Plant Molecular Biology 51: 483–492, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Membrane localization of Arabidopsis acyl-CoA binding protein ACBP2
Hong-Ye Li and Mee-Len Chye
Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, China. (
correspondence; e-mail: email@example.com)
Received 27 November 2001; accepted in revised form 18 July 2002
Key words: acyl-CoA transfer, green ﬂuorescencent protein, lipid metabolism, protein targeting, subcellular
fractionation, transmembrane domain
Cytosolic acyl-CoA binding proteins bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters
and pool formers. Recently, we have characterized Arabidopsis thaliana cDNAs encoding novel forms of ACBP,
designated ACBP1 and ACBP2, that contain a hydrophobic domain at the N-terminus and show conservation at
the acyl-CoA binding domain to cytosolic ACBPs. We have previously demonstrated that ACBP1 is membrane-
associated in Arabidopsis. Here, western blot analysis of anti-ACBP2 antibodies on A. 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 fur-
ther investigate the subcellular localization of ACBP2, we fused ACBP2 translationally in-frame to GFP. By means
of particle gene bombardment, ACBP2-GFP and ACBP1-GFP fusion proteins were observed transiently expressed
at the plasma membrane and at the endoplasmic reticulum in onion epidermal cells. GFP fusions with deletion
derivatives of ACBP1 or ACBP2 lacking the transmembrane domain were impaired in membrane targeting. Our
investigations also showed that when the transmembrane domain of ACBP1 or that of ACBP2 was fused with GFP,
the fusion protein was targeted to the plasma membrane, thereby establishing their role in membrane targeting.
The localization of ACBP1-GFP is consistent with our previous observations using immunoelectron microscopy
whereby ACBP1 was localized to the plasma membrane and vesicles. We conclude that ACBP2, like ACBP1, is a
membrane protein that likely functions in membrane-associated acyl-CoA transfer/metabolism.
Cytosolic acyl-CoA binding proteins (ACBPs) are typ-
ically 10 kDa cytosolic proteins conserved at the acyl-
CoA binding domain and bind long-chain acyl-CoAs.
Cytosolic ACBPs have been characterized in rat (Alho
et al., 1985; Mocchetti et al., 1986), man (Marquardt
et al., 1986; Gray et al., 1986; Webb et al., 1987),
cow (Marquardt et al., 1986; Webb et al., 1987), frog
(Lihrmann et al., 1994), yeast (Rose et al., 1992) and
plants (Hills et al., 1994). Cytosolic ACBPs play an
important role in mammalian acyl-CoA metabolism
by binding acyl-CoA esters (Mikkelsen and Knudsen,
1987; Mikkelsen et al., 1987; Rasmussen et al., 1990)
and are involved in the storage and intracellular trans-
port of acyl-CoAs for β-oxidation and glycerolipid
biosynthesis (Knudsen, 1991; Rasmussen et al., 1994;
Færgeman and Knudsen, 1997). Comparison of cy-
tosolic ACBPs from different sources shows that they
consist of 86–92 amino acids and are highly con-
served across species, suggesting that their physio-
logical roles have been preserved through evolution.
Analysis of promoter sequences of genes encoding cy-
tosolic ACBP from rat (Mandrup et al., 1992; Kolmer
et al., 1993), Drosophila (Kolmer et al., 1994) and
man (Swinnen et al., 1996) show that they lack a TATA
box, implicating that they are housekeeping genes.
We have isolated and characterized cDNAs and
their corresponding genes encoding ACBPs from
A. thaliana, designated ACBP1 (Chye, 1998; Chye
et al., 1999) and ACBP2 (Chye et al., 2000) that are
distinct from cytosolic ACBPs by the presence of an