Skip to main content
. 2016 Sep 15;30(18):2119–2132. doi: 10.1101/gad.285775.116

Figure 1.

Figure 1.

Phenotypes of the med10-196 mutant. (A) Location of the mutations in med10-196. The Med10 conserved domains 1, 2, and 3 (signature-specific motif [SSM]) from Saccharomyces cerevisiae (Sc) and Homo sapiens (Hs) with the consensus generated by WebLogo were adapted from Bourbon (2008). The mutated residues corresponding to identical (for L53S, E82D, and N108I) or functionally close (I79T) amino acids in the human Med10 protein are indicated in red and blue, respectively. (B) Thermosensitive growth phenotype of the med10 mutant. Cultures of wild-type and mutant med10 yeast strains were serially diluted, spotted on YPD agar plates, and incubated for 3 d at permissive (30°C) or nonpermissive (37°C) temperatures. (C) Two-hybrid interactions of the Med10-196 protein with its partners. Wild-type or mutant Med10 was fused to the Gal4 DNA-binding domain (GDB-Med10), and Med4, Med7, Med14, Med21, and Med31 were fused to the Gal4 activation domain (GAD-Med4, GAD-Med7, GAD-Med14, GAD-Med21, and GAD-Med31). (D) Quantitative analysis of two-hybrid interactions between Med10 and Med14. Wild-type or mutant Med10 was fused to the Gal4 DNA-binding domain (GDB-Med10), and Med14 or Med21 was fused to the Gal4 activation domain (GAD-Med14 and GAD-Med21). β-Galactosidase was assayed according to the Miller method, as described in the Supplemental Material. The mean values and standard deviation (indicated by error bars) of three independent experiments are shown. Asterisk represents a significant difference between the wild type and the mutant at P-value <0.002 in a Student's t-test. (E) Silver stain SDS-PAGE analysis of purified Mediator complex from wild-type and med10 mutant strains. Cells were grown at 30°C, and core Mediator complex was purified as described in the Supplemental Material. Mediator subunits with low molecular weight were not detectable by silver staining after SDS-PAGE. (F) Mass spectrometry analysis of Mediator integrity in the mutant grown at 30°C or transferred to 37°C. Core Mediator complex containing head, middle, and tail modules was purified from the med10-196 mutant and a wild-type strain. For each Mediator subunit identified by mass spectrometry analysis in wild-type and the med10 mutant, the number of identified peptides is indicated.