Biochem. J. (2009) 421
(357–369) (Printed in Great Britain)
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 Trenchi1, Guillermo A. Gomez1 and Jose L. Daniotti2
Centro 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
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.
Key words: acylation, Golgi complex, growth-associated protein-43 (GAP-43), membrane traffic, palmitoyl-acyl transferase, recycling endosome.
Abbreviations used: aa, amino acids; AOTF, acoustical optical transmission filter; A phase, aqueous upper phase; Arf, ADP-ribosylation factor; ATB, antibiotics; 2-BP, 2-bromo hexadecanoic acid; BFA, brefeldin A; CaM, calmodulin; CFP, cyan fluorescence protein; CHO, Chinese hamster ovary; D phase, detergent-enriched lower phase; ECFP, enhanced cyan fluorescence protein; ER, endoplasmic reticulum; EYFP, enhanced yellow fluorescent protein; CHX, cycloheximide; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; FLIP, fluorescence loss in photobleaching; FRAP, fluorescence recovery after photobleaching; GalNAc-T, UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase; Gal-T2, UDP-Gal: GA2/GM2/GD2/GT2 galactosyltransferase; GAP-43, growth-associated protein-43; GAP-43full, full-length wild-type GAP-43; N13GAP-43, GAP-43 only containing the acylation motif (aa 1–13); GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; NA, numerical aperture; PAT, palmitoyl-acyl transferase; GFP–PH-PLCδ1, chimeric protein containing the PIP2-specific pleckstrin homology domain of phospholipase Cδ1 fused to GFP; HA, haemagglutinin; PIP2, phosphatidylinositol (4,5)-bisphosphate; PKC, protein kinase C; PM, plasma membrane; RE, recycling endosome; ROI, region of interest; Tf, transferrin; TGN, trans-Golgi network; TX-114, Triton X-114; YFP, yellow fluorescent protein.
1These authors contributed equally to this article.
2To whom correspondence should be addressed (email daniotti@dqb.fcq.unc.edu.ar).
Received 24 March 2009/6 May 2009; accepted 14 May 2009
Published as BJ Immediate Publication 14 May 2009, doi:10.1042/BJ20090484
© The Authors Journal compilation © 2009 Biochemical Society
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