Conformational changes of the yeast mitochondrial adenosine diphosphate/adenosine triphosphate carrier studied through its intrinsic fluorescence. 1. Tryptophanyl residues of the carrier can be mutated without impairing protein activity.
During the transport process the mitochondrial adenine nucleotide carrier (Ancp) undergoes conformational changes which result in modifications of the intrinsic fluorescence of the carrier. To further study these changes by a fluorometric approach, the three tryptophanyl residues (Trp87, Trp126, and Trp235) of the Saccharomyces cerevisiae Anc2p were individually mutated to their tyrosine counterparts. The resulting mutated genes (two-Trp, one-Trp or Trp-less variants) were integrated at the ANC2 locus. A prerequisite for such studies is that all the engineered carrier molecules are still able to catalyze ADP/ATP exchange. The cellular characteristics of the strains expressing the mutated Anc2p and the biochemical properties of the variant Anc2p in mitochondria were examined. Although Trp87 is absolutely conserved in all 30 available Ancp sequences, none of the tryptophanyl residues is essential to the carrier protein folding and the transport activity. The mutated and wild-type Anc2p were expressed to the same level, as evidenced by both ligand binding and immunochemical analyses. When isolated in the presence of detergent, all the variant Anc2p preparations contained ergosterol in similar amounts (9 mol/mol of 35 kDa Anc2p) but no specific interaction was revealed. Our results show that the tryptophanmutated Anc2p are suitable for fluorescence studies, which are reported in the accompanying paper by Roux et al. [(1996) Biochemistry 35, 16125-16131].
Le Saux
A
A
Laboratoire de Physiologie Moléculaire et Cellulaire, IBGC, Centre National de la Recherche Scientifique, Bordeaux, France.
Roux
P
P
Trézéguet
V
V
Fiore
C
C
Schwimmer
C
C
Dianoux
A C
AC
Vignais
P V
PV
Brandolin
G
G
Lauquin
G J
GJ
D001278:Atractyloside / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004926:Escherichia coli; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan
D017931:DNA Primers; D011994:Recombinant Proteins; D001278:Atractyloside; D014364:Tryptophan; D000226:Mitochondrial ADP, ATP Translocases