University of Groningen The yeast mitochondrial ADP/ATP ... fileSI Text SI Text SI Results SI...
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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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