Abstract
Histone acetylation is one of many posttranslational modifications that affect nucleosome accessibility. Vps75 is a histone chaperone that stimulates Rtt109 acetyltransferase activity toward histones H3-H4 in yeast. In this study, we use sedimentation velocity and light scattering to characterize various Vps75-Rtt109 complexes, both with and without H3-H4. These complexes were previously ill-defined because of protein multivalency and oligomerization. We determine both relative and absolute stoichiometry and define the most pertinent and homogeneous complexes. We show that the Vps75 dimer contains two unequal binding sites for Rtt109, with the weaker binding site being dispensable for H3-H4 acetylation. We further show that the Vps75-Rtt109-(H3-H4) complex is in equilibrium between a 2:1:1 species and a 4:2:2 species. Using a dimerization mutant of H3, we show that this equilibrium is mediated by the four-helix bundle between the two copies of H3. We optimize the purity, yield, and homogeneity of Vps75-Rtt109 complexes and determine optimal conditions for solubility when H3-H4 is added. Our comprehensive biochemical and biophysical approach ultimately defines the large-scale preparation of Vps75-Rtt109-(H3-H4) complexes with precise stoichiometry. This is an essential prerequisite for ongoing high-resolution structural and functional analysis of this important multi-subunit complex.
Highlights
The nucleosome is the repeating unit of chromatin and consists of 146 bp of DNA wrapped around a histone octamer
We have investigated the stoichiometry of Vps75–Rtt109 using both reconstituted and co-purified complexes
We were prompted to do this as multiple crystal structures were reported showing that Vps75 can bind to Rtt109 at 2:2 or 2:1 (Su et al, 2011; Tang et al, 2011)
Summary
The nucleosome is the repeating unit of chromatin and consists of 146 bp of DNA wrapped around a histone octamer. Several lysine residues in the histones are acetylated by enzymes that transfer an acetyl group from an acetylCoA cofactor. This acetylation modulates the chromatin structure to regulate processes such as DNA replication, repair, and transcription (Shahbazian & Grunstein, 2007; Bannister & Kouzarides, 2011). H3-K56 acetylation is important in fungal pathogenicity, making Rtt109 an attractive antifungal therapeutic target (Wurtele et al, 2010; Dahlin et al, 2014). We study the complex between Vps, Rtt109, and H3-H4
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
