GAP-43 (growth-associated protein-43) is a dually palmitoylated protein, at cysteine residues at positions 3 and 4, that mostly localizes in plasma membrane both in neural and non-neural cells. In the present study, we have examined membrane association, subcellular distribution and intracellular trafficking of GAP-43 in CHO (Chinese hamster ovary)-K1 cells. Using biochemical assays and confocal and video microscopy in living cells we demonstrated that GAP-43, at steady state, localizes at the recycling endosome in addition to the cytoplasmic leaflet of the plasma membrane and TGN (trans-Golgi network). Pharmacological inhibition of newly synthesized GAP-43 acylation or double mutation of Cys3 and Cys4 of GAP-43 completely disrupts TGN, plasma membrane and recycling endosome association. A combination of selective photobleaching techniques and time-lapse fluorescence microscopy reveals a dynamic association of GAP-43 with recycling endosomes in equilibrium with the plasma membrane pool. Newly synthesized GAP-43 is found mainly associated with the TGN, but not with the pericentriolar recycling endosome, and traffics to the plasma membrane by a brefeldin A-insensitive pathway. Impairment of plasma membrane fusion and internalization by treatment with tannic acid does affect the trafficking of GAP-43 from plasma membrane to recycling endosomes which reveals a vesicle-mediated retrograde trafficking of GAP-43. Here, we also show that internalization of GAP-43 is regulated by Arf (ADP-ribosylation factor) 6. Taken together, these results demonstrate that dual acylation is required for sorting of peripheral membrane-associated GAP-43 to recycling endosome via an Arf6-associated endocytic vesicular pathway.
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Research Article|
July 15 2009
Dual acylation is required for trafficking of growth-associated protein-43 (GAP-43) to endosomal recycling compartment via an Arf6-associated endocytic vesicular pathway
Alejandra Trenchi;
Alejandra Trenchi
1
1Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC, UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, Córdoba X5000HUA, Argentina
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Guillermo A. Gomez;
Guillermo A. Gomez
1
1Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC, UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, Córdoba X5000HUA, Argentina
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Jose L. Daniotti
Jose L. Daniotti
2
1Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC, UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, Córdoba X5000HUA, Argentina
2To whom correspondence should be addressed (email daniotti@dqb.fcq.unc.edu.ar).
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Publisher: Portland Press Ltd
Received:
March 24 2009
Revision Received:
May 06 2009
Accepted:
May 14 2009
Accepted Manuscript online:
May 14 2009
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem J (2009) 421 (3): 357–369.
Article history
Received:
March 24 2009
Revision Received:
May 06 2009
Accepted:
May 14 2009
Accepted Manuscript online:
May 14 2009
Citation
Alejandra Trenchi, Guillermo A. Gomez, Jose L. Daniotti; Dual acylation is required for trafficking of growth-associated protein-43 (GAP-43) to endosomal recycling compartment via an Arf6-associated endocytic vesicular pathway. Biochem J 1 August 2009; 421 (3): 357–369. doi: https://doi.org/10.1042/BJ20090484
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