The redox domain of the Yap1p transcription factor contains two disulfide bonds.
The subcellular localization of the Saccharomyces cerevisiae transcription factor Yap1p is regulated by oxidation and reduction. We purified Yap1p from yeast and characterized its properties in vitro. Electrophoretic mobility shift assays showed that the purified protein can specifically bind the TRX2 target promoter. Yap1p was purified under reducing conditions, but removal of reducing agents resulted in the formation of an oxidized Yap1p species with properties similar to in vivo oxidized Yap1p. MALDI-TOF mass spectrometry analysis revealed that the oxidized form of Yap1p contains two disulfide bonds between C303-C598 and C310-C629. A stable domain of approximately 15 kDa was detected upon limited proteolysis of oxidized but not reduced Yap1p. This Yap1p protease resistant domain was purified, and MALDI-TOF mass spectrometry analysis showed that it was comprised of two separate cysteine-containing peptides of Yap1p. These peptides are separated by 250 amino acids and are joined by the C303-C598 and C310-C629 disulfide bonds. Taken together, these data suggest that the domain that controls Yap1p subcellular localization is modular and contains a redox center comprised of four cysteine residues.
Wood
Matthew J
MJ
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-5430, USA.
Andrade
Erika C
EC
Storz
Gisela
G
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D004220:Disulfides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C086677:YAP1 protein, S cerevisiae; D006639:Histidine; D003545:Cysteine