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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 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-p150glued. 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.
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fig3: 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-p150glued. 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.

Mentions: Although dynein associates with budding vesicles on the Golgi (Fath et al., 1997), it has not been implicated directly in COPI-vesicle–mediated trafficking. Hence, we tested whether COPI vesicles cofractionate with dynein upon flotation through an isopycnic sucrose gradient (Fig. 3 A). COPI vesicles fractionate with a buoyant density equivalent to ∼42% sucrose (fractions 5–8). Addition of either GTPγS or ARF1(Q71L) activates COPI vesicle formation as indicated by the presence of coatomer in the center of the gradient (Fig. 3 A and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200501157/DC1). Dynein cofractionates with COPI vesicles when vesicle formation is activated by ARF1(Q71L). However, when ARF and Cdc42 are activated simultaneously with GTPγS, dynein remains in the bottom load fractions.


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 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-p150glued. 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.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: 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-p150glued. 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.
Mentions: Although dynein associates with budding vesicles on the Golgi (Fath et al., 1997), it has not been implicated directly in COPI-vesicle–mediated trafficking. Hence, we tested whether COPI vesicles cofractionate with dynein upon flotation through an isopycnic sucrose gradient (Fig. 3 A). COPI vesicles fractionate with a buoyant density equivalent to ∼42% sucrose (fractions 5–8). Addition of either GTPγS or ARF1(Q71L) activates COPI vesicle formation as indicated by the presence of coatomer in the center of the gradient (Fig. 3 A and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200501157/DC1). Dynein cofractionates with COPI vesicles when vesicle formation is activated by ARF1(Q71L). However, when ARF and Cdc42 are activated simultaneously with GTPγS, dynein remains in the bottom load fractions.

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