Emerging new paradigms for ABCG transporters

Abstract

Every cell is separated from its external environment by a lipid membrane. Survival depends on the regulated and selective transport of nutrients, waste products and regulatory molecules across these membranes, a process that is often mediated by integral membrane proteins. The largest and most diverse of these membrane transport systems is the ATP binding cassette (ABC) family of membrane transport proteins. The ABC family is a large evolutionary conserved family of transmembrane proteins (>250 members) present in all phyla, from bacteria to Homo sapiens, which require energy in the form of ATP hydrolysis to transport substrates against concentration gradients. In prokaryotes the majority of ABC transporters are involved in the transport of nutrients and other macromolecules into the cell. In eukaryotes, with the exception of the cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7), ABC transporters mobilize substrates from the cytoplasm out of the cell or into specific intracellular organelles. This review focuses on the members of the ABCG subfamily of transporters, which are conserved through evolution in multiple taxa. As discussed below, these proteins participate in multiple cellular homeostatic processes, and functional mutations in some of them have clinical relevance in humans.

Figure 1

Structure of a typical ABCG half-transporter dimer. ABCG proteins contain an amino-terminal ABC domain followed by six transmembrane helices. The functional transporter is thought to be dependent on the formation of homo- or heterodimers, in which the resultant twelve transmembrane helices configure a channel in the membrane phospholipid bilayer. The energy necessary for the translocation of the substrate is derived from the hydrolysis of two ATP molecules at the ABC domain. Studies have shown that the Walker A, Walker B and Signature motifs within the ABC domain are not functionally equivalent (see text for details).

Figure 2

Complementation experiments in Drosophila suggest that neither mammalian ABCG1 nor ABCG4 are the true orthologs of white. A. P-element vectors containing a Gal4 cassette under transcriptional control of a heat shock-inducible promoter (hs-Gal4), or containing the gene of interest (GOI, murine Abcg1 or Abcg4, CD8GFP (a negative control) or Drosophila white) under transcriptional control of UAS sequences were introduced into Drosophila yw¹¹¹⁸ embryos using standard germ line transformation procedures. The F1 progeny of these crosses were heat shocked at 37°C for 30 min daily starting at the third instar larval stage. B. Expression of the exogenous Abcg1 and Abcg4 after heat shock was confirmed by real time PCR using RNA harvested from 10–15 F1 progeny of each genotype, and gene specific primers. C. Effectiveness of the heat shock process was also confirmed in flies carrying an inducible GFP cassette. D. Eye color was assessed in new emerged adult flies and pictures were taken at 5× magnification with a SPOT camera attached to a Zeiss dissecting microscope. Detailed experimental protocols have been published [111]. Only flies carrying both UAS-white and hs-Gal4 exhibited red eyes following heat shock treatment (second panel).

Figure 3

Phylogenetic analysis of Drosophila and mammalian ABCG genes. Amino acid sequences for the 15 Drosophila and 5 Mus musculus ABCG proteins were retrieved from the NCBI protein database. Sequences were aligned using the multiple alignment algorithms in the ClustalX program version 1.83 with the BLOSUM 62 protein weight matrix [112, 113]. Pairwise identity and similarity scores were generated using the MatGAT v2.02 program [114]. Three different phylogenetic methods were applied to aligned sequences to obtain tree topologies: the Bayesian inference of phylogeny method, maximum likelihood, and distance matrix methods. The final tree presented was obtained from the Bayesian inference analysis that represented the best fit for the data generated from the MrBayes program v3.1 [115]. Detailed parameters used in the Bayesian analysis are available [111]. The tree shows the distant relationship of Drosophila pigmentation genes with the other members of this family in Drosophila and mice. The Drosophila members most closely related to ABCG1 and ABCG4 are Atet and CG3164 and are highlighted. The posterior probabilities of the tree are indicated at each branch point.