Biochem. J. (2008) 410
(463–472) (Printed in Great Britain)
Acyl-CoA-binding protein (ACBP) localizes to the endoplasmic reticulum and Golgi in a ligand-dependent manner in mammalian cells
Jesper S. HANSEN*, Nils J. FÆRGEMAN*, Birthe B. KRAGELUND† and Jens KNUDSEN*1
*Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark, and †Department of Molecular Biology, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark
In the present study, we microinjected fluorescently labelled liver bovine ACBP (acyl-CoA-binding protein) [FACI-50 (fluorescent acyl-CoA indicator-50)] into HeLa and BMGE (bovine mammary gland epithelial) cell lines to characterize the localization and dynamics of ACBP in living cells. Results showed that ACBP targeted to the ER (endoplasmic reticulum) and Golgi in a ligand-binding-dependent manner. A variant Y28F/K32A-FACI-50, which is unable to bind acyl-CoA, did no longer show association with the ER and became segregated from the Golgi, as analysed by intensity correlation calculations. Depletion of fatty acids from cells by addition of FAFBSA (fatty-acid-free BSA) significantly decreased FACI-50 association with the Golgi, whereas fatty acid overloading increased Golgi association, strongly supporting that ACBP associates with the Golgi in a ligand-dependent manner. FRAP (fluorescence recovery after photobleaching) showed that the fatty-acid-induced targeting of FACI-50 to the Golgi resulted in a 5-fold reduction in FACI-50 mobility. We suggest that ACBP is targeted to the ER and Golgi in a ligand-binding-dependent manner in living cells and propose that ACBP may be involved in vesicular trafficking.
Key words: acyl-CoA-binding protein (ACBP), confocal microscopy, endoplasmic reticulum, Golgi, trafficking, two-photon excitation fluorescence recovery after photobleaching (FRAP).
Abbreviations used: ACBP, acyl-CoA-binding protein; badan, 6-bromoacetyl-2-dimethylaminonaphthalene; BMGE, bovine mammary gland epithelial; BODIPY®, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; DAPI, 4´,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; DMEMpen/strep, DMEM with 50 units/ml penicillin and 50 mg/ml streptomycin; DOPC, 1,2-dioleoyl-sn-3-glycerophosphocholine; ER, endoplasmic reticulum; FACI, fluorescent acyl-CoA indicator; FAFBSA, fatty-acid-free BSA; FBS, foetal bovine serum; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; GP, generalized polarization; ICA, intensity correlation analysis; ICQ, intensity correlation quotient; L-ACBP, liver-type ACBP; NA, numerical aperture; OA, oleic acid; PDM, product of the differences from the mean; SNARE, soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor.
1To whom correspondence should be addressed (email jkk@bmb.sdu.dk).
Received 25 April 2007/4 October 2007; accepted 22 October 2007
Published as BJ Immediate Publication 22 October 2007, doi:10.1042/BJ20070559
© The Authors Journal compilation © 2008 Biochemical Society
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