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Coatomer-bound Cdc42 regulates dynein recruitment to COPI vesicles.

Chen JL, Fucini RV, Lacomis L, Erdjument-Bromage H, Tempst P, Stamnes M - J. Cell Biol. (2005)

Bottom Line: Biol.Dynein recruitment was found to involve actin dynamics and dynactin.By contrast, dynein-independent transport to the Golgi complex is insensitive to mutant Cdc42.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA 52242, USA.

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.

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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.
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fig2: 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.

Mentions: Multiple GTP-binding proteins are candidates to function in the secretory pathway or in motor protein recruitment (Hammer and Wu, 2002; Symons and Rusk, 2003). To test whether Cdc42 is sufficient to mediate the effects of GTPγS on dynein recruitment, we examined the effects of adding recombinant Cdc42 to the vesicle budding reaction in addition to ARF1(Q71L) (Fig. 2, A and B). Addition of the constitutively active Cdc42(Q61L) greatly decreased the amount of dynein in the vesicle fraction. Cdc42(Q61L) and GTPγS were found to reduce dynein levels to the same extent (Fig. 2 A) indicating that Cdc42 is the predominant GTP-binding protein that negatively regulates dynein recruitment in the budding reaction.


Coatomer-bound Cdc42 regulates dynein recruitment to COPI vesicles.

Chen JL, Fucini RV, Lacomis L, Erdjument-Bromage H, Tempst P, Stamnes M - J. Cell Biol. (2005)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171931&req=5

fig2: 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.
Mentions: Multiple GTP-binding proteins are candidates to function in the secretory pathway or in motor protein recruitment (Hammer and Wu, 2002; Symons and Rusk, 2003). To test whether Cdc42 is sufficient to mediate the effects of GTPγS on dynein recruitment, we examined the effects of adding recombinant Cdc42 to the vesicle budding reaction in addition to ARF1(Q71L) (Fig. 2, A and B). Addition of the constitutively active Cdc42(Q61L) greatly decreased the amount of dynein in the vesicle fraction. Cdc42(Q61L) and GTPγS were found to reduce dynein levels to the same extent (Fig. 2 A) indicating that Cdc42 is the predominant GTP-binding protein that negatively regulates dynein recruitment in the budding reaction.

Bottom Line: Biol.Dynein recruitment was found to involve actin dynamics and dynactin.By contrast, dynein-independent transport to the Golgi complex is insensitive to mutant Cdc42.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA 52242, USA.

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.

Show MeSH
Related in: MedlinePlus