Fol. Biol. 2019, 65, 203-211
Proteomic Interactome of C. elegans Mediator Complex Subunit 28 (MDT-28) Reveals Predominant Association with a Restricted Set of Core Mediator Subunits and an Affinity to Additional Structural and Enzymatic Proteins
Transcription factors exert their regulatory potential on RNA polymerase II machinery through a multiprotein complex called Mediator complex or Mediator. The Mediator complex integrates regulatory signals from cell regulatory cascades with the regulation by transcription factors. The Mediator complex consists of 25 subunits in Saccharomyces cerevisiae and 30 or more subunits in multicellular eukaryotes. Mediator subunit 28 (MED28), along with MED30, MED23, MED25 and MED26, belong to presumably evolutionarily new subunits that seem to be absent in unicellular eukaryotes and are likely to have evolved together with multicellularity and cell differentiation. Previously, we have shown that an originally uncharacterized predicted gene, F28F8.5, is the true MED28 orthologue in Caenorhabditis elegans (mdt-28) and showed that it is involved in a spectrum of developmental processes. Here, we studied the proteomic interactome of MDT-28 edited as GFP::MDT-28 using Crispr/Cas9 technology or MDT-28::GFP expressed from extrachromosomal arrays in transgenic C. elegans exploiting the GFPTRAP system and mass spectrometry. The results show that MDT-28 associates with the Head module subunits MDT-6, MDT-8, MDT-11, MDT-17, MDT- 20, MDT-22, and MDT-30 and the Middle module subunit MDT-14. The analyses also identified additional proteins as preferential MDT-28 interactants, including chromatin-organizing proteins, structural proteins and enzymes. The results provide evidence for MDT-28 engagement in the Mediator Head module and support the possibility of physical (direct or indirect) interaction of MDT-28 with additional proteins, reflecting the transcription-regulating potential of primarily structural and enzymatic proteins at the level of the Mediator complex.
Keywords
Caenorhabditis elegans, GPF-TRAP, interactome, Head module, mass spectrometry, Mediator complex, MED28, MDT-28, proteomic.
Funding
This work was supported by the European Regional Development Fund “BIOCEV — Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University in Vestec” (CZ.1.05/1.1.00/02.0109) (the Start-Up Grant to the group Structure and Function of Cells in Their Normal State and in Pathology – Integrative Biology and Pathology (5.1.10)) and the LQ1604 National Sustainability Programme II (Project BIOCEV-FAR) from the Ministry of Education, Youth and Sports of the Czech Republic; grants PROGRES Q26/LF1 and PROGRES Q28/LF1 and LF3 “Oncology” from Charles University; grant SVV 260 377 from Charles University, Czech Republic; and by project OPPK No. CZ.2.16/3.1.00/24024, awarded by the European Fund for Regional Development (Prague & EU – We Invest for Your Future).
References
Copyright
This is an open-access article distributed under the terms of the Creative Commons Attribution License.
