Structural Approaches of the Mitochondrial Carrier Family
The transport of solutes across the inner mitochondrial membrane is highly selective and necessitates membrane proteins mainly from the mitochondrial carrier family (MCF). These carriers are required for the transport of a variety of metabolites implicate
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1. Introduction Mitochondria are organelles in which major metabolic cycles, Krebs cycle, degradation of fatty acids, and others take place. They also provide the environment to produce ATP, the main fuel for cellular processes. The molecular complexes of the respiratory chain located in the inner membrane create a proton gradient that is further used by the ATP-synthase, a membrane machinery, which catalyzes the production of ATP from ADP and inorganic phosphate. The structures of several components of the respiratory chain, including domains of the ATP-synthase (1, 2), are now known at quasi-atomic resolution and have shed light Jean-Jacques Lacapère (ed.), Membrane Protein Structure Determination: Methods and Protocols, Methods in Molecular Biology, vol. 654, DOI 10.1007/978-1-60761-762-4_6, © Springer Science+Business Media, LLC 2010
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Fig. 1. The MCF motif. The characteristic MCF motif is found within each repeat of 100 amino acids present three-times in MCF carriers. The limits of the helices determined in the AAC structure are superimposed on the consensus MCF sequence. It spans over the second part of an odd-numbered helix (Hn) to the first part of the following evennumbered helix (Hn+1). “a” represents an aromatic amino acid.
into major mechanisms, such as the rotary mechanism of ATP-synthase. The production of ATP as well as the function of all the major cycles is strongly connected to the import and export of metabolites between the cytosol and the mitochondrial matrix. The metabolites are small molecules with a large variety of size and chemical characteristics. They are able to cross the outer membrane relatively easily via a porin-like membrane protein, VDAC, which structure has been solved in 2008 by NMR (3) and X-ray crystallography (4). The transport through VDAC is rather unspecific and mainly limited by the size of the substrate with a slight preference for anionic molecules. On the contrary, the inner membrane is impermeable, and molecules are only transported through very specific membrane carriers. Many of these carriers belong to the MCF characterized by a specific motif of amino acids (Fig. 1) first identified by Walker et al. (5) and further described in (6). More than 40 carriers were described in human. They are encoded by the SLC25 gene family, and several are related to diseases as reviewed in (7). An overview of plant mitochondrial carriers is presented in (8). The ADP/ATP carrier (AAC) is the most abundant – it represents 10% of the proteins in bovine heart mitochondria – and was therefore studied extensively in the last 30 years (9). It serves as a model for other MCF carriers. This chapter first details the structural studies of the bovine AAC, discusses possible implications for the transport mechanism, and in particular, the import of ADP, and finally discusses general implications for the whole MCF.
2. Methods to Solve the Structure of Bovine AAC 2.1. General Strategy
The 3D structure of the bovine AAC was solved by X-ray crystallography (10). The same y
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