In vitro characterization of the Mig1 repressor from Saccharomyces cerevisiae reveals evidence for monomeric and higher molecular weight forms.
The Mig1 DNA-binding protein of Saccharomyces cerevisiae was expressed and purified from yeast and the physical properties were characterized by several methods, including gel filtration, sucrose gradient sedimentation and native gel electrophoresis. Purified Mig1 exists as a monomer with a Stokes' radius of 48 A and a sedimentation coefficient of 3.55 S. Mig1 has an elongated shape with a frictional coefficient of 1.83. The K(d) of purified Mig1 for the SUC2 A site is 2.8 nM and for SUC2 B site 25.8 nM; these values were similar for Mig1 purified from repressed and derepressed cells. Full-length Mig1 expressed in yeast binds more tightly to SUC2 B than bacterially expressed GST-Mig1. Sucrose gradient sedimentation resolved a larger molecular weight form of Mig1 in whole-cell extracts that was not seen in purified samples and may represent a complex with another protein. This complex is found within the nucleus and is seen only in repressed cells. Mig1 exists in multiple phosphorylation states and only less phosphorylated forms of Mig1 are associated with this complex.
Needham
Patrick G
PG
Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, 3035 Arlington Avenue, Toledo, OH 43614, USA. needhamp@niddk.nih.gov
Trumbly
Robert J
RJ
D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004586:Electrophoresis / Q000379:methods; D015966:Gene Expression Regulation, Fungal; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043324:beta-Fructofuranosidase / Q000235:genetics
D004268:DNA-Binding Proteins; C085976:MIG1 protein, S cerevisiae; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C468437:SUC2 protein, S cerevisiae; D043324:beta-Fructofuranosidase