Coatomer-bound Cdc42 regulates dynein recruitment to COPI vesicles

Abstract

Cytoskeletal dynamics at the Golgi apparatus are regulated in part through a binding interaction between the Golgi-vesicle coat protein, coatomer, and the regulatory GTP-binding protein Cdc42 (Wu, W.J., J.W. Erickson, R. Lin, and R.A. Cerione. 2000. Nature. 405:800-804; Fucini, R.V., J.L. Chen, C. Sharma, M.M. Kessels, and M. Stamnes. 2002. Mol. Biol. Cell. 13:621-631). The precise role of this complex has not been determined. We have analyzed the protein composition of Golgi-derived coat protomer I (COPI)-coated vesicles after activating or inhibiting signaling through coatomer-bound Cdc42. We show that Cdc42 has profound effects on the recruitment of dynein to COPI vesicles. Cdc42, when bound to coatomer, inhibits dynein binding to COPI vesicles whereas preventing the coatomer-Cdc42 interaction stimulates dynein binding. Dynein recruitment was found to involve actin dynamics and dynactin. Reclustering of nocodazole-dispersed Golgi stacks and microtubule/dynein-dependent ER-to-Golgi transport are both sensitive to disrupting Cdc42 mediated signaling. By contrast, dynein-independent transport to the Golgi complex is insensitive to mutant Cdc42. We propose a model for how proper temporal regulation of motor-based vesicle translocation could be coupled to the completion of vesicle formation.

Figure 1.

Dynein is present on Golgi vesicles assembled without Cdc42. (A) Shown is a Coomassie blue–stained gel of Golgi vesicle-enriched fractions obtained from budding incubations performed in the presence of 20 μM GTPγS with or without 250 μM p23 peptide as indicated. The identities of the coatomer subunits, α-COP, β-COP, β′-COP, and γ-COP were confirmed by Western blotting (not depicted). (B) Immunoblots of the vesicle-enriched fractions were probed with the indicated antibodies. (C) Shown is a blot of Golgi-binding assays performed in the presence of 20 μM GTPγS or 25 μg/ml of recombinant ARF1(Q71L) and probed as indicated.

Figure 2.

Cdc42 and actin affect dynein localization. (A) A Western blot of COPI-vesicle–enriched fractions isolated by flotation from Golgi-budding reactions was probed with the indicated antibodies. Incubations were performed in the presence of 25 μg/ml ARF1(Q71L), 20 μg/ml Cdc42(Q61L), and GTPγS as indicated. (B) Plotted are the average levels of dynein and coatomer found in the COPI-vesicle enriched fraction isolated as in A. 20 μg/ml of recombinant mutant Cdc42 proteins were added as indicated. The error bars represent the SEM (n = 3). (C) NRK cells that had been transfected (asterisk) with GFP-Cdc42(Q61L) (inset image) were labeled with an antibody against the dynein light chain (red). Bars, 20 μm. (D) Golgi-binding assays were used to determine the levels of bound dynein and actin (inset, graph) at various concentrations of cytochalasin D. The error bars represent the SEM (n = 3). (E) A Golgi-binding assay were performed adding ARF1(Q71L) when indicated and probed with the indicated antibodies.

Figure 3.

Dynein is recruited to COPI vesicles. (A) Vesicle extracts from budding reactions performed with GTPγS or ARF1(Q71L) were fractionated on a sucrose gradient. Shown are immunoblots of the fractions probed as indicated. (B) Shown is a blot of proteins precipitated with the anti-dynein IC antibody from a vesicle extract. Coatomer levels were determined using anti–ɛ-COP and anti–β-COP. Dynein levels were inferred using anti-p150^glued. The blot on the left indicates the total amount of COPI vesicles in the extract isolated by sedimentation. (C) Vesicles were precipitated with the anti–ζ-COP antibody as in B. The amounts of coatomer and dynein were determined by probing immunoblots with the appropriate antibodies. (D and E) Cryosections were taken from Vero cells and decorated with anti–ɛ-COP, large gold particles, and anti-dynein IC, small gold particles. The large arrows indicate structures labeled with both antibodies and the small arrows indicate structures labeled only with anti-dynein. Bar, 300 nm.

Figure 4.

Reclustering of Golgi membranes is sensitive to Cdc42 function. (A) NRK cells were transfected with a plasmid for the expression of myc-Cdc42(Q61L). The cells were treated with nocodazole and washed for the indicated times. The Golgi were labeled using an anti-GM130 antibody (red). Transfected cells (asterisks) were identified using an anti-myc antibody (green). (B) Before nocodazole treatment, NRK cells were transfected (asterisks) with HA–wild-type Cdc42 (WT), myc-Cdc42(Q61L), or HA-Cdc42(F28L) as indicated (green). The cells were allowed to recover for 60 min after the nocodazole washout as in A. The Golgi apparatus was labeled with anti-GM130 (red). Bar, 10 μm.

Figure 5.

Translocation of VTCs is sensitive to Cdc42 function. (A) Vero cells were cotransfected with vectors expressing GFP-VSVG(ts045) and myc-tagged Cdc42(Q61L). VSVG was accumulated in the ER. Where indicated, 20 μM nocodazole was added for 6 h before incubating at 32°C for 15 min. The cells were lysed and digested with endoglycosidase H (EndoH) where indicated. VSVG levels were determined by Western blotting. (B) Cells expressing Cdc42(Q61L) and GFP-VSVG(ts045) were treated as in A then decorated with antiGM130. (C) Cells expressing GFP-VSVG(ts045) were treated with nocodazole (for 6 h) and either BAPTA-AM or DMSO (for 1 h) before the shift to 32°C for 15 min. The amount of endoH-sensitive VSVG was determined by Western blotting.