Functional characterisation of the mitochondrial ABC transport complex MDL1 from Saccharomyces cerevisiae

Für den mitochondrialen ABC-Transporter MDL1 (multidrug resistance like) aus Saccharomyces cerevisiae wurde eine Funktion als intrazellulärer Peptidexporter vorhergesagt. MDL1 ist wahrscheinlich am Export von Degradationsprodukten der m-AAA (matrixoriented ATPases associated with a variety of cellular activities) Protease in den Intermembranraum beteiligt (Young et al., 2001). Das MDL1-Homodimer besteht aus zwei Transmembrandomänen mit jeweils sechs potentiellen α-Helices und zwei Nukleotidbindedomänen. Eine Überexpression des ABC-Transporters in E. coli und L. lactis ist nicht möglich. Nur im homologen Expressionssystem kann eine bis zu 100-fach gesteigerte MDL1-Konzentration in Anwesenheit des induzierbaren GAL1-Promotors gegenüber dem endogenen Protein erreicht werden. Differentielle Zentrifugation, Immunogold-Markierungen und Proteasezugänglichkeitsexperimente zeigen, dass MDL1 ausschließlich in der mitochondrialen Innenmembran lokalisiert ist und die Nukleotidbindedomänen zur Matrix orientiert vorliegen. Mit Hilfe von Edman Sequenzierung des gereinigten His-getaggten MDL1 wurde eine 59 Aminosäuren lange mitochondriale Leitsequenz identifiziert. Die Deletionsvariante MDL1(60-695) wird ausschließlich in den Membranen des Endoplasmatischen Retikulums exprimiert. Ihre Motordomänen liegen zytosolisch orientiert vor. Beide MDL1-Varianten bilden homooligomere Komplexe vergleichbarer Größe und weisen ähnliche ATPase Aktivitäten auf. Die physiologischen Konsequenzen der Lokalisation in unterschiedlichen Membranen wurden in Zellen näher untersucht, deren mitochondrialer ABC-Transporter ATM1 (ABC transporter of mitochondria) deletiert ist. ATM1 ist von essentieller Bedeutung für die Biogenese zytosolischer Eisen/Schwefel-Proteine (Lill und Kispal, 2000). Der mitochondriale MDL1-Komplex kann zum Teil die ATM1-Funktion übernehmen, wohingegen ER-ständiges MDL1, als auch ATP Binde- und Hydrolyse inaktive Mutanten, den Δatm1 Wachstumsphänotyp nicht komplementieren können. Die physiologische Funktion von MDL1 ist somit eng mit der mitochondrialen Innenmembran und der Funktionalität des Proteins verbunden. Durch in vivo Komplementationsstudien wurden zwei mitochondriale ABC-Transporter ABCB10 und Pa_2_9660 aus H. sapiens bzw. P. anserina als funktionelle MDL1-Homologe identifiziert.The mitochondrial ABC transporter MDL1 (multidrug resistance like) of Saccharomyces cerevisiae is postulated to be involved in the export of peptides, derived from proteolysis of non-assembled or misassembled respiratory chain complexes out of the mitochondrial matrix (Young et al., 2001). MDL1 forms a homodimeric complex consisting of two transmembrane domains with six putative α-helices and two nucleotide-binding domains. We could not achieve heterologous over-expression of MDL1 in the organisms E. coli and L. lactis. On the other hand a 100-fold homologous over-expression was possible in S. cerevisiae. In this case a plasmidborne copy of MDL1 under the control of the inducible GAL1-promoter was used. Subcellular fractionation, immunogold labeling and protease protection assays identified that MDL1 exclusively resides in the inner mitochondrial membrane with its motor domains focus to the matrix. By Edman sequencing of purified His-tagged MDL1 an N-terminal mitochondrial leader sequence of 59 residues was identified, which is cleaved off in the matrix. Strikingly, MDL1 which lacks its presequence is directed to tubulo-vesicular membranes resembling the endoplasmic reticulum, where the NBDs are located in the cytosol. Within both targeting routes, the ABC transporter maintains a default membrane insertion and assembly pathway leading to homooligomeric complexes of comparable sizes and similar activities in ATP hydrolysis. The physiological consequences of both targeting routes were elucidated in cells lacking the mitochondriale ABC transporter ATM1 (ABC transporter of mitochondria), which is essential for biogenesis of cytosolic iron-sulfur proteins (Lill und Kispal, 2000). The mitochondrial MDL1 complex can complement ATM1 function, whereas the ER-targeted version as well as MDL1 mutants deficient in ATP binding and hydrolysis cannot overcome the Δatm1 growth phenotyp. These findings demonstrate that the physiological function of the ABC transporter MDL1 is intimately linked to its correct targeting to the inner mitochondrial membrane. Furthermore, the human mitochondrial ABC-transporter ABCB10 as well as the MDL1 homolog in P. anserina can complement the ATM1 growth phenotyp

The ATP hydrolysis cycle of the nucleotide-binding domain of the mitochondrial ATP-binding cassette transporter Mdl1p

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 Km 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 1.7), indicating that the functional state is a dimer. Dimeric catalytic transition states could be trapped either by incubation with orthovanadate or beryllium fluoride, or by mutagenesis of the NBD. The nucleotide composition of trapped intermediate states was determined using [alpha-32P]ATP and [gamma-32P]ATP. Three different dimeric intermediate states were isolated, containing either two ATPs, one ATP and one ADP, or two ADPs. Based on these experiments, it was shown that: (i) ATP binding to two NBDs induces dimerization, (ii) in all isolated dimeric states, two nucleotides are present, (iii) phosphate can dissociate from the dimer, (iv) both nucleotides are hydrolyzed, and (v) hydrolysis occurs in a sequential mode. Based on these data, we propose a processive-clamp model for the catalytic cycle in which association and dissociation of the NBDs depends on the status of bound nucleotides

Structural and functional fingerprint of the mitochondrial ATP-binding cassette transporter Mdl1 from Saccharomyces cerevisiae

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. cerevisiae. Based on detergent screens, Mdl1 was solubilized and purified to homogeneity. Mdl1 showed a high binding affinity for MgATP (Kd = 0.26 μm) and an ATPase activity with a Km of 0.86 mm (Hill coefficient of 0.98) and a turnover rate of 2.6 ATP/s. Mutagenesis of the conserved glutamate downstream of the Walker B motif (E599Q) or the conserved histidine of the H-loop (H631A) abolished ATP hydrolysis, whereas ATP binding was not affected. Mdl1 reconstituted into liposomes showed an ATPase activity similar to the solubilized complex. By single particle electron microscopy, a first three-dimensional structure of the mitochondrial ATP-binding cassette transporter was derived at 2.3-nm resolution, revealing a homodimeric complex in an open conformation