Functional Analysis of Acbp2, an Arabidopsis Acyl-Coa Binding Protein

This dissertation, "Functional Analysis of ACBP2, an Arabidopsis Acyl-CoA Binding Protein" by Hongye, Li, 李宏業, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled FUNCTIONAL ANALYSIS OF ACBP2, AN Arabidopsis ACYL-COA BINDING PROTEIN Submitted by LI Hongye for the Degree of Doctor of Philosophy at The University of Hong Kong in October 2002 Cytosolic acyl-CoA binding proteins (ACBP) bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and pool formers. Recently, Arabidopsis thaliana cDNAs encoding novel forms of ACBP, designated ACBP1 and ACBP2, have been characterized. Both ACBP1 and ACBP2 show conservation at the acyl- CoA binding domain to cytosolic ACBPs but are distinct from cytosolic ACBPs by the presence of a hydrophobic domain at the N-termini and of ankyrin repeats at the C-termini. It has been previously demonstrated that ACBP1 is membrane-associated in Arabidopsis. In this study, ten mutant forms of recombinant ACBP2 with single amino acid substitutions at conserved residues within the acyl-CoA binding domain were generated by in vitro site-directed mutagenesis. Four mutant forms of recombinant ACBP2 with single amino acid substitutions at conserved residues within the acyl-CoA binding domain corresponding to residues in recombinant bovine cytosolic ACBP, that have been shown to interact with palmitoyl-CoA, were found to be less 14 effective in binding [C]palmitoyl-CoA. However, six other mutant forms of recombinant ACBP2 with single amino acid substitutions at other conserved residues within the acyl-CoA binding domain were not altered in binding efficiency to 14 [C]palmitoyl-CoA. Western blot analysis using anti-ACBP2 antibodies on Arabidopsis thaliana protein showed that ACBP2 is located in the microsome-containing membrane fraction and in the subcellular fraction containing large particles (mitochondria, chloroplasts and peroxisomes), resembling the subcellular localization of ACBP1. To further investigate subcellular localization, ACBP2 was fused translationally in- frame to GFP (green fluorescent protein). Using particle gene bombardment of onion epidermal cells, ACBP2-GFP and ACBP1-GFP fusion proteins were transiently expressed at the plasma membrane and at the endoplasmic reticulum. GFP fusions with deletion derivatives of ACBP1 or ACBP2 lacking the transmembrane domain were impaired in membrane targeting. Investigations also showed that when the N- terminal hydrophobic domain of ACBP1, or that of ACBP2, was fused with GFP, the fusion protein was targeted to the plasma membrane, thereby establishing the role of this domain in membrane targeting. The localization of ACBP1-GFP is consistent with previous observations using immunoelectron microscopy whereby ACBP1 was localized to the plasma membrane and vesicles. It can be concluded that ACBP2, like ACBP1, is also a membrane protein that likely functions in membrane-associated acyl-CoA transfer or metabolism. The presence of ankyrin repeats in the C-terminus of ACBP2 suggests that it interacts with other proteins, since ankyrin repeats are known to mediate protein-protein interaction. To identify these interacting proteins, yeast two-hybrid analysis using an Arabidopsis thaliana cDNA library was carried out with a bait containing a DNA sequence encoding ACBP2. Results from yeast two-hybrid screens and in vitro binding assays show that ACBP2 interacts with AtEBP, an A. thaliana ethylene- responsive element binding factor protein, which belongs to a superfamily of transcription