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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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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.
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fig5: 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.

Mentions: Molecular motor proteins and actin dynamics may play multiple roles in trafficking between the Golgi and the ER (Allan et al., 2002; Luna et al., 2002; Matanis et al., 2002; Short et al., 2002; Stamnes, 2002; Duran et al., 2003). The translocation of vesiculotubular clusters (VTCs) from ER exit sites to the juxtanuclear Golgi complex is a trafficking step where coatomer (Aridor et al., 1995; Scales et al., 1997; Stephens et al., 2000; Presley et al., 2002), Cdc42 (Wu et al., 2000; Fucini et al., 2002), and dynein (Burkhardt et al., 1997; Presley et al., 1997) have each been implicated. As with Golgi positioning (Fig. 4), we anticipated that Cdc42 might regulate the dynein-based motility of coatomer-coated VTCs. Cdc42(Q61L) expression led to an increase in non-Golgi processed (endoglycosidase H [endoH]-sensitive) VSVG(ts045) after release from the ER (Fig. 5 A and Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200501157/DC1) confirming the previous studies (Wu et al., 2000; Fucini et al., 2002). In Cdc42(Q61L)-transfected cells, VSVG appeared to be adjacent to SEC23-positive ER exit sites (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200501157/DC1). The results indicate that Cdc42(Q61L) expression leads to immobile VTCs.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: 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.
Mentions: Molecular motor proteins and actin dynamics may play multiple roles in trafficking between the Golgi and the ER (Allan et al., 2002; Luna et al., 2002; Matanis et al., 2002; Short et al., 2002; Stamnes, 2002; Duran et al., 2003). The translocation of vesiculotubular clusters (VTCs) from ER exit sites to the juxtanuclear Golgi complex is a trafficking step where coatomer (Aridor et al., 1995; Scales et al., 1997; Stephens et al., 2000; Presley et al., 2002), Cdc42 (Wu et al., 2000; Fucini et al., 2002), and dynein (Burkhardt et al., 1997; Presley et al., 1997) have each been implicated. As with Golgi positioning (Fig. 4), we anticipated that Cdc42 might regulate the dynein-based motility of coatomer-coated VTCs. Cdc42(Q61L) expression led to an increase in non-Golgi processed (endoglycosidase H [endoH]-sensitive) VSVG(ts045) after release from the ER (Fig. 5 A and Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200501157/DC1) confirming the previous studies (Wu et al., 2000; Fucini et al., 2002). In Cdc42(Q61L)-transfected cells, VSVG appeared to be adjacent to SEC23-positive ER exit sites (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200501157/DC1). The results indicate that Cdc42(Q61L) expression leads to immobile VTCs.

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