Conformational Changes of the Yeast Mitochondrial Adenosine Diphosphate/Adenosine Triphosphate Carrier Studied through Its Intrinsic Fluorescence. 1. Tryptophanyl Residues of the Carrier Can Be Mutate
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- 作者：Agnè ; s Le Saux ; Pierre Roux ; Vé ; ronique Tré ; zé ; guet ; Christelle Fiore ; Christine Schwimmer ; Anne-Christine Dianoux ; Pierre V. Vignais ; Gé ; rard Brandolin ; and Guy J.-M. La
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：December 17, 1996
- 年：1996
- 卷：35
- 期：50
- 页码：16116 - 16124
- 全文大小：440K
- 年卷期：v.35,no.50(December 17, 1996)
- ISSN：1520-4995
文摘
During the transport process the mitochondrial adenine nucleotidecarrier (Ancp) undergoesconformational changes which result in modifications of the intrinsicfluorescence of the carrier. Tofurther study these changes by a fluorometric approach, the threetryptophanyl residues (Trp87, Trp126,and Trp235) of the Saccharomyces cerevisiae Anc2p wereindividually mutated to their tyrosinecounterparts. The resulting mutated genes (two-Trp, one-Trp orTrp-less variants) were integrated at theANC2 locus. A prerequisite for such studies is that allthe engineered carrier molecules are still able tocatalyze ADP/ATP exchange. The cellular characteristics of thestrains expressing the mutated Anc2pand the biochemical properties of the variant Anc2p in mitochondriawere examined. Although Trp87 isabsolutely conserved in all 30 available Ancp sequences, none of thetryptophanyl residues is essential tothe carrier protein folding and the transport activity. Themutated and wild-type Anc2p were expressedto the same level, as evidenced by both ligand binding andimmunochemical analyses. When isolated inthe presence of detergent, all the variant Anc2p preparations containedergosterol in similar amounts (9mol/mol of 35 kDa Anc2p) but no specific interaction was revealed.Our results show that the tryptophan-mutated Anc2p are suitable for fluorescence studies, which are reportedin the accompanying paper byRoux et al. [(1996) Biochemistry 35,16125-16131].