Understanding the role of specific bilayer components in controlling the function of G-protein coupled receptors (GPCRs) will be a key factor in the development of novel pharmaceuticals. Cholesterol-dependence in particular has become an area of keen interest with respect to GPCR function; not least since the 2.6Å crystal structure of the β2 adrenergic receptor revealed a putative cholesterol binding motif conserved throughout class-A GPCRs. Furthermore, experimental evidence for cholesterol-dependent GPCR function has been demonstrated in a limited number of cases. This modulation of receptor function has been attributed to both direct interactions between cholesterol and receptor, and indirect effects caused by the influence of cholesterol on bilayer order and lateral pressure. Despite the widespread occurrence of cholesterol binding motifs, available experimental data on the functional involvement of cholesterol on GPCRs are currently limited to a small number of receptors. Here we investigate the role of cholesterol in the function of the neurotensin receptor 1 (NTS1) a class-A GPCR. Specifically we show how cholesterol, and the analogue cholesteryl hemisuccinate, influence activity, stability, and oligomerisation of both purified and reconstituted NTS1. The results caution against using such motifs as indicators of cholesterol-dependent GPCR activity.

The Na+-coupled betaine symporter BetP shares a highly conserved fold with other sequence unrelated secondary transporters, e.g., with neurotransmitter symporters. Recently, we obtained atomic structures of BetP in distinct conformational states, which elucidated parts of its alternating access mechanism. Here, we report for the first time a structure of BetP in a new outward-open state in complex with an anomalous scattering substrate, adding a fundamental piece to an unprecedented set of structural snapshots for a secondary transporter. In combination with molecular dynamics simulations these structural data highlight important features of the sequential formation of the substrate and sodium-binding sites, in which coordinating water molecules play a crucial role. We observe a strictly interdependent binding of betaine and sodium ions during the coupling process. All three sites undergo progressive reshaping and dehydration during the alternating-access cycle, with the most optimal coordination of all substrates found in the closed state.

A small number of physiologically important ATP-binding cassette (ABC) transporters are found in mitochondria. Most are half transporters of the B group forming homodimers and their topology suggests they function as exporters. The results of mutant studies point towards involvement in iron cofactor biosynthesis. In particular, ABC subfamily B member 7 (ABCB7) and its homologues in yeast and plants are required for iron-sulfur (Fe-S) cluster biosynthesis outside of the mitochondria, whereas ABCB10 is involved in haem biosynthesis. They also play a role in preventing oxidative stress. Mutations in ABCB6 and ABCB7 have been linked to human disease. Recent crystal structures of yeast Atm1 and human ABCB10 have been key to identifying substrate-binding sites and transport mechanisms. Combined with in vitro and in vivo studies, progress is being made to find the physiological substrates of the different mitochondrial ABC transporters. Sequence analysis of mitochondrial ABC transportersMitochondria of most eukaryote species harbour 2-4 different ABC transporters that belong to the B subfamily of half transporters. The proteins contain an N-terminal transmembrane domain (TMD) and a C-terminal nucleotidebinding domain (NBD), which form homodimers for full functionality [1]. Plant and algal genomes additionally encode a unique ABC transporter, termed cytochrome c maturation (Ccm)AB, in which the TMD and NBD are encoded by separate genes. CcmAB is placed in the ABCI subfamily, see section 'CcmAB in plant mitochondria'. Phylogenetic relationship of mitochondrial transporters of the ABCB subfamilyAs a starting point for the discussion of recent crystal structures and functional data, we have reanalysed the phylogenetic relationships of B-type mitochondrial ABC transporters and their closest prokaryotic homologues (Figure 1). The CcmAB proteins were excluded from the phylogenetic analysis due to their large evolutionary distance from the other mitochondrial ABC transporters.Key words: ATP-binding cassette (ABC) transporter, glutathione, haem, iron, mitochondria, oxidative stress. Abbreviations: ABC, ATP-binding cassette; Aft, Activator of ferrous transport; ATM, ABC transporter of the mitochondria; Ccm, cytochrome c maturation; dALA, δ-amino levulinic acid; IRP1, iron regulatory protein 1; Mdl, multi-drug resistance-like; Mfrn, mitoferrin; NBD, nt-binding domain; PPIX, protoporphyrin IX; TMD, transmembrane domain; TMH, transmembrane helix. 1 To whom correspondence should be addressed (email janneke.balk@jic.ac.uk).The ABCB7 group, which includes the ABC transporters of the mitochondria Atm1 in yeast and ATM3 in Arabidopsis, can be found in virtually all eukaryotic species. Human ABCB7 and Arabidopsis ATM3 can largely rescue the phenotypes of yeast atm1 mutants [2][3][4], providing experimental support that members of this subfamily may have the same function. Human ABCB6 can also partially complement a yeast atm1 mutant [5] but differences in the intracellular localization of ABCB6 and Atm1 may point to distinct phys...