Bamber et al. 10.1073/pnas.0703969104. |
Fig. 4.
Amino acid sequence alignment of bovine AAC1 and yeast AAC2. The arrows indicate the position of the cysteines in AAC2, which were replaced by alanines. Note that the Cys73 and Cys271, which mediate the effect of MTSES on the transport activity, are conserved in the bovine and yeast ADP/ATP carrier.
Fig. 5.
Expression of wild-type AAC2, cysteine-less AAC2, single and double cysteine mutants of AAC2. (A) Coomassie blue-stained sodium-dodecylsulfate polyacrylamide and (B) Western blot of mitochondrial membranes probed with α-AAC2 primary antibody, expressing wild-type AAC2 (CCCC), cysteine-less AAC2 (AAAA), and the single cysteine mutants of AAC2. (C and D) As in SI Fig. 5 A and B, but with the double cysteine mutants of AAC2. The single and double cysteine mutants of the cysteine-less carrier (AAAA) are named by the cysteine replacement in the four positions (Fig. 1). The wild-type AAC2 is called CCCC accordingly. Approximately 12 μg and 1 μg of total protein were used per lane of the gel and Western blot, respectively. Closed arrowheads indicate the approximate molecular mass of AAC2.
Fig. 6.
The residual initial transport rate of the wild-type, cysteine-less, and single- and double-cysteine replacement AAC2 after the addition of MTSES. The residual initial transport rate after the addition of MTSES was expressed as a percentage of the rate in the absence of MTSES. The specific rate in the absence of MTSES (100%, dotted line) was approximately 20 nmol×min-1×mg-1 of AAC2. The nomenclature is described in the legend to SI Fig. 5.
Fig. 7.
Correlation between the fraction of cysteine-less AAC2 and the residual transport rate after the addition of MTSES, assuming a functional monomer and dimer. The cysteine-less and wild-type AAC2 are shown in orange and yellow, respectively. The blue ball indicates the poly-histidine tag, which is required for the separation of tagged and untagged AAC2 by sodium-dodecylsulfate electrophoresis. The wild-type AAC2 is inhibited by MTSES, whereas the cysteine-less carrier is not. (A) The fraction of cysteine-less AAC2 is 0.25. The residual initial uptake rate after addition of MTSES is 0.25, if the carrier functions as a monomer, whereas it is (0.25)2 = 0.0625 if the carrier functions as a dimer. (B) The fraction of cysteine-less AAC2 is 0.75. The residual initial uptake rate after addition of MTSES is 0.75, if the carrier functions as a monomer, whereas it is (0.75)2 = 0.5625 if the carrier functions as a dimer. In general, the correlation of dependent and independent functional association is described by Eqs. 1 and 2.
SI Text
SI Results
The yeast ADP/ATP carrier AAC2 has cysteines at residue position 73 in matrix α-helix h12, at 244 in transmembrane α-helix H5, at 271 in matrix α-helix h56, and at 288 in transmembrane α-helix H6 (Fig. 1). The wild-type and cysteine-less AAC2 are designated as CCCC and AAAA, respectively, referring to the position of the four cysteines and four alanines in the wild-type AAC2 and cysteine-less AAC2, respectively. The contribution of each cysteine to the inhibitory effect on the transport activity of AAC2 was determined. Single cysteines were introduced into the cysteine-less AAC2, generating mutant carriers CAAA, ACAA, AACA and AAAC. The resulting mutant carriers were expressed in yeast mitochondrial membranes to approximately the same levels as AAC2 (SI Fig. 6). The effect of MTSES on transport activity of the single cysteine mutant carriers was determined (SI Fig. 7). The transport activities of CAAA and AACA were affected most by the addition of MTSES. These two cysteines are present in the α-helices of the matrix loops h12 and h56 in the first and third domain of the carrier, respectively (Fig. 1). The transport activities of the single cysteine mutant carriers ACAA and AAAC were not significantly affected by MTSES (SI Fig. 7), and these cysteines are present in the transmembrane α-helices H5 and H6 (Fig. 1). Two double cysteine mutant carriers were created (CACA and ACAC) and expression trials showed that they were expressed to similar levels as the wild-type and cysteine-less carriers as well (SI Fig. 6). Transport assays indicated that the transport activity of mutant carrier with Cys73 and Cys271 (CACA) was inhibited by MTSES, whereas the mutant carrier with Cys244 and Cys288 (ACAC) was slightly, but not significantly, affected by MTSES (Fig. 7). Thus, the effect of MTSES on the transport activity can be attributed mainly to Cys73 and Cys271 and to a much lesser extent to Cys244 and Cys288. In the structure of the bovine AAC1 in the cytoplasmic state (1), the equivalent residues of Cys73 and Cys271 are buried inside the protein and they are inaccessible to the water phase and reagents. Cross-linking reagents that target the same cysteines, do not react with the ADP/ATP carrier when it is locked in the cytoplasmic-state by carboxy-atractyloside, whereas they do when the carrier is locked in the matrix-state by bongkrekic acid (2). Thus, these cysteines may become accessible when the carrier is in the matrix-state. Once MTSES modification has taken place, the carrier may be prevented from returning to the cytoplasmic-state to complete the transport cycle.
SI Methods
Growth of Yeast Strains. Two 50-ml cultures of synthetic complete medium minus tryptophan (SC-Trp) supplemented with 3% (vol/vol) glycerol and 0.05% (wt/vol) glucose were inoculated with a single colony from a SC-Trp plus 3% (vol/vol) glycerol plate. The cultures were incubated at 30ºC with shaking overnight. Two 2-liter flasks containing 500 ml of YPG medium (10 g/liter yeast extract, 20 g/liter peptone, 30 ml/liter glycerol) were inoculated with the overnight cultures to give an A600 of »0.05. The cells were harvested by centrifugation (3,000 ´ g for 5 min) and washed twice with deionised water.
Destabilization of Liposomes for Reconstitution.
The amount of Triton X-100 required for destabilization of the liposomes to facilitate insertion of the purified protein was determined. Extruded liposomes (10 mg) were added to 2 ml of KPi buffer with 0.05% (vol/vol) Triton X-100 and mixed. After 2 min, the A600 was measured and an additional 0.05% Triton X-100 was added. This process was repeated until the absorbance began to decrease.
Sodium-dodecylsulfate-polyacrylamide Gel Electrophoresis and Western Blot Analysis.
Proteins were separated by sodium-dodecylsulfate polyacrylamide gel electrophoresis in gels consisting of 15% polyacrylamide (Severn Biotech, Kidderminster, U.K.) at 30 mA for 90 min. Protein bands were visualized with Coomassie stain (50% (vol/vol) methanol, 10% (vol/vol) acetic acid, 0.1% (wt/vol) Coomassie blue R250) followed by destaining with 15% (vol/vol) methanol and 10% (vol/vol) acetic acid. Proteins were transferred electrophoretically to a polyvinylidenefluoride membrane (Immobilon-P; Millipore, Billerica, MA) at 120 mA for 1 h. Before transfer, the polyvinylidenefluoride membrane was activated with methanol and washed in transfer buffer consisting of 0.025 M Tris×HCl, 192 mM glycine, and 10% (vol/vol) methanol. Nonspecific binding of the antibody to the membrane was prevented by incubating the blot for »16 h in blocking buffer, consisting of phosphate-buffered saline (PBS), 0.1% (vol/vol) Tween 20, and 5% (wt/vol) skimmed milk powder (Chivers Ireland, Dublin, Ireland. Proteins were detected with chick α-AAC primary antibody (custom-made by AgriSera, Vδnnäs, Sweden) at 1:25,000. Primary antibody was incubated for 4 h with agitation. Then, the membrane was washed 3 times with PBS and 0.1% (vol/vol) Tween for 10 min. The membrane was incubated for 2 h with rabbit α-chick IgY peroxidase conjugate (Sigma) at a titer of 1:25,000 in PBS and 0.1% (vol/vol) Tween. The membrane was washed three times with PBS and 0.1% (vol/vol) Tween for 10 min and the labeled protein was detected by ECL (G.E. Healthcare).
1. Pebay-Peyroula E, Dahout-Gonzalez C, Kahn R, Trezeguet V, Lauquin GJ, Brandolin G (2003) Nature 426:39-44.
2. Majima E, Ikawa K, Takeda M, Hashimoto M, Shinohara Y, Terada H (1995) J Biol Chem 270:29548-29554.