Chimers of two fused ADP/ATP carrier monomers indicate a single channel for ADP/ATP transport.
The mitochondrial ADP/ATP carrier (AAC) is generally believed to function as a homodimer (Wt. Wt). It remains unknown whether the two monomers possess two independent but fully anticooperative channels or they form a single central channel for nucleotide transport. Here we generated fusion proteins consisting of two tandem covalent-linked AAC monomers and studied the kinetics of ADP/ATP transport in reconstituted proteoliposomes. Functional 64-kDa fusion proteins Wt-Wt and Wt-R294A (wild-type AAC linked to a mutant having low ATP transport activity) were expressed in mitochondria of yeast transformants. Compared to homodimer Wt. Wt, the fusion protein Wt-Wt retained the transport activity and selectivity of ADP versus ATP. The strongly divergent selectivities of Wt and R294A were partially propagated in the Wt-R294A fusion protein, suggesting a limited cooperativity during solute translocation. The rates of ADP or ATP transport were significantly higher than those predicted by the two-channel model. Fusion proteins for Wt-R204L (Wt linked to an inactive mutant) and R204L-Wt were not expressed in aerobically grown yeast cells, which contained plasmid rearrangements that regenerated the fully active 32-kDa homodimer Wt. Wt, suggesting that these fusion proteins are inactive in ADP/ATP transport. These results favor a single binding center gated pore model [Klingenberg, M. (1991) in A Study of Enzymes, Vol. 2: pp. 367-388] in which two AAC subunits cooperate for a coordinated ADP/ATP exchange through a single channel.
Huang
S G
SG
Institute of Physical Biochemistry, University of Munich, Schillerstrasse 44, Munich, D-80336, Germany. shuang@tularik.com
Odoy
S
S
Klingenberg
M
M
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001692:Biological Transport; D015153:Blotting, Western; D019281:Dimerization; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics; D011487:Protein Conformation; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
C112225:PET9 protein, S cerevisiae; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C020485:proteoliposomes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases