Regulation of protein transport from the Golgi complex to the endoplasmic reticulum by CDC42 and N-WASP

Abstract

Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and non-coated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgi-to-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(Delta WA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-N-WASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP-dependent manner.

Figure 1

GFP-Cdc42 is localized in the Golgi complex. (A) NRK cells were microinjected into the nucleus with expressing vectors for wild-type (WT), dominant-negative (N17), and dominant-positive (V12) GFP-Cdc42 forms. After microinjection (3–4 h), the cells were processed for immunofluorescence microscopy and examined by confocal microscopy. The organization of the Golgi complex was visualized with anti–Mannosidase II (ManII) antibodies. Bar, 10 μm. (B) Microinjected NRK cells (asterisks) with GFP-Cdc42V12 construct were, after 3–4 h of expression, treated with nocodazole, which causes fragmentation and dispersal of Golgi fragments throughout the cell. The Golgi-associated Cdc42 colocalizes with ManII in small punctate Golgi structures. Bar, 10 μm.

Figure 2

Activated GFP-Cdc42 is enriched in the lateral portions of Golgi cisternae and in peri-Golgi transport intermediates. Transfected HeLa cells with GFP-Cdc42V12 vector were fixed and processed for cryoimmunogold electron microscopy using polyclonal antibodies to GFP. Note the gold decoration for GFP-Cdc42V12 in the cytoplasmic part of the plasma membrane in a transfected cell (asterisk), whereas no gold particles are observed in the neighboring nontransfected cell (A). In the Golgi area (B and C), activated GFP-Cdc42 is predominantly located in the lateral portions of the Golgi cisternae (B) and in COPI-coated (arrowheads) and noncoated peri-Golgi transport intermediates (C). G, Golgi stack; m, mitochondria. Bars, 200 nm.

Figure 3

The ER-to-Golgi transport is unaltered in cells expressing Cdc42. HeLa cells were transfected with GFP-Cdc42V12 or GFP-Cdc42N17 vectors and incubated for 12 h. Subsequently, the cells were infected with the thermosensitive ts045 VSV-G mutant incubated at restrictive temperature (40°C). At this temperature VSV-G protein is retained in the ER (A and B). When cells were transferred to the permissive temperature of 32°C, the VSV-G glycoprotein exited the ER and was transported to the Golgi complex (C–F). At indicated transport times (C–F), cells were processed for indirect immunofluorescence for VSV-G glycoprotein. In this and the other panels, the GFP-Cdc42-expressing cells were detected by GFP fluorescence, as marked by asterisks (*). Note that both the dominant-positive (A, C, and E, asterisks) and dominant-negative Cdc42 mutants (B, D, and F, asterisks) show the same kinetics of transport of the VSV-G from the ER to the Golgi complex as the nontransfected neighboring cells. Bar, 10 μm.

Figure 4

The kinetics of Golgi disassembly is slowed in cells expressing activated Cdc42. HeLa cells were incubated for 12 h after transfection with the GFP-Cdc42 vectors. The cells were treated with BFA (5 μg/ml) and the kinetics of the fusion of Golgi membranes with the ER was monitored by immunofluorescence using antibodies against the Golgi protein giantin. After BFA treatment, the pericentriolar Golgi complex can be visualized in transfected cells with GFP-Cdc42V12 (C and E, asterisks) but not in cells expressing the GFP-Cdc42N17 (D and F, asterisks) or in neighboring nontransfected cells (C–F). Inset in E shows the characteristic BFA-induced Golgi tubulation in a transfected cell with GFP-Cdc42V12 mutant (asterisk), whereas the neighbor nontransfected cells show a more ER-like staining pattern. (G) Quantitative analysis of the morphological observations. Data represent the average of two independent experiments, in which at least 200 cells were counted for each experiment. (C, nontransfected cells; WT, cells transfected with the wild-type form of Cdc42; V12, cells transfected with Cdc42V12 mutant; N17, cells transfected with Cdc42N17). Bar, 10 μm.

Figure 5

Cdc42 blocks the redistribution of the KDEL receptor when cells are incubated at 15°C. HeLa cells were transfected as described in the legends of Figures 3 and 4. At 37°C, the steady state distribution of the KDEL receptor (KDELr) shows both the Golgi-like and a diffuse punctate staining. When cells were incubated at 15°C for various times, the steady state distribution of KDELr changes to exclusive punctate staining in nontransfected (C–H) and GFP-Cdc42N17–transfected cells (asterisks in D, F, and H). In contrast, in GFP-Cdc42V12–transfected cells, the KDELr shows the Golgi-like staining pattern (C, E, and G; asterisks). (I) A quantitative visual analysis of the percentage of neighboring nontransfected (control, C) and GFP-Cdc42–transfected cells (WT, N17, and V12 forms) with a Golgi-like staining pattern. Results are the average of two independent experiments, in which at least 200 cells were counted for each experiment. Bar, 10 μm.

Figure 6

Cdc42 blocks the Sar1^dn-induced ER accumulation of Golgi enzymes and the KDEL receptor. Sar1^dn construct alone (A and D; asterisks) and Sar1^dn plus GFP-Cdc42V12 (B, E; asterisks) or GFP-Cdc42N17 mutant constructs (C and F; asterisks) were microinjected into the nucleus of HeLa cells. After 7 h of expression, the cells were fixed and processed for indirect immunofluorescence with antibodies against galactosyltransferase (Gal-T; A–C) or the KDEL receptor (KDELr; D–F). In comicroinjected cells with Sar1^dn plus activated GFP-Cdc42, Gal-T (B, asterisks), and KDELr (E, asterisk) reveal a Golgi-like staining pattern like the neighboring control (nonmicroinjected) cells. In contrast, cells microinjected with Sar1^dn alone (A and D; asterisks) or with Sar1^dn plus the dominant-negative GFP-Cdc42N17 (C and F; asterisks) show the characteristic ER accumulation of both Gal-T and KDELr induced by Sar1^dn. (G) Microinjected cells were visually quantified for the presence of Gal-T in the Golgi complex. Results are the mean of two independent experiments, and the number of counted microinjected cells is indicated (n). Bar, 10 μm.

Figure 7

Cdc42 blocks transport of native Shiga toxin B-fragment from Golgi to the ER. HeLa cells were microinjected as described in Figure 6. After 1.5 h of expression, cells were incubated with cy3-tagged native Shiga toxin (ST-B) for 45 min at 37°C, rinsed, and transferred to 20°C for 2 h to allow internalized ST-B to be retained in the early/recycling endosomes (A and B). Cells were then shifted to 37°C to elicit retrograde transport to the ER via the Golgi complex (C–F). Unlike Sar1^dn-expressing cells (C and E; asterisks), ST-B remained accumulated in the Golgi complex in GFP-Cdc42V12 expressing cells (D and F; asterisks). After 60 min of transport, the Golgi complex (stained to giantin) remained virtually unaltered in Sar1^dn-expressing cells (E, insert; asterisk), but ST-B (asterisk in E) showed the expected ER-like staining pattern as a consequence of its transport from Golgi to the ER. Cells shown in E and its inset were double-stained with antibodies to giantin and ST-B. (G) Quantitative validation of the morphological data in cells microinjected with different vectors. Results are the mean of two independent experiments and the number of counted microinjected cells is indicated (n). Bar, 10 μm.

Figure 8

Activated Cdc42 recruits N-WASP to the Golgi complex. HeLa cells were coinjected with GFP-tagged wild-type N-WASP vector alone (A) or together with untagged Cdc42V12 construct (B). After 4 h of expression, cells were stained with antibodies to Gal-T and examined by confocal microscopy. Unlike cells expressing GFP-N-WASP alone (A, A′, and A"), GFP fluorescence corresponding to N-WASP (B) in cells that coexpress Cdc42V12 partially colocalize with Gal-T in the Golgi complex (B′) by the appearance of yellow color when images were superimposed (B", overlay). Bars, 10 μm.

Figure 9

N-WASP blocks the Sar1^dn-induced ER accumulation of Golgi enzymes and the retrograde transport of Shiga toxin from Golgi to the ER. (A) Sar1^dn plus wild-type GFP-N-WASP (left; asterisks) or Sar1^dn plus GFP-N-WASP(ΔWA) (right; asterisks) constructs were coinjected into the nucleus of HeLa cells. After 3–4 h, cells were processed for indirect immunofluorescence with anti–Gal-T antibodies. Unlike N-WASP(ΔWA), N-WASP prevents the Sar1^dn-induced ER accumulation of Gal-T. These morphological observations were quantified and results are shown in Figure 7G. Bar, 10 μm. (B) The experimental procedure is described in the legend to Figure 7. N-WASP blocks transport of ST-B from Golgi to the ER (left; asterisks). However, in cells expressing GFP-N-WASP(ΔWA), ST-B accumulates in the ER (right; asterisks). Nonmicroinjected cells show the characteristic steady state Golgi localization of ST-B. The inset shows the double immunostaining to giantin, which reveals of the presence of a virtually intact Golgi complex at this time of Sar1^dn expression. Bar, 10 μm.

Figure 10

N-WASP(ΔWA) alleviates the blocking effect of activated Cdc42 on the Sar1^dn-induced ER accumulation of Gal-T. Sar1^dn plus untagged Cdc42V12 plus wild-type GFP-N-WASP or Sar1^dn plus untagged Cdc42V12 plus GFP-N-WASP(ΔWA) constructs were coinjected into the nucleus of HeLa cells (A and B, respectively; asterisks). After 7 h, cells were processed for indirect immunofluorescence with anti–Gal-T antibodies. Unlike cells that overexpress wild-type N-WASP (A), those expressing the mutant form of N-WASP lacking the Arp2/3 binding domain (N-WASP(ΔWA)) showed that Gal-T was redistributed to the ER by Sar1^dn despite Cdc42V12 (B). These morphological observations were quantified and results are shown in Figure 6G. Bar, 10 μm.