Volume 23, Issue 6, Pages 1078-1086 (June 2015) The Elp2 Subunit Is Essential for Elongator Complex Assembly and Functional Regulation  Chunming Dong, Zhijie Lin, Wentao Diao, Dan Li, Xinlei Chu, Zheng Wang, Hao Zhou, Zhiping Xie, Yuequan Shen, Jiafu Long  Structure  Volume 23, Issue 6, Pages 1078-1086 (June 2015) DOI: 10.1016/j.str.2015.03.018 Copyright © 2015 Elsevier Ltd Terms and Conditions

Structure 2015 23, 1078-1086DOI: (10.1016/j.str.2015.03.018) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 1 Structure of Elp2 (A–C) Ribbon diagram of Elp2 showing two covalently linked seven-bladed β propellers. (A) Side view of Elp2 with the conventionally named top faces of each propeller indicated. (B) View down the axis of the N-terminal β propeller I (colored green). (C) View down the axis of the C-terminal β propeller II (colored cyan). The nomenclature used to describe the blades and strands is shown. (D) Schematic diagram of the secondary structure and domain arrangement of Elp2. The top surface loops of each propeller, which connect interblade strands d-a and b-c, are indicated by solid lines, whereas the bottom surface loops, which link intrablade strands a-b and c-d, are plotted as dashed lines. β Propellers I and II are shown in the same color as in (B) and (C). See also Figure S1. Structure 2015 23, 1078-1086DOI: (10.1016/j.str.2015.03.018) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 2 The Conserved Alkaline Residues of Elp2 Contribute to Its Binding to Microtubules In Vitro and In Vivo (A) Elp2 can bind microtubules in high-speed spin-down assays. Co-pelleting assays of purified Elp2 (2 μM) with buffer (lanes 3 and 4) and purified αβ-tubulin (1.8 μM) (lanes 5 and 6), or the purified mutant Elp2-4(R-A) (2 μM) with buffer (lanes 7 and 8) and purified αβ-tubulin (1.8 μM) (lanes 9 and 10). The mixtures were ultracentrifuged, and equal fractions of the supernatants (S) and pellets (P) were analyzed by SDS-PAGE. The bottom panel is plotted with the percentages of Elp2 protein (pellet fraction) in total loading. Error bars indicate SEM (n = 3, separate experiments). ∗∗∗P < 0.001. (B) The solvent-accessible electrostatic surface representation of Elp2. The surfaces are colored according to the electrostatic potential, ranging from deep blue (positive charge, +5 kT/e) to red (negative charge, −5 kT/e). The electrostatic potentials were calculated using ABPS tools (Baker et al., 2001) with the default settings. The alkaline residues involved in forming the positively charged surface are labeled and the universally conserved residues are underlined. (C) The alkaline residues involved in MTs binding are highly conserved in different species. The amino acids involved in forming the positive surface are marked by stars, and the four universally conserved residues that were mutated to alanine in Elp2-4(R-A) or Myc-hElp2(R-A) constructs are marked by black-bordered boxes. These alkaline residues are all located in the top surface of β-propeller II on blades 11, 12, and 13. The GenBank numbers are shown at the end of each alignment. Species abbreviations: D.m, Drosophila melanogaster; D.r, Danio rerio; H.s, Homo sapiens; S.c, Saccharomyces cerevisiae; X.t, Xenopus tropicalis. (D) Co-IP experiments in yeast lysate. Fusion proteins were prepared from yeast cells after a homologous recombination as indicated in the Experimental Procedures, immunoprecipitated with agarose-conjugated anti-GFP, and immunoblotted with anti-GFP or anti-α-tubulin as indicated. The lower panels represent 8% of the input material for each immunoprecipitation (IP). The mobility of α-tubulin and various versions of Elp2-GFP are indicated in the left margin. WT, wild-type. (E) Co-IP experiments in HEK293T cell lysate. Extracts that were prepared from HEK293T cells transfected with the indicated plasmids were immunoprecipitated with agarose-conjugated anti-Myc and subsequently immunoblotted with anti-Myc or anti-α-tubulin as indicated. The lower panels represent 8% of the input material for each IP. (F) Co-IP experiments in yeast lysate. Fusion proteins were prepared from yeast cells after a homologous recombination as indicated in the Experimental Procedures, immunoprecipitated with agarose-conjugated anti-GFP, and immunoblotted with anti-GFP or anti-Myc as indicated. The lower panels represent 8% of the input material for each IP. See also Figures S2 and S3. Structure 2015 23, 1078-1086DOI: (10.1016/j.str.2015.03.018) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 3 The WD40 Fold Integrity of Elp2 Is Essential for the Core Subcomplex Assembly in Yeast and Humans (A and B) Co-IP experiments of the yeast core subcomplex formation by Elp2 wild-type (WT) and mutants. Extracts were prepared from yeast cells after a homologous recombination as indicated in the Experimental Procedures, immunoprecipitated with agarose-conjugated anti-Myc, and immunoblotted with anti-Myc or anti-HA as indicated. The upper panels show the IP results. The lower panels represent 10% of the input material for each IP. The mobility of Elp1-Myc, Elp3-Myc, and various versions of Elp2-HA are indicated in the left margin. (C) Co-IP experiments of the human core subcomplex formation by human Elp2 (hElp2) wild-type (WT) and mutants. Extracts that were prepared from HEK293T cells transfected with the indicated combinations of plasmids were immunoprecipitated with agarose-conjugated anti-Myc and subsequently immunoblotted with anti-Myc or Anti-GFP or anti-hElp1 as indicated. The upper panels show the IP results. The lower panels represent 8% of the input material for each IP. The mobility of endogenous hElp1, GFP-hElp3, and various versions of Myc-hElp2 are indicated in the left margin. See also Figures S3 and S4. Structure 2015 23, 1078-1086DOI: (10.1016/j.str.2015.03.018) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 4 Elp2 Is Essential for the Integrity of a Functional Elongator (A–D) Elp2 is essential for yeast cell viability. Strains of the indicated genotype were grown to late log phase/stationary phase (overnight) and plated in serial dilutions on a YPD plate. The plate is shown after 2–3 days of incubation at 30°C (A), at 37°C (B), on YPD containing 0.5 M NaCl (C), and on YPD containing 100 mM hydroxyurea (HU) (D). (E) Bar graph of H3K14-acetylation level from various indicated yeast strains. Representative immunoblot with specific H3K14Ac antibody to detect acetylation strength (lower panel), or with H3 antibody to demonstrate equal loading (upper panel). Error bars indicate SEM (n = 3, separate experiments). ∗∗P < 0.01, ∗∗∗P < 0.001. Structure 2015 23, 1078-1086DOI: (10.1016/j.str.2015.03.018) Copyright © 2015 Elsevier Ltd Terms and Conditions