The ABC transporter Mdl1p, a structural and functional homologue of the transporter associated with antigen processing (TAP) plays an important role in intracellular peptide transport from the mitochondrial matrix of Saccharomyces cerevisiae. To characterize the ATP hydrolysis cycle of Mdl1p, the nucleotide-binding domain (NBD) was overexpressed in Escherichia coli and purified to homogeneity. The isolated NBD was active in ATP binding and hydrolysis with a turnover of 25 ATP per minute and a K m of 0.6 mM and did not show cooperativity in ATPase activity. However, the ATPase activity was non-linearly dependent on protein concentration (Hill coefficient of 1. ATP-binding cassette (ABC)1 transporters comprise a large family of membrane proteins that catalyze the active transfer of a variety of solutes across biological membranes (1). The function of ABC transporters is central to various human pathologies such as cystic fibrosis, adrenoleukodystrophy, retinal dystrophies, and multidrug resistance. The transporter associated with antigen processing (TAP) is an ABC transporter in vertebrates, which translocates peptides from the cytosol into the ER and performs a key function in the antigen presentation and adaptive immune response (2). Recently, a close homologue, Mdl1p (multidrug resistance like), localized in the inner mitochondrial membrane of Saccharomyces cerevisiae, has been identified as an intracellular peptide transporter (3). This transporter exports peptides derived from the degradation of non-assembled membrane proteins. These peptides are generated by ATP-dependent m-AAA (matrix-oriented ATPases associated with a variety of cellular activities) proteases, which mediate the degradation and turnover of inner mitochondrial membrane proteins and short-lived regulatory proteins in an ubiquitin/proteasome-independent manner (4). Protein fragments with a length of 6 -21 amino acids are released by Mdl1p into the intermembrane space (3).Half-size ABC transporters, like the heterodimeric TAP and homodimeric Mdl1p, have a common molecular architecture consisting of two polytopic transmembrane domains (TMD) and two nucleotide-binding domains (NBD). The transmembrane domains interact with the substrates and form the substrate translocation pore across the membrane. The TMDs generally share little homology (5), probably caused by the broad substrate spectrum of the ABC transporter family. Binding and hydrolysis of nucleotides drive the transport process by transducing conformational changes from the NBDs to the TMDs. The similarity of different NBDs is significantly higher compared with the TMDs, suggesting that even in transporters of unrelated function the structure and function of the NBDs be highly conserved. Each NBD contains a highly conserved Walker A and Walker B motif (6) characteristic of ATP-binding P-loop proteins, as well as the C-loop motif (LSGGQ) unique to ABC proteins, which is also known as the ABC signature motif. The crystal structures of bacterial ABC transporters (e.g. MsbA, BtuCD) and of is...

The ATP-binding cassette half-transporter Mdl1 from Saccharomyces cerevisiae has been proposed to be involved in the quality control of misassembled respiratory chain complexes by exporting degradation products generated by the m-AAA proteases from the matrix. Direct functional or structural data of the transport complex are, however, not known so far. After screening expression in various hosts, Mdl1 was overexpressed 100-fold to 1% of total mitochondrial membrane protein in S.

ATP-binding cassette (ABC) transporters belong to a large family of membrane proteins found in all three kingdoms of life. The chemical energy of ATP is used to drive uphill transport of a broad range of solutes across membranes [1][2][3]. ABC transporters have a conserved domain organization consisting of two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). The TMDs form a translocation pore, whereas the NBDs catalyze ATP hydrolysis.The ABC half-transporter multidrug resistance like protein 1 (MDL1), composed of a TMD followed by a NBD, is located in the inner mitochondrial membrane (IMM) of Saccharomyces cerevisiae. It has been suggested to be involved in the export of 6-mer to 20-mer peptides, derived from proteolysis of nonassembled inner membrane proteins by the m-AAA (i.e. matrixoriented ATPase associated with a variety of cellular activities) protease [4]. It has been further reported that MDL1 mediates resistance against oxidative stress and can partially complement the function of ABC transporter of mitochondria (ATM) 1 [5]. Deletion of ATM1 in S. cerevisiae results in a severe growth defect because ATM1 is essential for the biogenesis of cytosolic iron-sulfur (Fe-S) proteins [6]. The ATP-binding cassette transporter MDL1 of Saccharomyces cerevisiae has been implicated in mitochondrial quality control, exporting degradation products of misassembled respiratory chain complexes. In the present study, we identified an unusually long leader sequence of 59 amino acids, which targets MDL1 to the inner mitochondrial membrane with its nucleotidebinding domain oriented to the matrix. By contrast, MDL1 lacking this leader sequence is directed into the endoplasmic reticulum membrane with the nucleotide-binding domain facing the cytosol. Remarkably, in both targeting routes, the ATP-binding cassette transporter maintains its intrinsic properties of membrane insertion and assembly, leading to homooligomeric complexes with similar activities in ATP hydrolysis. The physiological consequences of both targeting routes were elucidated in cells lacking the mitochondrial ATP-binding cassette transporter ATM1, which is essential for biogenesis of cytosolic iron-sulfur proteins. The mitochondrial MDL1 complex can complement ATM1 function, whereas the endoplasmic reticulumtargeted version, as well as MDL1 mutants deficient in ATP binding and hydrolysis, cannot overcome the Datm1 growth phenotype.Abbreviations ABC, ATP-binding cassette; ATM, ABC transporter of mitochondria; ER, endoplasmic reticulum; 5-FOA, 5-fluoroorotic acid; IMM, inner mitochondrial membrane; MDL1, multidrug resistance like protein 1; MTS, mitochondrial targeting signal; NBD, nucleotide-binding domain; SC, synthetic complete; TIM, translocase of the inner mitochondrial membrane; TOM, translocase of the outer mitochondrial membrane; TMD, transmembrane domain.

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