with siRNAs for LacZ and ArfGAP1+ArfGAP2+ArfGAP3 was estimated using an IPLab 4.0 software (Solution Systems) and expressed as a Pearson coefficient. ^**, p < 0.0001; ^*, p < 0.001.

FIGURE 5.

Block in Golgi-to-ER transport in cells triple-depleted of ArfGAPs. HeLa cells were treated with siRNAs for LacZ (A, a-c) or ArfGAP1+ArfGAP2+ArfGAP3 (A, d-f, and B, f-f″) and transfected with the expression vector for the EGFP-VSVG-KDELR chimera. After transfection, the cells were incubated at 40 °C overnight (A, a and d), successively incubated at 20 °C for 3 h (A, b and e) and 40 °C for 2 h (A, c and e, and B) in the presence of 50 μg/ml cycloheximide, and processed for staining for ERGIC-53. Note that panels Af and Bf represent the same single image. C, after the final 40 °C incubation, the cells were scored for ER distribution of the EGFP-VSVG-KDELR chimera. The bar graph represents the percentage of cells with ER distribution of the chimera among all cells expressing the chimera. The number (n) is the sum of counted cells in two independent experiments. D, the cells triple-knocked down of ArfGAPs were classified as having normal, stage 1, and stage 2 distributions of ERGIC-53 (for example, see a cell indicated by an asterisk in B). The bar graph represents the percentage of cells with ER distribution of the chimera in each cell population. The number (n) is the sum of counted cells in two independent experiments.

FIGURE 6.

Block in anterograde transport through the Golgi in cells triple-depleted of ArfGAPs. HeLa cells were treated with siRNAs for LacZ (A, a and b) or ArfGAP1+ArfGAP2+ArfGAP3 (A, c and d, and B, d-d″) and transfected with the expression vector for EGFP-VSVG. After transfection the cells were incubated at 40 °C overnight (A, a and c), and subsequently at 32 °C for 60 min in the presence of 50 μg/ml cycloheximide (A, b and d, and B). The cells were then stained for ERGIC-53. Note that panels Ad and Bd represent the same single image. C, after the 32 °C incubation, the cells were scored for cell surface delivery of EGFP-VSVG. The bar graph represents the percentage of cells with surface EGFP-VSVG expression among all cells expressing EGFP-VSVG. The number (n) is the sum of counted cells in two independent experiments. D, the cells triple-knocked down of ArfGAPs were classified as having normal, stage 1, and stage 2 distributions of ERGIC-53 (for example, see the cell indicated by an asterisk in B). The bar graph represents the percentage of cells in each cell population with surface expression of EGFP-VSVG. The number (n) is the sum of counted cells in two independent experiments.

FIGURE 7.

Cells depleted of COPI show phenotypes similar to those of cells triple-depleted of ArfGAPs. HeLa cells were treated with siRNAs for LacZ (A, lane 1, and Ba) or β-COP (A, lane 2, Bb, C, and D) for 24 h. A, cell extracts were prepared and subjected to immunoblot analysis using antibody against β-COP, ArfGAP1, ArfGAP2, or ArfGAP3. B, the cells were immunostained for β-COP. C, β-COP-depleted cells were doubly stained for either syntaxin 5 (a), GM130 (b), or golgin-97 (c) and ERGIC-53 (a′-c′). D, β-COP-depleted cells were subjected to retrograde transport assay as described under the legend for Fig. 5, A and B. Asterisks indicate cells showing stage 1 and 2 distribution of ERGIC-53.

FIGURE 8.

Electron microscopic analyses of cells triple-depleted of ArfGAPs and those depleted of β-COP. HeLa cells treated for 120 h with siRNAs for LacZ (A) or ArfGAP1 + ArfGAP2 + ArfGAP3 (B) or for 48 h with siRNAs for β-COP (C) were processed for electron microscopic analysis. Boxed regions are enlarged and shown in A′, B′, and C′. Bars:5 μm in A-C; 1 μm in A′-C′. D, the number of vesicular and tubular structures surrounded by vacuoles was counted. ^*, p < 0.001 (n = 40).

FIGURE 9.

Association of β-COP with vacuolar structures in cells triple-depleted of ArfGAPs. HeLa cells treated for 120 h with siRNAs for LacZ (A) or ArfGAP1+ArfGAP2+ArfGAP3 (B-D) were processed for immunoelectron microscopic analysis. Ultrathin cryosections were stained with polyclonal anti-β-COP antibody (1:10) followed by goat anti-rabbit IgG conjugated with 10-nm colloidal gold. Note that β-COP is localized on vesicles near the Golgi (Go) in the control cells (A), whereas it is found on rims (arrowhead) of the vacuoles (asterisks) and vesicular structures in the triple knockdown cells (B-D). Bars, 0.2 μm.