title: Conformational changes play a role in regulating the activity of the proline utilization pathway-specific regulator in Saccharomyces cerevisiae.
abstract: In Saccharomyces cerevisiae, the ability to use proline as a nitrogen source requires the Put3p transcriptional regulator, which turns on the expression of the proline utilization genes, PUT1 and PUT2, in the presence of the inducer proline and in the absence of preferred nitrogen sources. Changes in target gene expression occur through an alteration in activity of the DNA-bound Put3p, a member of the Zn(II)2Cys6 binuclear cluster family of proteins. Here, we report that the 'on' conformation can be mimicked in the absence of proline by the insertion of an epitope tag in several different places in the protein, as well as by specific amino acid changes that suppress a put3 mutation leading to non-inducibility of the pathway. In addition, the presence of proline causes a conformational change in the Put3 protein detected by increased sensitivity to thrombin or V8 protease. These findings suggest that Put3p shifts from an inactive to an activate state via conformational changes.
authors.affiliation: Department of Microbiology and Molecular Genetics, Room MSB F-607, UMDNJ - New Jersey Medical School, 185 S. Orange Ave., Newark, NJ 07103, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: Des Etages
authors.forename: D
authors.initials: D
authors.lastname: Saxena
authors.forename: H L
authors.initials: HL
authors.lastname: Huang
authors.forename: D A
authors.initials: DA
authors.lastname: Falvey
authors.forename: D
authors.initials: D
authors.lastname: Barber
authors.forename: M C
authors.initials: MC
authors.lastname: Brandriss
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism; D000939:Epitopes / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors
chemical_list: D004271:DNA, Fungal; D000939:Epitopes; D005656:Fungal Proteins; C068399:PUT3 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D011392:Proline; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D013917:Thrombin