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Stoichiometry of Rtt109 complexes with Vps75 and histones H3-H4

View ORCID ProfileNoushin Akhavantabib, Daniel D Krzizike, View ORCID ProfileVictoria Neumann, View ORCID ProfileSheena D’Arcy  Correspondence email
Noushin Akhavantabib
1Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX, USA
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  • ORCID record for Noushin Akhavantabib
Daniel D Krzizike
2Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA
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Victoria Neumann
1Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX, USA
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Sheena D’Arcy
1Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX, USA
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  • ORCID record for Sheena D’Arcy
  • For correspondence: sheena.darcy@utdallas.edu
Published 10 September 2020. DOI: 10.26508/lsa.202000771
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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.

  • Received May 11, 2020.
  • Revision received August 31, 2020.
  • Accepted September 1, 2020.
  • © 2020 Akhavantabib et al.
Creative Commons logoCreative Commons logohttps://creativecommons.org/licenses/by/4.0/

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Stoichiometry of Vps75–Rtt109 complexes
Noushin Akhavantabib, Daniel D Krzizike, Victoria Neumann, Sheena D’Arcy
Life Science Alliance Sep 2020, 3 (11) e202000771; DOI: 10.26508/lsa.202000771

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Stoichiometry of Vps75–Rtt109 complexes
Noushin Akhavantabib, Daniel D Krzizike, Victoria Neumann, Sheena D’Arcy
Life Science Alliance Sep 2020, 3 (11) e202000771; DOI: 10.26508/lsa.202000771
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Volume 3, No. 11
November 2020
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