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 microm) 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.
Hofacker
Matthias
M
Institute of Biochemistry, Biocenter, Johann Wolfgang Goethe University, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany.
Gompf
Simone
S
Zutz
Ariane
A
Presenti
Chiara
C
Haase
Winfried
W
van der Does
Chris
C
Model
Kirstin
K
Tampé
Robert
R
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology / Q000648:ultrastructure; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019281:Dimerization; D008081:Liposomes; D024101:Mitochondrial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology / Q000648:ultrastructure; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D011487:Protein Conformation; D011510:Proteolipids / Q000737:chemistry / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology / Q000648:ultrastructure; D012995:Solubility; D013329:Structure-Activity Relationship
D018528:ATP-Binding Cassette Transporters; D008081:Liposomes; C490069:MDL1 protein, S cerevisiae; D024101:Mitochondrial Proteins; D011510:Proteolipids; D029701:Saccharomyces cerevisiae Proteins; C020485:proteoliposomes; D000251:Adenosine Triphosphatases