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...