Structure-function analysis of the protein-binding domains of Mac1p, a copper-dependent transcriptional activator of copper uptake in Saccharomyces cerevisiae.
The Mac1 protein in Saccharomyces cerevisiae is essential for the expression of yeast high affinity copper uptake. A positive transcription factor, Mac1p binds via its N-terminal domain to GCTC elements in the promoters of CTR1 and FRE1, encoding a copper permease and metal reductase, respectively. Mac1p-dependent transcriptional activation is negatively regulated by copper. We have mapped the domains in Mac1p responsible for its nuclear localization and for the protein-protein interactions that underlie its transcriptional activity. Immunofluorescence studies indicate that Mac1p contains two nuclear localization signals, one each in the N- and C-terminal halves of the protein. Yeast one-hybrid analysis demonstrates that the copper-dependent transcriptional activity in Mac1p resides primarily in a cysteine-rich element encompassing residues 264-279. Two-hybrid analysis indicates that a copper-independent Mac1p-Mac1p interaction linked to DNA binding is due primarily to a predicted helix in the C-terminal region of the protein encompassing residues 388-406. Point mutations within this putative helix abrogate the Mac1-Mac1 interaction in vivo and formation of a ternary (Mac1p)(2).DNA complex in vitro. When produced in normal abundance, Mac1pI396D and Mac1pF400D helix mutants do not support transcriptional activation in vivo consistent with an essential Mac1p dimerization in transcriptional activation. Lastly, the one- and two-hybrid data indicate that an intramolecular interaction between the DNA-binding and transactivation domains negatively modulates Mac1p activity.
Serpe
M
M
Department of Biochemistry, School of Medicine, State University of New York at Buffalo, Buffalo, New York 14214, USA.
Joshi
A
A
Kosman
D J
DJ
D001665:Binding Sites; D027682:Cation Transport Proteins; D003300:Copper / Q000378:metabolism / Q000494:pharmacology*; D000079923:Copper Transporter 1; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics
C490078:CTR1 protein, S cerevisiae; D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C084954:MAC1 protein, S cerevisiae; D008565:Membrane Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D003300:Copper