Article

The Elp2 Subunit Is Essential for Elongator Complex Assembly and Functional Regulation Graphical Abstract

Authors Chunming Dong, Zhijie Lin, ..., Yuequan Shen, Jiafu Long

Correspondence [email protected] (J.L.), [email protected] (Y.S.)

In Brief Elongator complex (Elp1–6) plays vital roles in gene regulation. Dong et al. show that the Elp2 subunit folds into a two seven-bladed b-propeller structure, which is important for Elongator assembly and microtubules association, and highlight that Elp2 functions as a hub for formation of various complexes.

Highlights d

The structure of the WD40 domains in Elp2 consists of two seven-bladed b propellers

d

Elp2 binds to microtubules via its conserved alkaline residues

d

The WD40 fold integrity of Elp2 is essential for the Elongator complex assembly

d

Elp2 acting as a hub for various complexes formation is crucial for yeast viability

Dong et al., 2015, Structure 23, 1–9 June 2, 2015 ª2015 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.str.2015.03.018

Accession Numbers 4XFV

Please cite this article in press as: Dong et al., The Elp2 Subunit Is Essential for Elongator Complex Assembly and Functional Regulation, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.03.018

Structure

Article The Elp2 Subunit Is Essential for Elongator Complex Assembly and Functional Regulation Chunming Dong,1,2,5 Zhijie Lin,1,2,5 Wentao Diao,1,2 Dan Li,3 Xinlei Chu,1,2 Zheng Wang,1,2 Hao Zhou,1,2 Zhiping Xie,3 Yuequan Shen,1,2,4,* and Jiafu Long1,2,* 1State

Key Laboratory of Medicinal Chemical Biology, Nankai University, 94 Weijin Road, Tianjin 300071, China of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, China 3School of Medicine, Nankai University, Tianjin 300071, China 4Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, China 5Co-first author *Correspondence: [email protected] (J.L.), [email protected] (Y.S.) http://dx.doi.org/10.1016/j.str.2015.03.018 2College

SUMMARY

Elongator is a highly conserved multiprotein complex composed of six subunits (Elp1–6). Elongator has been associated with various cellular activities and has attracted clinical attention because of its role in certain neurodegenerative diseases. Here, we present the crystal structure of the Elp2 subunit revealing two seven-bladed WD40 b propellers, and show by structure-guided mutational analyses that the WD40 fold integrity of Elp2 is necessary for its binding to Elp1 and Elp3 subunits in multiple species. The detailed biochemical experiments indicate that Elp2 binds microtubules through its conserved alkaline residues in vitro and in vivo. We find that both the mutually independent Elp2-mediated Elongator assembly and the cytoskeleton association are important for yeast viability. In addition, mutation of Elp2 greatly affects the histone H3 acetylation activity of Elongator in vivo. Our results indicate that Elp2 is a necessary component for functional Elongator and acts as a hub in the formation of various complexes.

INTRODUCTION The Elongator complex is composed of six subunits (Elp1–6) and was initially identified in Saccharomyces cerevisiae as a component that preferentially associates with hyperphosphorylated forms of RNA polymerase II (Otero et al., 1999; Svejstrup, 2007). Within the Elongator complex, the Elp1, -2, and -3 subunits form the core subcomplex (Otero et al., 1999) and Elp4, -5, and -6 subunits compose the accessory subcomplex, which assembles into a heterohexameric ring-like structure involved in substrate recognition (Glatt et al., 2012; Lin et al., 2012). Elp3 is the catalytic subunit of the Elongator complex, and acetylates histones (Hawkes et al., 2002; Kim et al., 2002; Winkler et al., 2002) and a-tubulin (Creppe et al., 2009; Solinger et al., 2010). In addition to its lysine acetyltransferase activity, the

Elongator complex has been associated with other functions of tRNA processing (Esberg et al., 2006; Huang et al., 2005), zygotic paternal DNA demethylation (Okada et al., 2010), and exocytosis (Rahl et al., 2005). Elp2 is also known as StIP1 (Stat3-interacting protein 1) in humans (Collum et al., 2000; Suaud et al., 2011) and contains multiple WD40 repeats (Fellows et al., 2000). Yeast cells lacking the elp2 gene display typical elp phenotypes including temperature, salt, and 6-azauracil sensitivity, and slow adaptation to growth on carbon sources such as galactose (Fellows et al., 2000; Krogan and Greenblatt, 2001; Otero et al., 1999; Winkler et al., 2001). The deletion of any of the Elongator subunits leads to a loss of Elongator complex integrity and to its dysfunction, resulting in almost identical phenotypes in S. cerevisiae, Caenorhabditis elegans, Arabidopsis thaliana, and Drosophila melanogaster (Chen et al., 2009; Frohloff et al., 2001; Huang et al., 2005; Mehlgarten et al., 2010; Singh et al., 2010; Walker et al., 2011). In humans, the Elongator complex seems to primarily affect the development and maintenance of the nervous system. Some individuals suffer from familial dysautonomia (FD) disease, which is caused by a mutation in the IKBKAP gene (encoding IKAP, human Elp1) (Anderson et al., 2001; Slaugenhaupt et al., 2001). The WD40 repeat is an abundant domain that often contains a seven-bladed b-propeller structure, and is also one of the most promiscuous interactors in eukaryotic genomes (Xu and Min, 2011). WD40 repeat-containing proteins often act as stable structural hubs that can confer structural integrity to a large variety of protein complexes (Stirnimann et al., 2010; Xu and Min, 2011). The WD40 repeat-containing protein Elp2 affects the structural integrity of the functional Elongator complex. The structure of S. cerevisiae Elp2 subunit solved here reveals that Elp2 consists of two seven-bladed WD40 b propellers. We demonstrate that Elp2 binds to microtubules (MTs) via its conserved alkaline residues and that the WD40 fold integrity of Elp2 is essential for the Elongator assembly. We further show that both the mutually independent Elp2-mediated Elongator assembly and cytoskeleton association are important for yeast cell viability, indicating that Elp2 is a necessary component for functional Elongator and acts as a hub in the formation of various complexes.

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Figure 1. Structure of Elp2 (A–C) Ribbon diagram of Elp2 showing two covalently linked seven-bladed b 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 b propeller I (colored green). (C) View down the axis of the C-terminal b 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. b Propellers I and II are shown in the same color as in (B) and (C). See also Figure S1.

tural motif, sometimes referred to as ‘‘molecular velcro,’’ has been observed in the structures of other WD40 domain proteins and other b-propeller proteins (Adams et al., 2000). These interactions may help to keep propeller II tightly folded, and are believed to be a major contributor to the stability of the WD40 fold of Elp2 (see later).

RESULTS Overall Structure of Elp2 Prior studies have shown that yeast cells lacking the elp2 gene display the same typical phenotypes as other Elongator subunit deletions (Fellows et al., 2000; Krogan and Greenblatt, 2001; Winkler et al., 2001), providing genetic evidence that Elp2 works together with other subunits in a functional Elongator complex. To understand the molecular mechanism of the Elp2 subunit in the assembly of the Elongator complex, we solved the crystal structure of yeast Elp2 using molecular replacement-based single anomalous dispersion at a resolution of 3.2 A˚. The structure of the WD40 domains in Elp2 consists of two seven-bladed b propellers, which are slightly twisted with respect to each other (Figure 1; Figure S1). The N-terminal b-propeller domain (propeller I) contains blades 1–7 (residues 20–380), and the C-terminal b-propeller domain (propeller II) contains blades 8–14 (residues 1–10 and 380–788). These propellers are oriented such that the top surfaces of the propellers, which are formed by the d-a and b-c loops of each blade, face one another (Figures 1A and 1D). The two b-propeller domains are similar in size and can be superimposed with a 2.4 A˚ root-mean-square deviation for 266 Ca pairs. The only significant difference in the makeup of the two individual domains is that propeller I consists of a continuous string of residues, while the N-terminal ten residues of Elp2 form the final b strand of blade 14 in propeller II (Figure 1D). This struc-

Elp2 Binds Microtubules WD40 domains can bind to various targets (Stirnimann et al., 2010; Xu and Min, 2011), and the Elongator complex has been shown to acetylate histones (Winkler et al., 2002) and a-tubulin (Creppe et al., 2009; Solinger et al., 2010). Therefore, we speculated that Elp2 might bind to these substrates. To test this hypothesis, we used different approaches to test whether Elp2 can bind these substrates. In a dot-blot overlay assay, Elp2 did not interact with histone octamers or nucleosomes, whereas the Paf1/Leo1 subcomplex of Paf1 complex specifically bound to both histone octamers and nucleosomes, serving as a positive control (Figures S2A and S2B; Chu et al., 2013). Interestingly, we detected Elp2 protein in the MTs precipitate in a spin-down assay (lane 6 in Figure 2A). Because a-tubulin contains a C-terminal acidic tail, we hypothesized that charge-charge interactions may contribute to the association between Elp2 and MTs. As expected, the solvent-accessible electrostatic surface representation of Elp2 shows its surface is positively charged at the top of b-propeller II, in large part because of residues Arg-626, Arg-628, Arg-654, Arg-675, Lys-677, Lys-702, and Lys-754 (Figure 2B). Among these positively charged residues, the first four are universally conserved from yeast to humans (Figure 2C; Figure S3). To evaluate the importance of these four residues in interactions between Elp2 and MTs, we mutated all four Arg residues in Elp2 to alanine (hereafter referred as Elp2-4(R-A)). In the spin-down assay, the amounts of the Elp2-4(R-A) mutant precipitated with MTs was reduced dramatically (lane 10 in Figure 2A). We used circular dichroism to confirm similar behavior between wildtype and mutant Elp2, which ensured that any loss in MTs

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binding activity was not because of decreased Elp2 protein stability (Figure S2C). Next, we wanted to test the MTs binding activity of Elp2 in vivo. In a co-immunoprecipitation (co-IP) assay, GFP-tagged wild-type Elp2 (Elp2-GFP) specifically co-precipitated with MTs (lane 1 in Figure 2D) in yeast lysate, whereas the amount of MTs was reduced dramatically in co-precipitation with the mutant Elp2-4(R-A)-GFP (lane 2 in Figure 2D). To distinguish Elp2 binding to a-tubulin in the context of MTs or a heterodimer with b-tubulin, we used the well-known drug nocodazole to disrupt MTs formation in the co-IP assay. Accordingly, HEK293T cells were transfected with Myc-tagged Elp2 (MycElp2). 24 hours after transfection, HEK293T cells were treated with nocodazole or DMSO and subsequently were lysed for co-IP assays. As a control, the endogenous a-tubulin from HEK293T cells after DMSO treatment specifically co-precipitated with Myc-Elp2 in cell lysate (lane 1 in Figure 2E), whereas the amount of a-tubulin was reduced dramatically in co-precipitation with Myc-Elp2 in the cell lysate after nocodazole treatment (lane 2 in Figure 2E), indicating that Elp2 binds a-tubulin in the context of MTs. Furthermore, we deleted five residues of the very C-terminal acidic tail of a-tubulin (referred to as a-tubulin(C5D)) and found that the Myc-tagged a-tubulin(C5D) (a-tubulin(C5D)-Myc) mutant cannot bind to MTs (lane 2 in Figure 2F). Together, these data indicate that the Elongator complex may recognize the cytoskeleton through the Elp2 subunit binding to MTs via charge-charge interactions. We attempted to purify human recombinant Elp2, which was unsuccessful, so it was not possible to test whether human Elp2 binds to MTs in vitro. Nevertheless, sequence alignments indicate that the conserved alkaline residues of yeast Elp2 that are involved in MTs binding are also present in human Elp2 (Figure 2C), and it is safe to assume that human Elp2 binds to MTs using a mechanism similar to that of yeast Elp2. The WD40 Fold Integrity of Elp2 Is Essential for the Core Subcomplex Assembly The structure of Elp2 reveals that b-propeller II utilizes the very N terminus of the protein stretching across the interdomain interface to form the outer (d) strand of blade 14 (b14d; Figure 1D), which is likely to be important for the integrity of the WD40 fold in Elp2. Based on this observation and the fact that Elp1, -2, and -3 subunits form the core subcomplex (Otero et al., 1999), we hypothesized that the WD40 fold integrity of Elp2 is essential for the core subcomplex assembly in Elongator. To test our hypothesis, the first 14 amino acids of Elp2, which are located in b14d of b-propeller II, were deleted (named Elp2-b14dD) (Figure S3). In a co-IP assay, wild-type HA-tagged Elp2 (Elp2-HA), specifically co-precipitated with Elp3-Myc (lane 2 in Figure 3A) and Myc-Elp1 (lane 2 in Figure 3B) in yeast lysate, whereas binding by the Elp2-b14dD-HA mutant was completely lost (lane 6 in Figure 3A and lane 6 in Figure 3B). This suggests that the integrity of the WD40 fold in Elp2 is necessary for Elp2 binding to Elp1 and Elp3. Interestingly, the mutant Elp2-4(R-A), which is incapable of binding to MTs co-precipitated with equal amounts of Elp3-Myc and Elp1-Myc as the wild-type Elp2 (lane 4 comparing to lane 2 in Figures 3A and 3B, respectively). Cross-species complementation studies indicate that the structural features of these Elongator subunits are highly conserved among all eukaryotes (Li et al., 2005; Mehlgarten

et al., 2010). To this end, the first 17 amino acids of human Elp2, which are predicted to fold into the corresponding b14d strand in yeast Elp2 (Figure S3), were deleted, and the mutant was named hElp2(N17D). In a co-IP assay, wild-type Myctagged human Elp2 (Myc-hElp2) specifically co-precipitated with GFP-hElp3 and endogenous hElp1 in HEK293T cells (lane 1 in Figure 3C), whereas the Myc-hElp2(N17D) mutant coprecipitated with negligible amounts of GFP-hElp3 and hElp1 (lane 3 in Figure 3C). Another mutant, hElp2(R-A), has the positive charged residues Arg-634, Arg-636, Arg-670, and Arg-689 of human Elp2, corresponding to the residues of yeast Elp2 involved in MTs binding (Figure 2C) mutated to alanine. It coprecipitated with the same amounts of GFP-hElp3 and hElp1 as wild-type hElp2 (lane 2 compared with lane 1 in Figure 3C). Together, these data indicate that the structural features of Elp2 mediating the core subcomplex assembly are highly conserved from yeast to humans. Elp2 Is Important for Yeast Cell Viability The biochemical and structural data described thus far demonstrated that Elp2 is important for Elongator assembly and association with the cytoskeleton. Next, we wanted to use yeast as a host to investigate the role of Elp2 in vivo. To this end, an elp2D strain was made with the ELP2 gene replaced by S. cerevisiae URA3. Yeast strains with elp2D displayed typical elp phenotypes (e.g., the slow-start phenotype, and sensitivity to temperature, salt, and hydroxyurea [HU]) (Figures 4A–4D), which is consistent with results in earlier studies (Fellows et al., 2000; Krogan and Greenblatt, 2001; Otero et al., 1999; Winkler et al., 2001). To further explore the role of Elp2 in vivo, we tested the importance of Elp2-mediated Elongator assembly and MTs association with a complementation assay. To test their ability to complement elp phenotypes, the plasmids pP1K-ELP2(404), pP1K-ELP2b14dD(404), and pP1K-ELP2-4(R-A)(404) and the corresponding empty vectors were linearized and integrated into the elp2D strain. Interestingly, expression of the wild-type ELP2 gene (ELP2-WT), but neither of the mutant genes (ELP2-4(R-A) and ELP2-b14dD), was able to fully rescue the elp2D phenotype (Figures 4A–4D). Taken together, these results indicate that Elp2mediated core subcomplex assembly and MTs association are crucial for the growth of yeast. Elp2 Is Necessary for Activity of Elongator Histone Acetyltransferase In Vivo It has been shown that in elp3D yeast cells, histone acetylation drops below a critical level required for normal growth and that the predominant site of acetylation in vitro is Lys-14 of histone H3 (Winkler et al., 2002; Wittschieben et al., 2000). Our results show that Elp2-mediated core subcomplex assembly and MTs association are important for the growth of yeast. This, together with the fact that the holo-Elongator complex is required for histone acetyltransferase (HAT) activity (Winkler et al., 2002), led us to consider whether the elp2D strain displays the typical elp phenotypes because of a decreased level of histone acetylation. To test this hypothesis, we examined the level of histone acetylation by analyzing the strength of the H3K14 acetylation signal in whole yeast lysate. As expected, the average level of H3K14 acetylation of elp2D decreased to 67% of the wild-type level (lane 2 compared with lane 1 in Figure 4E). The expression of

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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 mM) with buffer (lanes 3 and 4) and purified ab-tubulin (1.8 mM) (lanes 5 and 6), or the purified mutant Elp2-4(R-A) (2 mM) with buffer (lanes 7 and 8) and purified ab-tubulin (1.8 mM) (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 b-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-a-tubulin as indicated. The lower panels (legend continued on next page)

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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) wildtype (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.

the ELP2-WT gene, but not the mutant gene ELP2-b14dD (incapable of core subcomplex assembly), restored the acetylation level of H3K14 against an elp2D background (lanes 3 and 4 in Figure 4E). Interestingly, expression of the mutant gene ELP24(R-A) (incapable of binding to MTs) fully restored the acetylation level of H3K14 (lane 5 in Figure 4E), indicating that an Elongator with functional HAT activity assembles in the ELP2-4(R-A) mutant strain. This observation is supported by our data showing that the Elp2-4(R-A) mutant still forms a complex with Elp1 and -3 (Figure 3). Together, these data indicate that Elp2 may act as a hub for the formation of various protein complexes. DISCUSSION The crystal structure of Elp2 shown in this study provides structural insight into the roles of Elp2 in Elongator assembly and activity. The structure of Elp2 reveals a novel arrangement of two seven-bladed WD40 b propellers. This arrangement of the b-propeller domains in Elp2 may provide multiple binding surfaces for adjacent proteins oriented at specific angles with respect to one another. One example is revealed by the evidence that the Elp24(R-A) mutant (incapable of binding to MTs) still binds to Elp1 and Elp3 subunits. Notably, related proteins with multiple WD40 repeat-containing domains (e.g., Aip1 [Voegtli et al., 2003], DDB1 [Angers et al., 2006; Li et al., 2006], and Sro7 [Hattendorf et al., 2007]) are particularly advantageous for binding to large substrates, such as protein complexes, or for the simultaneous binding of multiple protein targets. Furthermore, the structure-based mutagenesis and biochemical and genetic analyses indicate that Elp2 is essential for the integrity of a functional Elon-

gator, and may function as a sophisticated regulator of Elongator activity by mediating the physical connection between Elongator and different signaling cascades. Although the sequence identity between yeast and humans is low (Figure S3), data shown in Figure 3 indicate that the architecture of the WD40 domains in Elp2 is almost certainly conserved among species. Therefore, it is reasonably safe to assume that the structural and biochemical features of Elp2 described here are shared by the orthologs of Elp2 in other species. The Elp3 subunit of the Elongator complex is a conserved member of the GNAT (Gcn5-related N-acetyltransferase) protein family, which has been shown to have HAT activity and is involved in gene transcription (Close et al., 2006; Nelissen et al., 2010; Winkler et al., 2002). In our study, we find that deletion of the Elp2 subunit displays typical elp phenotypes (e.g., the slow-start phenotype, sensitivity to salt, temperature, and HU) and that two mutant strains harboring the ELP2-b14dD and ELP2-4(R-A) mutations display similar phenotypes to the elp2D strain (Figures 4A–4D). In addition, our data indicate that Elp2 may not be involved in histone and nucleosome binding (at least for non-postmodified histone and nucleosome), although we observed that several histone-modifying complexes contain WD40 proteins, which often directly participate in histone recognition and nucleosome binding (Suganuma et al., 2008). In fact, the Elp4-6 subcomplex of Elongator has been shown to bind histones for Elongator substrate recognition in earlier studies (Lin et al., 2012). Furthermore, we show that the level of H3K14 acetylation is decreased in both elp2D and ELP2-b14dD strains to a certain extent, which is consistent with the observation of functional redundancy among Elongator and Gcn5 in histone

represent 8% of the input material for each immunoprecipitation (IP). The mobility of a-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-a-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.

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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.

acetylation (Wittschieben et al., 2000). However, the H3K14 acetylation level in the ELP2-4(R-A) strain remains the same as in the wild-type strain (Figure 4E). A possible explanation is that the structural integrity of Elongator is lost in both Elp2D and Elp2-b14dD strains, leading to a reduction in Elongator HAT activity below a certain threshold, but that the Elongator integrity is intact in the Elp2-4(R-A) strain. Thus, both Elp2-mediated histone acetylation and cytoskeleton association are important for yeast cell viability. It is noted that obtaining purified recombinant Elp3 protein for preparing the holoenzyme or the core subcomplex Elp1-3 remains a technical hurdle to us and others in the field. In the past several years we have tried several expression systems (e.g., Escherichia coli and insect cells) to obtain the recombinant Elp3 protein, but all these efforts have failed. The most likely reason is that the catalytic subunit Elp3 of the Elongator contains an Fe-S cluster. It is extremely difficult to incorporate the Fe-S cluster in an aerobic culture environment. We are still trying very hard to overcome this challenge. In the future it will be important to reveal the molecular mechanism of the core subcomplex or the holoenzyme assembly using X-ray crystallography, cryo-electron microscopy, or small-angle X-ray scattering. The Elongator complex has been shown to acetylate neuronal a-tubulin, which is involved in cellular motility and is linked to human neurodegenerative diseases such as amyotrophic lateral sclerosis and FD (Creppe et al., 2009; Solinger et al., 2010). However, recent findings suggest that Elongator is not a major acetyltransferase for a-tubulin (Akella et al., 2010; Cheishvili et al., 2011; Miskiewicz et al., 2011) and may regulate a-tubulin acetylation indirectly through tRNA modification (Bauer et al., 2012). We also showed that loss of Elongator function does not result in reduced levels of acetylated a-tubulin in HEK293T cells (data not shown). Moreover, there is some disagreement about whether a-tubulin is acetylated in yeast (Alfa and Hyams, 1991; Campetelli et al., 2005), and we could not detect any acetylated tubulin using the antibody 6-11B-1 (Figure S4), even though Elp2 directly binds to MTs through its conserved alkaline residues in vitro and in vivo (Figure 2). More detailed biochemical assays in vitro are needed to clarify the physiological substrate(s) (e.g., histone, a-tubulin, tRNA, or others) of Elongator. Nevertheless, the association of Elp2 with the cytoskeleton is important for

yeast viability because yeast cells harboring the ELP2-4(R-A) mutation (incapable of MTs binding) display elp phenotypes (Figure 4). The cytoskeleton participates in the spatial organization and regulation of translation (Kim and Coulombe, 2010); this, together with the fact that Elongator is involved in modulating translation efficiency by tRNA modification (Esberg et al., 2006; Ferna´ndez-Va´zquez et al., 2013; Huang et al., 2005; Lin et al., 2013) leads us to hypothesize that Elongator plays vital roles in translational regulation. In the future, it will be important to investigate the molecular mechanism of cytoskeleton-based spatiotemporal translation by the Elongator complex. EXPERIMENTAL PROCEDURES Expression and Purification To express S. cerevisiae Elp2, DNA fragments were amplified by PCR and cloned into an in-house modified version of the pET32a vector (Novagen) (Lin et al., 2012). Mutations in Elp2 were made using a standard PCR-based mutagenesis method and were confirmed by DNA sequencing. All of the resulting proteins contained a thioredoxin (Trx)-His6 tag at their N terminus. BL21(DE3)-pUBS520 E. coli cells harboring the expression plasmid were grown in lysogeny broth (LB) medium at 37 C until the OD600 reached 2 and then induced with 0.4 mM isopropyl-b-D-thiogalactoside at 16 C overnight. The SeMet derivative protein was expressed in methionine auxotrophic E. coli B834 (DE3) cells grown in LeMaster medium. All of the recombinant proteins were purified by Ni2+-NTA agarose affinity chromatography followed by ion-exchange and size-exclusion chromatography. Crystallization and Data Collection Crystals of either the wild-type or the SeMet-substituted Elp2 were grown at 20 C with a protein concentration of 6.5 mg/ml using the sitting drop vapor diffusion method. The protein was equilibrated against a reservoir solution of 0.1 M citric acid (pH 4.8) and 3.4 M NaCl for 3 days. Both crystals were frozen in a cryo-protectant that consisted of the reservoir solution supplemented with 0.1 M citric acid (pH 3.5) and 3.0 M NaCl. The crystals of the wild-type Elp2 diffracted to 3.2 A˚ with a space group of P6122 and unit cell dimensions of a = b = 80.6 A˚, c = 535.8 A˚. The SeMet-substituted crystals diffracted to 3.3 A˚ in the same space group and with unit cell dimensions of a = b = 80.8 A˚, c = 536.6 A˚. Both data sets were processed and scaled using the HKL2000 software package (Otwinowski and Minor, 1997). Structure Determination and Refinement The initial phases of the Elp2 structures were determined by the PHASER program (McCoy et al., 2007) using the structure of WD40 protein Ciao1 (PDB code: 3FM0) as a template and then improved with the molecular replacement-based single anomalous dispersion phases by PHENIX (Zwart et al.,

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25 C for 30 min, after which each tube was centrifuged at 90,000 rpm at 25 C for 40 min. When the centrifuge stopped, the supernatant was carefully removed and 25 ml of 53 Laemmli sample buffer was added to each tube. The sediment was resuspended with the same volume of 13 Laemmli sample buffer, and 10 ml each of the supernatant and pellet samples were loaded for SDS-PAGE.

Table 1. Data Collection and Refinement Statistics SeMet Crystal

Wild-Type

Space group Unit cell (A˚)

P6122

P6122

a = b = 80.8, c = 536.6

a = b = 80.6, c = 535.8

Wavelength (A˚)

0.9792

0.9792

Resolution range (A˚)

50–3.30 (3.42–3.30)a

50–3.20 (3.31–3.20)a

No. of unique reflections

14,522

16,846

Redundancy

9.6 (2.7)a

23.5 (6.3)a

Rsym (%)b

15.2 (40.4)a a

I/s

12.1 (2.1)

Completeness (%)

85.9 (46.2)a

FOM

0.786

Yeast Culture and Growth Conditions Yeast cells were grown in rich (YPD; 1% yeast extract, 2% peptone, and 2% glucose) or synthetic minimal medium (SMD; 0.67% yeast nitrogen base, 2% glucose, and amino acids as needed). In some experiments, cells were also cultured in the presence of 0.5 M NaCl or 100 mM HU.

19.9 (42.2)a

Plasmids To construct a plasmid expressing wild-type Elp2 under the control of the endogenous promoter, a DNA fragment containing the ELP2 locus (including 1,000 base pairs of the ELP2 50 sequence and the ELP2 open reading frame) was amplified from S. cerevisiae genomic DNA. The corresponding Elp2b14dD and Elp2-4(R-A) variants were constructed by site-directed mutagenesis. All of the recombinant fragments were inserted into vectors pRS404 and pRS405 using BamHI and XhoI sites. The resulting plasmids pP1KElp2(404), pP1K-Elp2(405), pP1K-Elp2-b14dD(404), pP1K-Elp2-b14dD(405), pP1K-Elp2-4(R-A)(404), pP1K-Elp2-4(R-A)(405), pP1K-GFP-Elp2(405), and pP1K-GFP-Elp2-4(R-A)(405) were linearized by SalI before transforming into elp2D strain. C-Terminally 33 HA- or GFP-tagged Elp2 and its variants, 133 Myc-tagged Elp1 and Elp3, and 133 Myc-tagged a-tubulin and its mutant were constructed by homologous recombination. DNA fragments containing the homologous end sequences and appropriate HA and Myc tags were amplified from the plasmids pFA6a-3HA-TRP1 and pFA6a-13Myc-HIS3MX6, respectively.

14.9 (3.35)a 88.9 (59.2)a

Refinement Resolution range (A˚)

48.34–3.20

Rcrystal (%)c

19.9

Rfree (%)d

24.1

RMSDbond (A˚)

0.006

RMSDangle ( )

1.2

No. of protein atoms

5,375

No. of solvent atoms

5

Residues (%) Most favored

79.0

Additional allowed

20.2

Generously allowed

0.8

Disallowed

0

Average B factor (A˚2) of Protein

44.5

FOM, figure of merit; RMSD, root-mean-square deviation. The highest-resolution  P shell. P b Rsym = j hIi
Ij = hIi. P P c Rcrystal = hkl jFobs
Fcalc j= hkl Fobs . d Rfree is calculated as for Rcrystal, but from a test set with 5% of data excluded from the refinement calculation. a

2008). Nine of the 12 Se sites in one asymmetric unit were identified. The residues were built manually using Coot (Emsley and Cowtan, 2004), based on 2Fobs
Fcalc and Fobs
Fcalc difference Fourier maps. The structural model was refined using CNS (Brunger et al., 1998) and PHENIX (Zwart et al., 2008). The final structure had an Rcryst value of 19.9% and an Rfree value of 24.1%. The data collection and refinement statistics are summarized in detail in Table 1. Spin-Down Assay A microtubule (MT) protein binding assay was performed according to the instructions of the spin-down assay kit (Cytoskeleton, Inc.). In brief, the MTs assembly was left at room temperature (37 C) for 30 min. One 10-ml aliquot of tubulin protein (1 mg/ml) was defrosted by incubating for 30 min in a room-temperature water bath with 1 ml of Cushion buffer (80 mM Pipes [pH 7.0], 1 mM MgCl2, 1 mM EGTA, and 60% glycerol) and 0.5 ml 100 mM guanosine triphosphate. After 30 min incubation, the 100 ml of general tubulin buffer (80 mM Pipes [pH 7.0], 2 mM MgCl2, and 0.5 mM EGTA) was removed from 37 C, and 1 ml of Taxol stock solution was added and mixed well. The MTs were immediately removed from incubation and diluted with the 100 ml of general tubulin buffer plus Taxol, mixed thoroughly and gently, and left at room temperature. Five Beckman ultraclear ultracentrifuge tubes were labeled and set up for the microtubule binding reactions. After setting up the microtubule binding reaction systems in a 100-ml volume, the reactions were left at

Strains The S. cerevisiae strains used in this study are listed in Table S1. For gene disruptions, the ELP2 coding regions were replaced with the S. cerevisiae URA3 using PCR primers containing 59 bases complementary to the regions flanking the open reading frame. PCR-based integration of a 133 Myc tag at the 30 end of ELP1 and ELP3, a 33 HA or GFP tag at the 30 end of ELP2 wild-type and mutants, and a 133 Myc tag at the 30 end of a-TUBULIN and mutant were used to generate strains expressing C-terminal fusion proteins under the control of the native promoters. PCR verification was used to confirm all deletions and integrations. The resulting protein levels were tested by Western blotting. When necessary, empty integration plasmids were transformed into appropriate yeast strains to ensure that strains being compared had the same auxotrophic genotype. Phenotypic assays were performed as previously described (Fellows et al., 2000; Li et al., 2009; Winkler et al., 2001). Co-immunoprecipitation in Yeast Lysate Yeast cells were grown at 30 C in YPD medium until the OD600 reached 1. After centrifugation at 4,000 rpm for 1 min, yeast cells were collected and then resuspended in ice-cold cell lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 0.5% Nonidet P-40, 5% glycerol, 1 mM PMSF,1 mg/ml leupeptin, and 1 mg/ml antipain) with 1 ml of acid-washed glass beads in each sample. Yeast cells were lysed using a multi-shocker for 1 hr and clarified by centrifugation at 12,000 rpm for 15 min at 4 C. To test yeast Elongator complex formation, the supernatants were incubated with agarose beads conjugated to anti-Myc (clone PL14, Medical & Biological Laboratories) for 30 min at 4 C; or, to test microtubule binding of Elp2, the supernatants were incubated with agarose beads conjugated to anti-GFP (Medical & Biological Laboratories) for 30 min at 4 C. The agarose beads were then washed with cell lysis buffer three times and eluted with SDS sample buffer. The prepared samples were analyzed by SDS-PAGE and Western blotting. Co-immunoprecipitation of Human Elongator Complex HEK293T cells were transfected with the indicated combinations of plasmids containing the human Elongator subunits. Twenty-four hours after transfection, or subsequent treatment with nocodazole or DMSO, HEK293T cells were lysed with ice-cold cell lysis buffer (50 mM Tris [pH 7.5], 50 mM NaCl,

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0.5% Nonidet P-40, 3% glycerol, 1 mg/ml leupeptin, and 1 mg/ml antipain) and clarified by centrifugation at 13,000 rpm for 10 min at 4 C. The supernatant was incubated with agarose beads conjugated to anti-Myc for 20 min at 4 C. The agarose beads were washed three times with cell lysis buffer and eluted with SDS sample buffer. The prepared samples were analyzed by SDS-PAGE and Western blotting. Histone and Tubulin Acetylation In Vivo Yeast cells were grown at 30 C in YPD medium until the OD600 reached 1. After centrifugation at 12,000 rpm for 1 min, the yeast cells were resuspended in buffer including 50 mM Tris (pH 8.0) and 500 mM NaCl. The cells were then lysed by repeated freeze-thaws, and the whole-cell lysate was applied to SDS-PAGE followed by immunoblotting with anti-H3K14Ac (Millipore), histone H3 (Santa Cruz), tubulin (Santa Cruz), and acetylated tubulin (Sigma). ACCESSION NUMBERS The atomic coordinates and structure factors for the structure of Elp2 have been deposited in the PDB with accession code PDB: 4XFV. SUPPLEMENTAL INFORMATION Supplemental Information includes four figures and one table and can be found with this article online at http://dx.doi.org/10.1016/j.str.2015.03.018. AUTHOR CONTRIBUTIONS C.D., Z.L., Y.S., and J.L. conceived and designed the experiments. C.D., Z.L., W.D., D.L., X.C., Z.W., and H.Z. performed the experiments. C.D., Z.L., and J.L. analyzed the data and prepared the figures. Z.X. contributed reagents, materials, and analysis tools. J.L. revised the data and wrote the main manuscript text. All authors reviewed the manuscript. ACKNOWLEDGMENTS We are grateful to the staff at the beamline BL17U1 of the Shanghai Synchrotron Radiation Facility (SSRF) and the staff at the beamline NW3A at Photon Factory (Tsukuba, Japan) for excellent technical assistance during data collection. We thank Dr. Chunguan Wang and Maorong Wen at Tongji University for their assistance with the microtubules-protein binding assay. This work was supported by the 973 Program (2014CB910201 to J.L., 2012CB917201 and 2013CB910400 to Y.S.); National Natural Science Foundation of China (31270815 and 31470755 to J.L., and 31370826 to Y.S.); and the Fundamental Research Funds for the Central Universities (grant 65142007 to Y.S.). Received: October 4, 2014 Revised: March 20, 2015 Accepted: March 25, 2015 Published: May 7, 2015 REFERENCES Adams, J., Kelso, R., and Cooley, L. (2000). The kelch repeat superfamily of proteins: propellers of cell function. Trends Cell Biol. 10, 17–24. Akella, J.S., Wloga, D., Kim, J., Starostina, N.G., Lyons-Abbott, S., Morrissette, N.S., Dougan, S.T., Kipreos, E.T., and Gaertig, J. (2010). MEC17 is an [agr]-tubulin acetyltransferase. Nature 467, 218–222. Alfa, C.E., and Hyams, J.S. (1991). Microtubules in the fission yeast Schizosaccharomyces pombe contain only the tyrosinated form of alphatubulin. Cell Motil. Cytoskeleton 18, 86–93. Anderson, S.L., Coli, R., Daly, I.W., Kichula, E.A., Rork, M.J., Volpi, S.A., Ekstein, J., and Rubin, B.Y. (2001). Familial dysautonomia is caused by mutations of the IKAP gene. Am. J. Hum. Genet. 68, 753–758. Angers, S., Li, T., Yi, X., MacCoss, M.J., Moon, R.T., and Zheng, N. (2006). Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery. Nature 443, 590–593.

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