Yeast mitochondrial ADP/ATP carriers are monomeric in detergents as demonstrated by differential affinity purification.
Most mitochondrial carriers carry out equimolar exchange of substrates and they are believed widely to exist as homo-dimers. Here we show by differential tagging that the yeast mitochondrial ADP/ATP carrier AAC2 is a monomer in mild detergents. Carriers with and without six-histidine or hemagglutinin tags were co-expressed in defined molar ratios in yeast mitochondrial membranes. Their specific transport activity was unaffected by tagging or by co-expression. The co-expressed carriers were extracted from the membranes with mild detergents and purified rapidly by affinity chromatography. All of the untagged carriers were in the flow-through of the affinity column, whereas all of the tagged carriers bound to the column and were eluted subsequently, showing that stable dimers, consisting of associated tagged and untagged carriers, were not present. The specific inhibitors carboxyatractyloside and bongkrekic acid and the substrates ADP, ATP and ADP plus ATP were added during the experiments to determine whether lack of association might have been caused by carriers being prevented from cycling through the various states in the transport cycle where dimers might form. All of the protein was accounted for, but stable dimers were not detected in any of these conditions, showing that yeast ADP/ATP carriers are monomeric in detergents in agreement with their hydrodynamic properties and with their structure. Since strong interactions between monomers were not observed in any part of the transport cycle, it is highly unlikely that the carriers function cooperatively. Therefore, transport mechanisms need to be considered in which the carrier is operational as a monomer.
Bamber
Lisa
L
The Medical Research Council, Dunn Human Nutrition Unit, Hills Road, CB2 2XY, Cambridge, UK.
Slotboom
Dirk-Jan
DJ
Kunji
Edmund R S
ER
D002846:Chromatography, Affinity; D003902:Detergents / Q000494:pharmacology*; D004072:Digitonin / Q000494:pharmacology*; D015870:Gene Expression; D000226:Mitochondrial ADP, ATP Translocases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology*; D011485:Protein Binding; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D003902:Detergents; C112225:PET9 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D017830:Octoxynol; D000226:Mitochondrial ADP, ATP Translocases; D004072:Digitonin