title: Pichia pastoris 14-3-3 regulates transcriptional activity of the methanol inducible transcription factor Mxr1 by direct interaction.
abstract: The zinc-finger transcription factor, Mxr1 activates methanol utilization and peroxisome biogenesis genes in the methylotrophic yeast, Pichia pastoris. Expression of Mxr1-dependent genes is regulated in response to various carbon sources by an unknown mechanism. We show here that this mechanism involves the highly conserved 14-3-3 proteins. 14-3-3 proteins participate in many biological processes in different eukaryotes. We have characterized a putative 14-3-3 binding region at Mxr1 residues 212-225 and mapped the major activation domain of Mxr1 to residues 246-280, and showed that phenylalanine residues in this region are critical for its function. Furthermore, we report that a unique and previously uncharacterized 14-3-3 family protein in P. pastoris complements Saccharomyces cerevisiae 14-3-3 functions and interacts with Mxr1 through its 14-3-3 binding region via phosphorylation of Ser215 in a carbon source-dependent manner. Indeed, our in vivo results suggest a carbon source-dependent regulation of expression of Mxr1-activated genes by 14-3-3 in P. pastoris. Interestingly, we observed 14-3-3-independent binding of Mxr1 to the promoters, suggesting a post-DNA binding function of 14-3-3 in regulating transcription. We provide the first molecular explanation of carbon source-mediated regulation of Mxr1 activity, whose mechanism involves a post-DNA binding role of 14-3-3.
authors.affiliation: Department of Biochemistry, University of Washington, 1705 NE Pacific Street, Seattle, Washington 98195-7350, USA.
authors.forename: Pabitra K
authors.initials: PK
authors.lastname: Parua
authors.forename: Paul M
authors.initials: PM
authors.lastname: Ryan
authors.forename: Kayla
authors.initials: K
authors.lastname: Trang
authors.forename: Elton T
authors.initials: ET
authors.lastname: Young
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005816:Genetic Complementation Test; D000432:Methanol / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D014157:Transcription Factors; D000432:Methanol