A high resolution protein interaction map of the yeast Mediator complex

Abstract

Mediator is a large, modular protein complex remotely conserved from yeast to man that conveys regulatory signals from DNA-binding transcription factors to RNA polymerase II. In Saccharomyces cerevisiae, Mediator is thought to be composed of 24 subunits organized in four sub-complexes, termed the head, middle, tail and Cdk8 (Srb8-11) modules. In this work, we have used screening and pair-wise two-hybrid approaches to investigate protein-protein contacts between budding yeast Mediator subunits. The derived interaction map includes the delineation of numerous interaction domains between Mediator subunits, frequently corresponding to segments that have been conserved in evolution, as well as novel connections between the Cdk8 (Srb8-11) and head modules, the head and middle modules, and the middle and tail modules. The two-hybrid analysis, together with co-immunoprecipitation studies and gel filtration experiments revealed that Med31 (Soh1) is associated with the yeast Mediator that therefore comprises 25 subunits. Finally, analysis of the protein interaction network within the Drosophila Mediator middle module indicated that the structural organization of the Mediator complex is conserved from yeast to metazoans. The resulting interaction map provides a framework for delineating Mediator structure-function and investigating how Mediator function is regulated.

The Mediator Complex in Plants: Structure, Phylogeny, and Expression Profiling of Representative Genes in a Dicot (Arabidopsis) and a Monocot (Rice) during Reproduction and Abiotic Stress

“…4). Yeast two-hybrid screens support the interaction of Med17 with Med8C and -22 as well as Med6 (Guglielmi et al, 2004). That study also identified interactions between Med22 and -11 as well as Med16 and -5.…”

“…The yeast Med14N (amino acid residues 1-259) connects the tail to the middle module through Med10 (Guglielmi et al, 2004); moreover, in Arabidopsis, Med14N (amino acids 1-959) interacts with a transcription regulator (Gonzalez et al, 2007). The yeast and Arabidopsis Med14 sequences aligned only for 171 amino acids in the N-terminal region, showing only 21% identity and 41% similarity (data not shown).…”

“…2), with the fungal/metazoan Med7, -18, -21, and -31 being represented in all seven groups. In yeast, Med7 and -21 are important for cell viability and constitute the core along with eight other subunits upon which other Med subunits assemble (Björklund et al, 2001;Kang et al, 2001;Guglielmi et al, 2004). High diversity in Med subunits was observed for the plant-specific Meds and those with unknown functions (Fig.…”

“…Secondary structure comparison of Med4 highlighted the high level of homology these proteins shared, including in the regions that have been shown to interact with Med7 and -21 (Guglielmi et al, 2004;Fig. 4).…”

The Arabidopsis Mediator subunit MED16 regulates iron homeostasis by associating with EIN3/EIL1 through subunit MED25

“…The Mediator complex was first identified in yeast, in which the detailed proteinprotein interaction network of the Mediator subunits was established (Guglielmi et al, 2004). The Mediator complex is conserved in eukaryotes and some subunits are conserved across species, with some specific subunits in certain species.…”