University of Groningen

The yeast mitochondrial ADP/ATP carrier functions as a monomer in mitochondrialmembranesBamber, Lisa; Harding, Marilyn; Monné, Magnus; Slotboom, Dirk; Kunji, Edmund R.S.;Barber, LPublished in:Default journal

DOI:10.1073/pnas.0703969104

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Publication date:2007

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Citation for published version (APA):Bamber, L., Harding, M., Monné, M., Slotboom, D-J., Kunji, E. R. S., & Barber, L. (2007). The yeastmitochondrial ADP/ATP carrier functions as a monomer in mitochondrial membranes. Default journal, 104,10830 - 10834. DOI: 10.1073/pnas.0703969104

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The yeast mitochondrial ADP/ATP carrier functions as a monomer in mitochondrial membranes

Bamber et al. 10.1073/pnas.0703969104.

Supporting Information

Fig. 4.Fig. 4.Fig. 4.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.Fig. 5.Fig. 5.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.

FigFigFigFig. 6.. 6.. 6.. 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.Fig. 7.Fig. 7.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. 1111 and 2222.

SI TextSI TextSI TextSI Text

SI ResultsSI ResultsSI ResultsSI 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 MethodsSI MethodsSI MethodsSI 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 Destabilization of Liposomes Destabilization of Liposomes Destabilization of Liposomes for Reconstitution.for Reconstitution.for Reconstitution.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.

SodiumSodiumSodiumSodium----dodecylsulfatedodecylsulfatedodecylsulfatedodecylsulfate----polyacrylamide Gel Electrophoresis and Western Blot polyacrylamide Gel Electrophoresis and Western Blot polyacrylamide Gel Electrophoresis and Western Blot polyacrylamide Gel Electrophoresis and Western Blot

Analysis.Analysis.Analysis.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).

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