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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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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.
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fig1: 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.

Mentions: We analyzed the consequences of blocking Cdc42 recruitment to budding COPI vesicles using a peptide corresponding to the coatomer-binding motif of p23 (Wu et al., 2000; Fucini et al., 2002; Chen et al., 2004). Activating ARF with GTPγS in a cell-free Golgi-vesicle budding assay leads to COPI vesicle formation as indicated by the presence of coatomer in a vesicle-enriched fraction (Fig. 1, A and B). GTPγS also activates Cdc42 causing it to appear in the vesicle fraction (Fig. 1 B). As expected, the p23 peptide prevented Cdc42 from binding coatomer and appearing with the vesicles (Fig. 1 B). The levels of a high molecular weight protein was greatly increased in the presence of the peptide (Fig. 1 A). Subsequent analysis of tryptic fragments by mass spectrometry led to the identification of this protein as the heavy chain subunit of dynein.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171931&req=5

fig1: 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.
Mentions: We analyzed the consequences of blocking Cdc42 recruitment to budding COPI vesicles using a peptide corresponding to the coatomer-binding motif of p23 (Wu et al., 2000; Fucini et al., 2002; Chen et al., 2004). Activating ARF with GTPγS in a cell-free Golgi-vesicle budding assay leads to COPI vesicle formation as indicated by the presence of coatomer in a vesicle-enriched fraction (Fig. 1, A and B). GTPγS also activates Cdc42 causing it to appear in the vesicle fraction (Fig. 1 B). As expected, the p23 peptide prevented Cdc42 from binding coatomer and appearing with the vesicles (Fig. 1 B). The levels of a high molecular weight protein was greatly increased in the presence of the peptide (Fig. 1 A). Subsequent analysis of tryptic fragments by mass spectrometry led to the identification of this protein as the heavy chain subunit of dynein.

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