title: Membrane charge and curvature determine interaction with acyl-CoA binding protein (ACBP) and fatty acyl-CoA targeting.
abstract: Although acyl-CoA binding protein (ACBP) stimulates utilization of long-chain fatty acyl-CoA by a variety of membrane-bound enzymes, it is not known whether ACBP directly interacts with membranes. To test this hypothesis, mouse recombinant (mr) ACBP was engineered to contain the native mouse ACBP amino acid sequence expressed as a fusion protein at high levels (>150 mg/L) in Escherichia coli. Purification and cleavage of the fusion tag resulted in mrACBP identical to native ACBP as shown by mass (10000.5 Da) and amino acid sequence (peptide mapping after proteolysis) determined by matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectroscopy. The mrACBP was functionally active as shown by binding of cis-parinaroyl-CoA with high affinity, K(d) = 12 +/- 2 nM, at a single binding site, stimulating oleoyl-CoA utilization by microsomal glycerol-3-phosphate acyltransferase 3.2-fold and protecting oleoyl-CoA from microsomal acyl-CoA hydrolase. Direct interaction of mrACBP with membranes was demonstrated by two independent methods: (i) Circular dichroism showed an 8% increase in alpha-helix content of mrACBP in the presence of anionic phospholipid-rich, but not neutral, small unilamellar vesicles (SUV). (ii) Membrane filtration confirmed that mrACBP bound to anionic phospholipid-rich SUV but only weakly interacted with neutral SUV or large unilamellar vesicles (LUV), regardless of charge. (iii) The mrACBP-oleoyl-CoA complex transferred 2-3-fold more oleoyl-CoA to anionic phospholipid-rich SUV than to anionic phospholipid-rich LUV and neutral SUV or LUV. Conversely, mrACBP extracted less oleoyl-CoA from anionic phospholipid-rich SUV. Taken together, these data indicated for the first time that mrACBP interacted preferentially with anionic phospholipid-rich, highly curved membranes to facilitate transfer of ACBP-bound ligands.
authors.affiliation: Department of Pathobiology, Texas A&M University, TVMC, College Station, Texas 77843-4467, USA.
authors.forename: Hsu
authors.initials: H
authors.lastname: Chao
authors.forename: Gregory G
authors.initials: GG
authors.lastname: Martin
authors.forename: William K
authors.initials: WK
authors.lastname: Russell
authors.forename: Suryakant D
authors.initials: SD
authors.lastname: Waghela
authors.forename: David H
authors.initials: DH
authors.lastname: Russell
authors.forename: Friedhelm
authors.initials: F
authors.lastname: Schroeder
authors.forename: Ann B
authors.initials: AB
authors.lastname: Kier
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D026261:Diazepam Binding Inhibitor / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000378:metabolism; D006868:Hydrolysis; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008851:Micropore Filters; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010170:Palmitoyl-CoA Hydrolase / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D055672:Static Electricity; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000214:Acyl Coenzyme A; D026261:Diazepam Binding Inhibitor; D008081:Liposomes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D014364:Tryptophan; D005992:Glycerol-3-Phosphate O-Acyltransferase; D010170:Palmitoyl-CoA Hydrolase