Stoichiometry of Multi‐protein Complexes containing RTT109, Vps75, and Histone H3–H4

Abstract

RTT109 is a fungal‐specific acetyltransferase that acetylates lysine residues in histone H3–H4. These acetylations play a critical role in nucleosome dynamics as required for DNA transcription, replication and repair. RTT109 acetylation of specific histone H3 lysine residues requires Vps75. Vps75 is a histone chaperone belonging to the Nap‐family; it directly binds the RTT109 enzyme and the histone H3–H4 substrate. The goal of this study was to determine the absolute stoichiometry of the active complex containing RTT109, Vps75, and histone H3–H4. The stoichiometry was unclear as a Vps75 homodimer can crystalize with either one or two copies of RTT109. A high‐resolution structure of the complex between Vps75 and histone H3–H4 is not available, and both Vps75 and histone H3–H4 are known to form oligomers in solution. To measure absolute stoichiometry, we have used an elegant series of analytical ultracentrifugation experiments. We characterize all possible combinations of RTT109, Vps75, and histone H3–H4. These centrifugation experiments are complemented by other biophysical approaches. Our final stoichiometry, combined with targeted mutagenesis, reveals the organization of the active complex containing RTT109, Vps75, and histone H3–H4. Our results pave the way for high‐resolution structural studies and have repercussions for the mechanism and preferred substrate of RTT109 bound by Vps75.Support or Funding InformationStartup funds to S. D'Arcy from The University of Texas at Dallas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.