JBC Papers in Press. Published on November 24, 2015 as Manuscript M115.693259 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M115.693259 Interactions between mammalian WDR12 and Midasin

The Crystal Structure of the Ubiquitin-Like Domain of Assembly Factor Ytm1 and Characterization of its Interaction with the AAA-ATPase Midasin

Erin M. Romes, Mack Sobhany, and Robin E. Stanley*

From the Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, 111 T. W. Alexander Drive, Research Triangle Park, North Carolina 27709, USA

Running Title: Interactions between mammalian WDR12 and Midasin

*To whom correspondence should be addressed: Robin E. Stanley, Ph.D, Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health Building 101, Room F378, 111 T. W. Alexander Drive, Research Triangle Park, NC 27709, Telephone: (919)541-0270; E-mail: [email protected] Downloaded from Keywords: ribosome assembly, rRNA processing, crystallography, ATPases associated with diverse cellular activities (AAA), metal ion‐ interaction http://www.jbc.org/ ABSTRACT and the interaction is dependent upon metal The synthesis of eukaryotic ion-coordination as removal of the metal or is a complex, energetically-demanding process mutation of residues that coordinate the metal requiring the aid of numerous non-ribosomal ion diminishes the interaction. Mammalian factors such as the PeBoW complex. The WDR12 displays prominent nucleolar by guest on April 26, 2017 mammalian PeBoW complex, comprised of localization that is dependent upon active Pes1, Bop1, and WDR12, is essential for the rRNA transcription. Based upon these results processing of the 32S pre-ribosomal RNA. we propose that release of the PeBoW Previous work in Saccharomyces cerevisiae complex and subsequent release of Nle1 by has shown that release of the homologous Midasin is a well-conserved step in the in this complex (Nop7, Erb1, and ribosome maturation pathway in both yeast Ytm1, respectively) from pre-ribosomal and mammalian cells. particles requires Rea1 (Midasin or MDN1 in ______humans), a large dynein-like protein. Midasin Ribosomes are large macromolecular contains a C-terminal metal ion-dependent machines comprised of four pieces of adhesion site (MIDAS) domain that interacts ribosomal RNA (rRNA) and 80 (79 in yeast) with the N-terminal ubiquitin-like (UBL) associated ribosomal proteins (1,2). domain of Ytm1/WDR12 as well as the UBL Ribosomes are responsible for carrying out the domain of Rsa4/Nle1 in a later step in the synthesis of all proteins within a cell, and the ribosome maturation pathway. Here we eukaryotic ribosome is composed of two present the crystal structure of the UBL subunits known as the small subunit (40S) and domain of the WDR12 homologue from S. the large subunit (60S). The assembly of cerevisiae at 1.7 Å resolution and demonstrate ribosomes begins in the nucleolus with the that human Midasin binds to WDR12 as well transcription of the rRNAs. Three of the as Nle1 through their respective UBL rRNAs are transcribed as a single poly- domains. Midasin contains a well-conserved cistronic precursor (18S, 5.8S, and 25S), extension region upstream of the MIDAS which must then be modified, folded, domain required for binding WDR12 and Nle1 processed and exported to the cytoplasm in a

1

Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Interactions between mammalian WDR12 and Midasin carefully orchestrated manor (3,4). Ribosome in yeast, Nop7, Erb1, and Ytm1 are all biogenesis in eukaryotic cells is an incredibly essential proteins required for the processing complex and energetically demanding process of the 27SA3 pre-rRNA (17,18). All that requires more than 200 essential non- components of the PeBoW/Nop7 complex are ribosomal assembly factors (3-6). thought to be multifunctional proteins and Defects in the mammalian ribosome have been linked with roles in various cellular biogenesis pathway are linked to a group of processes including DNA replication, cell human diseases that are collectively called cycle regulation and cardiac function in ribosomopathies. These are all congenital, addition to (19,20). The inherited disorders with a broad clinical PeBoW/Nop7 complex is thought to primarily spectrum that have perplexed researchers for localize to the nucleolus. The complex is not years because they cause tissue-specific found on ribosomal particles within the effects even though ribosomes are essential in nucleoplasm or cytoplasm, indicating that the all cell types (7). Ribosome biogenesis has PeBoW complex must be released from pre- also been emerging as a new target for cancer ribosomal particles prior to their transport out therapy. Recent studies have shown that of the nucleolus (16,18,21). several important oncogenes, including One way in which ribosome cMYC, RAS and PI3K, play key roles in maturation factors are released from pre- Downloaded from promoting hyperactive ribosome biogenesis, ribosome particles is through the aid of three and deregulated ribosomal DNA transcription different AAA-ATPases, Rea1/Midasin, Rix7, is a requirement for the transformed and Drg1/Afg2, which all utilize both phenotype (8). Moreover, studies have also nucleotide binding and hydrolysis to drive http://www.jbc.org/ shown that the nucleolus is a key element in release of distinct ribosome maturation factors regulating the cellular stress response and is (reviewed in (22)). Rea1 is responsible for directly involved in regulating the activity of driving release of both the Nop7 complex and the tumor-suppressor p53 in response to stress the maturation factor Rsa4 from pre-60S (9-13). Furthering our understanding of the particles in S. cerevisiae. Rea1 is the largest by guest on April 26, 2017 ribosome biogenesis pathway is essential for protein in S. cerevisiae and contains an N- the development of new cancer therapeutics terminal domain, six concatenated ATPase and treatments for ribosomopathies. domains, a 260kDa linker domain, and a C- The majority of what is known about terminal MIDAS domain that is well eukaryotic ribosome biogenesis is based on conserved across all eukaryotes (23). Electron extensive studies in the budding yeast S. microscopy studies reveal that Rea1 has a cerevisiae (recently reviewed in (4)). While large AAA motor domain connected to a the overall process is thought to be well flexible tail-like structure, which contains the conserved among eukaryotes, much less is MIDAS domain at the end (24). Electron known about ribosome biogenesis in higher microscopy studies also indicate that Rea1 organisms (14). One complex that has been contacts pre-ribosome particles adjacent to the well characterized in both yeast and Rix1-Ipi1-Ipi3 subcomplex with the tail being mammalian cells is the PeBoW complex able to reach the pre-ribosomal factor Rsa4 (Nop7 complex in S. cerevisiae). The PeBoW (24). Further studies in S. cerevisiae complex was named for the mammalian demonstrated that Rea1 and Rsa4 interact with assembly proteins Pes1 (Pescadillo), Bop1 one another in vivo and in vitro through the (block of proliferation), and WDR12 (WD MIDAS domain of Rea1 and the conserved N- repeat domain 12), while the yeast terminal UBL domain of Rsa4, which was homologues are Nop7, Erb1, and Ytm1 previously referred to as the MIDO (MIDAS respectively. Knockdown of any of the interacting) domain (24). Interaction between proteins within the mammalian PeBoW Rea1 and Rsa4 coupled with ATP hydrolysis complex blocks processing of the large triggers release of Rsa4 from pre-ribosomal subunit 32S pre-RNA and triggers p53- particles, likely through a large-scale dependent cell cycle arrest (15,16). Similarly mechanical conformational change (22,24).

2 Interactions between mammalian WDR12 and Midasin

The mammalian homologue of Rsa4 is called structural information for WDR12, and Notchless (Nle1) and was so named because it suggest that interactions between Midasin with is a direct regulator of the Notch signaling the UBL domains of WDR12 and Nle1 are pathway in Drosophila melanogaster in important evolutionarily conserved addition to its role in ribosome maturation interactions in the ribosome assembly (25). pathway. In addition to driving release of Rsa4, Rea1 has also been shown to drive release of EXPERIMENTAL PROCEDURES the Nop7 complex from nucleolar pre- ribosome particles in S. cerevisiae. Reagents – Normal rabbit IgG Bioinformatic analysis revealed that the N- against Midasin and WDR12 for terminal UBL domain of Rsa4 is homologous use in immunohistochemistry were obtained to the N-terminal UBL domain of Ytm1, from Sigma Life Science. Normal mouse suggesting that Rea1 could interact with Ytm1 antibodies conjugated with horseradish through its MIDAS domain (21). Earlier work peroxidase against GST (B-14) and His (H3) in mice also suggested that the N-terminal for use in Western Blots were obtained from domain of WDR12 showed similarity to the Santa Cruz Biotechnology. Monoclonal N-terminal domain of Nle1, and it was mouse against the Strep-Tag peptide Downloaded from originally referred to in the literature as the and normal rabbit antibody against Actin for Nle1 domain (26). Subsequent experiments in use as primary antibodies in Western Blots S. cerevisiae demonstrated that Ytm1 interacts were obtained from Novagen and Sigma with Rea1 through its UBL domain. This Aldrich, respectively. Goat anti-rabbit IgG http://www.jbc.org/ interaction is essential for an earlier step in the and anti-mouse IgG antibodies conjugated 60S maturation pathway, and coupled with with horseradish peroxidase for use as ATP hydrolysis, it drives release of the Nop7 secondary antibodies in Western Blots were complex from pre-ribosome particles (21). obtained from Sigma Aldrich (anti-rabbit) and Rea1 is therefore able to act on two different Millipore (anti-mouse). Goat anti-rabbit IgG by guest on April 26, 2017 substrates at two different stages of assembly antibodies with Alexa Fluor 633 conjugate for of the large ribosomal subunit, including the fluorescence was obtained from Molecular nucleolar Nop7 complex and the Probes/Life Technologies. Normal goat serum nucleoplasmic Rsa4 (22). was obtained from Santa Cruz Biotechnology. Here we present the crystal structure Actinomycin D, paraformaldehyde, and of the UBL domain of the S. cerevisiae bovine serum albumin were obtained from homologue of WDR12 (Ytm1), which Sigma Aldrich. 4´,6-diamidino-2-phenylindole revealed that it is structurally homologous (DAPI) was purchased from Life with the UBL domain of Rsa4/Nle1. We Technologies. demonstrate that human Midasin and WDR12 Cloning, Protein Expression, and as well as Midasin and Nle1 interact through Protein Purification of the Ytm1 UBL domain their MIDAS and UBL domains, respectively. – Residues 1-94 of S. cerevisiae Ytm1 were The interaction between the MIDAS and UBL PCR amplified from genomic DNA and domains is reminiscent of integrin receptor inserted into the pGST2 parallel vector ligand binding and is dependent upon between the BamHI and NotI restriction sites coordination of a metal ion. However, the (27). The Ytm1-UBL plasmid was MIDAS domain of Midasin also contains a transformed into BL21(DE3) star cells (Life well-conserved extension region that we show Technologies). Cells were grown in the is required for binding WDR12 and Nle1. We presence of 100 µg/ml ampicillin in LB broth also demonstrate that nucleolar localization of at 37 °C to an OD600 of 0.6, and then protein WDR12 in U2OS cells is dependent upon expression was induced with 0.9 mM active rRNA transcription and that Midasin isopropyl-β-D-thiogalactopyranoside for 3 primarily localizes to the nucleoplasm. Taken hours at 37 °C. Cells were harvested and then together our data reveal the first piece of

3 Interactions between mammalian WDR12 and Midasin frozen at -20 °C until further use. Cells were on the SER-CAT 22-ID beamline (Chicago, resuspended in lysis buffer (50 mM Tris pH IL), and SeMet diffraction data were collected 8.0, 500 mM NaCl, 10 mM MgCl2, 0.01% at the Advanced Photon Source on the SER- Triton X-100) containing one EDTA-free CAT 22-BM beamline (Chicago, IL). The protease inhibitor tablet (Roche) and lysed by crystals are in the P1 space group with two sonication at 4 °C. Clarified lysate was applied copies in the asymmetric unit. The structure to a gravity flow column containing 5 ml of was initially solved by molecular replacement GST resin (GE Healthcare) and incubated for using a homology model derived from the 1 hour at 4 °C. Unbound protein was removed structure of the UBL domain of Rsa4 (PDB ID by successive washes with lysis buffer, and 4WJS) in the program MRage (28). Bucanneer the protein was eluted with 20 ml of elution (29) was used to build the model, followed by buffer (50 mM glutathione, 50 mM Tris pH 8, several rounds of manual model building with 200 mM NaCl). The sample was then COOT and refinement with Phenix (28). To concentrated and run over a Superdex 75 confirm that the assignment of the backbone 16/600 size exclusion column (GE Healthcare) was correct, we used the molecular pre-equilibrated with 20 mM Tris pH 8, 200 replacement phases to calculate an anomalous mM NaCl, 0.1 mM TCEP. Fractions difference map from the SeMet dataset, which Downloaded from containing the GST-tagged UBL domain were confirmed the correct placement of the 2 Met pooled and then the GST tag was cleaved residues. The structure was refined to a final overnight at 4 °C with TEV protease. The Rwork/Rfree of 17.5%/23.2%. cleaved sample was then run back over the Yeast Growth Assays – The S. cerevisiae strains used in the study are listed Superdex 75 16/600 size exclusion column to http://www.jbc.org/ remove GST and any uncleaved protein. in Table 2. The tetO7 parental strains were Fractions containing the UBL domain were obtained from Open Biosystems. Ytm1 concentrated to 14.4 mg/ml and used together with its endogenous promoter were immediately for crystallization. PCR amplified from S. cerevisiae genomic DNA and cloned into the centromeric plasmid Selenomethionyl (SeMet) incorporation was by guest on April 26, 2017 achieved by expression of the UBL domain in YCplac111 (30). Mutations to Ytm1 were BL21 (DE3) star cells grown in M9 minimal generated using QuikChange mutagenesis, and media that was supplemented with SeMet. The all mutations were verified by sequencing. To SeMet UBL domain was purified as described carry out the dilution plating assays, cells were above, and incorporation of SeMet was grown in YPD and then diluted to an OD600 of verified by mass spectrometry. 0.05. 1:10 serial dilutions were then plated on Crystallization of the UBL domain – YPD plates with and without 10 µg/ml The Ytm1 UBL domain was crystallized by doxycycline and incubated at 20, 30 and 37 °C hanging drop vapor diffusion using equal for 3 days. volumes of protein and well solution Mammalian Transfections and GST containing 0.1 M sodium cacodylate pH 6.2- Pull-Down Assays – Full length human Bop1 6.8 and 0.9-1.2M sodium citrate. Crystals and Pes1 were amplified from cDNAs (Open grew as plate clusters in 2-3 days. Crystal Biosystems) and cloned into the pLexM vector quality was improved by several rounds of (31) containing either an N-terminal 12-X His microseeding with seed beads (Hampton Tag or N-terminal GST tag. The DNA Research) to yield single plate crystals with encoding for residues 5287-5596, which dimensions up to 200 x 200 x 50 µm. Crystals includes the MIDAS domain of human were cryoprotected by the stepwise addition of Midasin, was codon optimized for expression ethylene glycol to a final concentration of 35% in HEK293 cells and cloned into the pLexM (v/v) and then flash frozen in liquid nitrogen. vector encoding an N-terminal GST tag and Data Collection and Structure the pCAG-OSF vector (32) encoding an N- Determination – Native x-ray diffraction data terminal OSF (One-Strep-Flag) tag. Full were collected at the Advanced Photon Source length human WDR12 and Nle1 were cloned into the pCAG-OSF vector encoding an N-

4 Interactions between mammalian WDR12 and Midasin terminal OSF tag. To create WDR12ΔUBL, IgG (1:10,000 dilution) or goat anti-rabbit IgG residues 84-423 of WDR12 were PCR (1:80,000 dilution) conjugated HRP antibodies amplified from cDNA encoding WDR12, and in 5% (w/v) nonfat milk and 1% (w/v) bovine cloned into the pCAG-OSF vector. To create serum albumin in TBST and incubated for at Nle1ΔUBL, residues 100-485 of Nle1 were least one hour at room PCR amplified from cDNA encoding Nle1 temperature. Subsequent to this incubation, and cloned into the pCAG-OSF vector. membranes were again vigorously washed Mutations were introduced using QuikChange three times with TBST. Protein bands were Mutagenesis (Stratagene). 40 ml cultures of visualized with ECL Plus Western blotting HEK293 cells adapted to grow in suspension detection reagent (GE Healthcare). were transfected with 1 µg/ml of maxi Cell Cultures – U2OS cells were prepped plasmid DNA and 2 µg/ml of PEI. cultured in Dulbecco’s modified Eagle’s Cells were harvested 72 hours after medium high glucose supplemented with 10% transfection, washed with PBS, and frozen at - (v/v) fetal bovine serum, antibiotics (100 80 °C until further use. Cells were lysed by units/ml penicillin and 100 µg/ml resuspension in lysis buffer (50 mM Tris 7.4, streptomycin) and 2 mM glutamine. Cells were incubated at 37 °C in a CO2 incubator.

500 mM NaCl, 10 mM MgCl2, 10 % (v/v) Downloaded from glycerol, 1% (v/v) Triton X-100, EDTA-free Immunohistochemistry/fluorescence – Protease Inhibitors (Roche) and 10 units of U2OS cells were cultured on 35mm glass Benzonase (Sigma)) with gentle rocking at 4 bottom culture dishes (MatTek Corporation) until 80-100% confluence was reached. Media °C for 1 hour. Lystates were clarified by was discarded, cells were rinsed with 1X centrifugation and then incubated with 100 µl http://www.jbc.org/ Phosphate Buffer Saline (PBS), pH 7.4, and of glutathione resin (GE Healthcare) for 1 cells were fixed in 3-4% paraformaldehyde hour at 4 C. Unbound protein was removed ° solubilized in 1X PBS for one hour. by 3 x 200 µl washes with lysis buffer, Paraformaldehyde solution was removed, the followed by 2 x 200 µl washes of lysis buffer cells were rinsed with 1x PBS, and cells were by guest on April 26, 2017 including 5 mM ATP and followed by a final permeablized with 0.5% Triton X-100/1X 200 µl wash with lysis buffer. Protein that was PBS (Sigma Aldrich) for 1.5 hours. Cells were retained on the resin was analyzed by both then washed three times (10 minutes each SDS-PAGE and western blotting. wash) with 1X PBS. Cells were then incubated Western Blots – Proteins were for a minimum of 1.5 hours with blocking separated on 4-15% mini-Protean TGX Stain buffer (1% bovine serum albumin, 4% normal Free gels (Bio-Rad) and transferred to goat serum and 0.4% Triton X-100 in 1X polyvinylidene difluoride membranes (Bio- PBS). Blocking buffer was then removed, and Rad). Following transfer, membranes were cells were incubated overnight at 4°C with a blocked for several hours in 5% (w/v) nonfat primary antibody from Sigma Life Science at milk in Tris-buffered saline, 0.1% Tween 20 manufacturer recommended dilutions. Cells (TBST). Membranes were then incubated were then washed three times (10 minutes overnight at 4 °C with either anti-GST (B-14) each wash) with 1X PBS, then protected from HRP conjugated antibody (1:200 dilution), light, and incubated with the Alexa Fluor anti-His (H3) HRP conjugated antibody (1:50 secondary antibodies for 1 hour at dilution), anti-Strep-Tag primary antibody manufacturer recommended dilutions. Cells (1:1000 dilution), or anti-Actin primary were then washed again with 1X PBS, as antibody (1:100 dilution) in 5% (w/v) nonfat previously described. Cells were finally milk and 1% (w/v) bovine serum albumin in incubated with 300 nM DAPI, washed once TBST. Following this overnight incubation, with 1X PBS, and then covered with 1X PBS membranes were vigorously washed three and visualized with a Zeiss Axio Observer Z1 times with TBST. Membranes requiring Epifluorescence Microscope with a Zeiss EC a secondary antibody for visualization were Plan-Neofluar 40X/0.9 objective and aperture then either incubated with goat anti-mouse lens. The Alexa Fluor 633 conjugate

5 Interactions between mammalian WDR12 and Midasin fluorochrome was imaged at room temperature the autophagy-associated ubiquitin-like on the Zeiss AxioCam MRm camera with Zen protein, Atg12, and the E3 ubiquitin-protein Pro 2012 (Blue Edition) software and ligase, RING2, highlighting the range of processed with ImageJ. diversity of biological activity of UBL domains. The Ytm1 UBL domain also RESULTS superimposes reasonably well with ubiquitin Structure of the UBL domain of S. (PDB ID 1UBQ) with an rmsd of 5.5 Å over cerevisiae Ytm1–To further our structural 71 Cα residues (Figure 2C). Overall, given the knowledge of Ytm1/WDR12 (Figure 1A), we structural similarity between Rsa4 and Ytm1 determined a crystal structure of the UBL along with the overlapping glutamate residues, domain (density was visible for residues 5-91) Rea1 undoubtedly interacts with both proteins of Ytm1 from S. cerevisiae at 1.7Å resolution in an analogous fashion. (Figure 1B). The structure was solved by Mutations in the UBL domain of Ytm1 molecular replacement with the recently cause growth defects in yeast–The UBL published UBL domain of Rsa4 (PDB ID domain of Ytm1 is essential for viability in S. 4WJS (33)) as a search model, and the cerevisiae and contains many well-conserved backbone assignment was confirmed by residues (Figure 2B). It has been previously calculation of an anomalous difference map shown in S. cerevisiae that truncation of the Downloaded from from a selenium methionine derivative. The UBL domain or mutation of residue E80A in structure was refined to an Rwork/Rfree of yeast is lethal (18,21). However, there are 17.5%/23.2% (See Figure 1C for a many other well-conserved residues within the representative section of unbiased electron UBL domain. To determine which of these http://www.jbc.org/ density and for further data collection and residues were essential for cell viability, we crystallography statistics see Table 1). There made mutations to the UBL domain and tested are two nearly identical copies of the domain the effects on growth. We transformed in the asymmetric unit, but there is no plasmids encoding wild type or mutant Ytm1 evidence to suggest that the domain forms a into cells harboring endogenous Ytm1 under a by guest on April 26, 2017 dimer in solution or in vivo. The structure Tet-promoter (Hughes Tet-Promoter revealed that the UBL domain of Ytm1 has a Collection, Openbiosystems) and assayed for classic, compact ubiquitin β-grasp fold, growth in the presence and absence of 10 µg comprised of 4 β-strands that form a central β- doxycycline at 20, 30 and 37 °C. Addition of sheet and two α-helices located on one side of doxycycline turns off expression of the β-sheet (Figure 1B). endogenous Ytm1. The E80A mutation was Despite little used as a positive control, and as expected, between the UBL domains of Rsa4 and Ytm1 both depletion of endogenous Ytm1 and (Figure 2B), the structures of the two domains expression of the E80A mutant conferred a superimpose well with one another with a root severe growth defect relative to growth of the mean square deviation (rmsd) of 4.5 Å over 80 plasmid expressing wild type Ytm1 (Figure Cα residues (Figure 2A). Both Rsa4 and Ytm1 2D). Surprisingly, mutation of the well- contain well-conserved glutamate residues that conserved residues R65 and E86 to alanine are thought to coordinate the metal ion with had no effect on growth of yeast, indicating the Rea1 MIDAS domain in S. cerevisiae that they are not essential for function (Figure (21,24). The conserved glutamate residue 2D). (residue E80 in S. cerevisiae) in Ytm1, Given the high structural similarity important for binding the Rea1 MIDAS between the UBL domain of Ytm1 and domain lies in an extended loop before the ubiquitin, we hypothesized that Ytm1 may final β-strand in the same structural location as contain an essential hydrophobic patch the conserved glutamate in Rsa4 (Figure 1B required for function. It has been well and 2A). A search of the Dali server revealed established in ubiquitin that residue I44 forms that the Ytm1 UBL domain is most similar to part of an essential hydrophobic patch along with residues L8 and V70 that is important for

6 Interactions between mammalian WDR12 and Midasin maintaining interactions with α-helical the absence of Midasin (Figure 3B). ubiquitin-associated (UBA) domains, such as Therefore the WDR12 UBL domain is S5a of the 26S proteasome (34). There are two necessary for interaction with Midasin. well-conserved hydrophobic residues in Ytm1, Midasin binds the PeBoW complex F57 and L58, that superimpose near I44 in through the UBL domain of WDR12–After Ubiquitin (Figure 2C). Individual mutation of demonstrating that WDR12 binds to Midasin F57 or L58 to serine was not lethal in yeast, through its UBL domain we wanted to but the double mutant of F57S-L58S confered determine how Midasin interacts with the a severe growth defect in yeast that is PeBoW complex. First we transfected comparable with depletion of the endogenous HEK293 cells with plasmids harboring full protein (Figure 2D), suggesting that retention length GST-tagged Bop1 as bait, full length of this hydrophobic patch is critical for Pes1, and full length WDR12 or function. F57-L58 in Ytm1 could be essential WDR12ΔUBL, to determine what effect the for interacting with another portion of Midasin UBL domain of WDR12 has on formation of and/or the Rix1 complex, which is known to the PeBoW complex. We assayed for binding be associated with Midasin pre-ribosome by GST pull down followed by western blot particles (24,35). analysis, which revealed that the UBL domain Human Midasin binds WDR12 of WDR12 is not required for formation of the Downloaded from through its UBL domain–After solving the PeBoW complex (Figure 3C). We do observe crystal structure of the UBL domain of Ytm1, differences in the amounts of WDR12 that is we probed the function of the UBL domain of pulled down. However, if we normalize

WDR12 in mammalian cells and demonstrated everything to the amount of Bop1, which was http://www.jbc.org/ that it is required for binding Midasin. used as bait, the levels of Pes1 and WDR12 Previous work has shown that WDR12ΔUBL are fairly uniform. All of our pull downs were inhibits cell proliferation, triggers carried out in cells that were transiently accumulation of p53, and disrupts rRNA transfected with multiple plasmids so we

processing, indicating that the UBL domain of attribute these differences to levels of by guest on April 26, 2017 WDR12 is essential for mammalian ribosome transfection efficiency. biogenesis (15). Given the importance of the After we confirmed that the UBL UBL domain in mammalian cells, we domain of WDR12 is not required for hypothesized that WDR12 binds to Midasin formation of the PeBoW complex we looked through its UBL domain like its yeast at binding to Midasin. HEK293 cells were counterpart. To determine if Midasin and transfected with plasmids harboring full length WDR12 are binding partners, HEK293 cells GST-tagged Bop1 as bait, full length Pes1, full were transfected with plasmids containing length WDR12 or WDR12ΔUBL, and human Midasin and human WDR12 and residues 5287-5596 of Midasin. GST-tagged assayed for binding by GST pull-downs Bop1 was able to pull-down the Midasin (Figure 3A). Our starting construct for the MIDAS domain only in the presence of full Midasin MIDAS domain was based on length WDR12 (Figure 3C). Therefore equivalent residues used to carry out in vitro Midasin can interact with WDR12 alone and pull-downs with the yeast homologues (21). within the PeBoW complex, however both GST-tagged residues 5287-5596 of Midasin, interactions are dependent upon the UBL which included the full MIDAS domain and domain of WDR12. about 100 residues upstream of the MIDAS Human Midasin binds Nle1 through domain, were able to pull-down full length its UBL domains–We further demonstrated WDR12 but not WDR12ΔUBL (Figure 3B). that human Nle1 also binds to Midasin Expression of both WDR12 and through its UBL domain. Since we showed WDR12ΔUBL was confirmed by western blot that the UBL domains of the yeast analysis, and full length WDR12 does not homologues of Nle1 (Rsa4) and WDR12 interact with GST-bound glutathione resin in (Ytm1) are structurally homologous (Figure 2A), we assumed that Nle1 would also bind

7 Interactions between mammalian WDR12 and Midasin

Midasin though its UBL domain in region is not part of the canonical MIDAS mammalian cells. Nle1 has previously been consensus sequence, the interaction between shown to bind to Midasin/Rea1 in several Midasin and WDR12 must require more than organisms including S. cerevisiae and the typical MIDAS/ligand metal-ion interface. Solanum chacoense (24,36). To determine if We repeated the same experiment with Nle1 mammalian Midasin and Nle1 are binding as well and got similar results (Figure 4C), partners, we transfected HEK293 cells with indicating the importance of the Midasin plasmids encoding residues 5287-5596 of MIDAS extension region for binding both Midasin and Nle1 with or without the UBL Nle1 and WDR12. domain and assayed for binding by GST pull- E78 of WDR12 is required for binding downs. GST-tagged residues 5287-5596 of Midasin–We mapped the interface between Midasin were able to pull-down full length Midasin and WDR12 and confirmed that the Nle1 but not Nle1ΔUBL (Figure 3D). conserved glutamate, residue E78 of WDR12 Expression of both Nle1 and Nle1ΔUBL was (E80 in S. cerevisiae Ytm1), is required for confirmed by western blot analysis, and full- binding Midasin. Mutation of either the length Nle1 does not interact with GST-bound conserved residues in the glutathione resin in the absence of Midasin MIDAS consensus motif or the conserved Downloaded from (Figure 3B). Our results confirm that, as in glutamate in the UBL domains of Ytm1 and yeast, mammalian Midasin is able to bind both Rsa4 abolishes the interactions between WDR12 and Nle1 through their respective Midasin and Ytm1/Rsa4 in yeast (21,24). To UBL domains. determine if this also holds true in mammalian cells, we mutated the corresponding glutamate The extension region of the Midasin http://www.jbc.org/ MIDAS domain is required to bind WDR12 in WDR12 (E78) to alanine and found that the and Nle1–After confirming that Midasin binds E78A mutation was unable to bind the to both WDR12 and Nle1, we mapped the Midasin MIDAS domain (Figure 5A). binding interface between the proteins and Mutation of S78L in yeast Ytm1 has been shown to be synthetic lethal when combined found that Midasin contains a well-conserved by guest on April 26, 2017 region required for binding WDR12 and Nle1. with a mutant of Rea1 (21). This serine Just upstream of the MIDAS consensus motif, residue is conserved in mammalian WDR12 there is a region that is highly conserved (46% (Figure 2B), and mutation of S76 to leucine invariant) in Midasin (Figure 4A) and is significantly reduced binding to the Midasin predicted to be α-helical, but shares no MIDAS domain (Figure 5A). Mutation of S76 significant homology with other known to a glutamate residue however, did not affect proteins (23). This will be referred to as the binding to Midasin (Figure 5A). Sequence MIDAS extension region for the remainder of alignments of WDR12 across numerous this manuscript. We hypothesized that given species (Figure 2B) revealed that higher its high sequence conservation the MIDAS organisms have a small insertion in the loop extension region must play an important role following the first α-helix, and deletion of in Midasin’s function and may be important residues 47-50 of mammalian WDR12 also for binding WDR12 and Nle1. To test this significantly reduced binding to the Midasin hypothesis, a series of N-terminal truncations MIDAS domain (Figure 5A). We also mutated to the MIDAS extension region were created the conserved aspartic acid residues within the and then assayed for binding to WDR12 and MIDAS consensus motif in Midasin, but were Nle1 by GST pull-down. Starting with the unable to determine if they abolish WDR12 Midasin truncation 5310-5596 we see a binding because we could not detect diminished capacity for WDR12 binding to expression of the Midasin-MIDAS double Midasin (Figure 4B). N-terminal truncations aspartic acid mutant when transfected in past residue 5320 were unable to pull-down HEK293 cells. WDR12, indicating that the entire MIDAS Metal Ion is required for Midasin- extension region is required for binding MIDAS and WDR12 binding–After verifying WDR12 (Figure 4B). Given that the extension that the residues thought to be important for

8 Interactions between mammalian WDR12 and Midasin coordinating the metal ion between Midasin experiments in U2OS osteoscarcoma cells and WDR12 are important for binding, we with specific antibodies for WDR12 and demonstrated that complex formation is Midasin. Endogenous WDR12 showed dependent upon the presence of a metal ion. prominent nucleolar localization, as expected MIDAS domains have been extensively (15) whereas endogenous Midasin localized to studied in integrins where they play important both the nucleoplasm and the cytoplasm roles in mediating ligand binding through (Figure 6). Interestingly, Midasin also appears coordination of a metal ion that is essential for to have prominent localization in a distinct ligand binding (22,37,38). We generated a location in the cytoplasm suggesting that it homology model for the WDR12 UBL domain may have additional non-ribosomal roles in and Midasin MIDAS domain interaction using the cell (Figure 6). The localization of our Ytm1 UBL structure for WDR12 and the endogenous WDR12 to the nucleolus is αL-β2 integrin (PDB ID 1T0P) MIDAS consistent with it playing a role in the early domain structure for Midasin (Figure 5B). stages of ribosome biogenesis that take place In the crystal structure of the αL-β2 integrin in the nucleolus, while Midasin’s localization bound to its ICAM ligand, the metal bound in the nucleoplasm is consistent with its role in between the two proteins is a magnesium ion. the later stages of ribosome biogenesis (40). Downloaded from The Midasin MIDAS domain contains the full Next we wanted to determine if the canonical MIDAS consensus sequence localization of WDR12 and Midasin was (hhhhDxSxS, ~70 residues T, and an affected by cellular stress and rRNA hhhh[S,T]DG in ~ 30 residues, where h is any transcription. Disruption of rRNA transcription by various cellular stressors can hydrophobic and x is any residue (Figure 4A)) http://www.jbc.org/ as is found in the αL-β2 integrin (39). WDR12 cause a relocalization of proteins involved in and Nle1 also contain the conserved glutamic ribosome biogenesis (41). For example Pes1, acid residue like the ICAM ligand. Thus all 6 one of the other proteins associated with residues that coordinate the metal ion are WDR12 in the PeBoW complex relocalizes from the nucleolus to the nucleoplasm upon conserved, so we believe the metal ion by guest on April 26, 2017 coordination will be the same as the αL-β2 induction of cellular stress (42). To mimic integrin bound to its ICAM ligand. Missing cellular stress we treated U2OS cells with a from our homology model is the MIDAS low concentration (10 µM) of actinomycin D extension region because we have no to inhibit rRNA transcription by Pol I, and we structural information for this region. To repeated our immunofluorescence experiments determine the importance of the metal ion for using dimethyl sulfoxide (DMSO) as a WDR12 binding to the Midasin MIDAS control. WDR12 loses its nucleolar domain, we transfected HEK293 cells with localization upon treatment with actinomycin plasmids containing human Midasin MIDAS D but not DMSO alone. It is instead found in and human WDR12. 72 hours after the nucleoplasm, confirming that cellular transfection the cells were harvested, split into stress affects the localization of WDR12 half, and then lysed in the presence or absence (Figure 6). In contrast, the nucleoplasmic of 10 mM EDTA to ablate metal coordination. localization of Midasin did not change upon EDTA greatly diminishes the amount of treatment of cells with actinomycin D or WDR12 that can be pulled-down by GST- DMSO (Figure 6). tagged Midasin-MIDAS (Figure 5C). This DISCUSSION result confirms that a metal ion is required to The assembly of eukaryotic ribosomes promote complex formation between the requires two essential proteins, Ytm1/WDR12 MIDAS domain of Midasin and WDR12. and Rsa4/Nle1 that both harbor UBL domains WDR12, but not Midasin localizes to followed by C-terminal WD40 β-propeller the nucleolus in U2OS cells–To further domains. Using x-ray crystallography we characterize mammalian WDR12 and determined the crystal structure of the UBL Midasin, we carried out immunofluorescence domain from the yeast homologue of Ytm1/WDR12. UBL domains mimic the

9 Interactions between mammalian WDR12 and Midasin properties of ubiquitin through incorporation (Figure 3D). These results suggest that release of the ubiquitin β-grasp fold within their of the nucleolar PeBoW complex and the protein coding regions. They are used in a nuclecoplasmic Nle1 by the large motor wide variety of cellular processes such as protein, Midasin, is a well-conserved step in protein degradation, DNA repair, cell division, the eukaryotic ribosome maturation pathway. and autophagy and are often part of protein- Roles of WDR12 and Midasin in protein interaction motifs (34,43,44). WDR12 mammalian ribosome biogenesis are also and Nle1 are the only proteins known to further supported by their endogenous contain a UBL domain in the ribosome localization. WDR12 displays prominent assembly pathway. The function of this nucleolar localization, however cellular stress domain is to recruit the tail of the large motor induced by treatment of cells with protein Midasin and drive release of both the actinomycin D halts rRNA transcription and PeBoW complex and Nle1 from pre-ribosome leads to a relocalization of WDR12 in the particles, presumably through factor-relay nucleoplasm (Figure 6). Human Midasin did mechanisms that cause conformation changes not display nucleolar localization but rather within the pre-rRNA (33). The recent crystal was found in the nucleoplasm and cytoplasm structure of the yeast homologue of Rsa4/Nle1 under both normal and stress conditions contained two copies of the protein in the (Figure 6). The localization of human Midasin Downloaded from asymmetric unit, and interestingly, the UBL is similar to that of its yeast homologue, which domain in each had a different orientation to is not found in the nucleolus except in the the β-propeller suggesting that the rotational presence of the E80A glutamate mutant of Ytm1 that halts ribosome maturation (21). flexibility of the UBL domain could be http://www.jbc.org/ important for function (33). During the How Midasin gets recruited to pre-ribosomal revision of this manuscript another group particles in the nucleolus is still unknown. published the crystal structure of Chaetomium Previous work has suggested that WDR12 and thermophilium Ytm1 bound to the C-terminal the Rix complex are the two most likely candidates for recruitment, although other end of Erb1. As was observed for Rsa4, they by guest on April 26, 2017 also found that the UBL domain of Ytm1 was trans-acting factors could be involved (21). found in different orientations emphasizing the Collectively these results suggest that Midasin flexible orientation of this domain with respect is only transiently associated with the to the β-propeller domain (45). Given the nucleolus to drive release of the PeBoW structural similarity of the UBL domain of complex. Ytm1/WDR12 presented here and Rsa4/Nle1 We also identified that the MIDAS (Figure 2A), including the overlapping extension region in mammalian Midasin is glutamate residues, the interactions between required for binding WDR12 and Nle1 (Figure the Midasin MIDAS domain and the UBL 4). The extension region is not part of the domains of WDR12 and Nle1 should be very canonical MIDAS domain but is highly similar. This is reminiscent of integrins, which conserved and essential for binding WDR12 can interact with a range of different ligands and Nle1, suggesting that it may also play an through their MIDAS domains (46). important role in metal ion-coordination. While WDR12 and Nle1 are well Integrins are dependent upon metal ions for conserved, little is known about their roles in their function, and their dependence upon mammalian ribosome biogenesis. In this study metal ions has been well studied (38). Some we present the first evidence that interactions integrin domains contain metal ion-binding with Midasin and WDR12 as well as Midasin sites in addition to the MIDAS domain. For and Nle1 are conserved in higher organisms. example the βI integrin domain is made up of As in yeast, the UBL domain of WDR12 is three interlinking metal ion-binding sites, required for binding Midasin, but not for including the MIDAS site, adjacent to MIDAS formation of the PeBoW complex along with site (AMIDAS) and synergistic metal ion- Pes1 and Bop1 (Figure 3C). The UBL domain binding site (SyMBS). The exact in vivo roles of Nle1 is also required for binding Midasin of all three sites are unclear, but in vitro all

10 Interactions between mammalian WDR12 and Midasin three sites appear to play important roles in both WDR12 and Nle1, two proteins that have mediating ligand binding (38). There is no interesting implications for human health. sequence similarity between the MIDAS Recent work has demonstrated that extension region and the SyMBS or AMIDAS overexpression of WDR12 is induced by metal ion-binding sites, but the MIDAS cardiac overload, and genome association extension region could represent a new type of studies have associated the WDR12 with metal ion-coordination site. Bioinformatic early onset and coronary analysis suggested that there is a weak artery disease (20,48). Up regulation of similarity between the MIDAS extension WDR12 has also been shown to lead to region and the D subunit of magnesium activation of the p38 MAPK pathway, an chelatases based upon the high density of increase in levels of Bop1, and ultimately basic and hydrophobic residues just upstream myocardial dysfunction, suggesting that of the MIDAS domain (23). Magnesium WDR12 could be a novel therapeutic target for chelatases, which play an important role in patients with failing hearts (20). Moreover, chlorophyll biosynthesis by inserting Mg2+ elevated levels of the proteins associated with into protoporphyrin, also contain C-terminal the PeBoW complex have been found in many MIDAS domains and two N-terminal AAA different types of human cancers, including domains (47). In addition to magnesium neuroblastoma, hepatocellular carcinoma and Downloaded from chelatases and Midasin, there is only one other breast, colon, and ovarian cancers (42,49-55). known protein, VWA8 (only found in higher Nle1 was also recently shown to be a crucial organisms and function is unknown), that factor for intestinal homeostasis and a contains the combination of AAA and MIDAS requirement for organogenesis, especially in http://www.jbc.org/ domains (37). Further work will be needed to axial skeleton formation in mice (56,57). determine the exact role of the MIDAS Further investigations into the roles of extension region in Midasin, but it will be WDR12 and Nle1 are needed to determine the interesting to see if there is a functional mechanisms by which they are involved in relationship between the AAA domains and cardiac function and intestinal homeostasis, by guest on April 26, 2017 the MIDAS domain, since this combination but our work identifies that the interactions also occurs in two other proteins. between the UBL domains of WDR12 and In this manuscript we present the first Nle1 with Midasin are both evolutionarily high-resolution structure of the UBL domain conserved interactions important for the of the yeast homologue of Ytm1/WDR12 and synthesis of eukaryotic ribosomes. demonstrate that mammalian Midasin binds to

Acknowledgements We thank Traci M. Tanaka Hall and Michael J. Ragusa for their critical reading of this manuscript. We would like to thank the NIEHS Protein Expression Core for their help with mammalian cell culture and the NIEHS X-ray Crystallography Core for their help with data collection. Diffraction data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID and 22-BM beamlines at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at www.ser-cat.org/members.html. We would also like to thank the NIEHS Fluorescence Microscopy and Imaging Center for their help with the immunofluorescence. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.

Conflict of Interest The authors declare that they have no conflict of interest with the contents of this article.

Author contributions: EMR purified and crystallized the UBL domain of Ytm1 and solved the structure. EMR designed and constructed vectors for pull-downs and characterized the complex

11 Interactions between mammalian WDR12 and Midasin interaction in mammalian cells. MS carried out the immunofluorescence experiments and all western blot analysis. RES designed the study and conceived the experiments. EMR, MS, and RES analyzed data, prepared figures, and wrote the paper.

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15 Interactions between mammalian WDR12 and Midasin

FOOTNOTES This work was supported in whole or in part by the intramural Research Program of the NIH, National Institute of Environmental Health Sciences. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38.

The abbreviations used are: rRNA, ribosomal RNA; MIDAS, metal ion-dependent adhesion site; UBL, ubiquitin-like;

FIGURE LEGENDS

Figure 1. The crystal structure of the UBL domain from S. cerevisiae Ytm1 (A) Domain diagram of Ytm1 and WDR12. The numbers on the top correspond to the amino acid residues from S. cerevisiae Ytm1, and numbers on the bottom correspond to the amino acid residues from Homo sapiens WDR12. (B) Ribbon diagram of the UBL domain of S. cerevisiae Ytm1. The N- terminus of the protein is labeled as well as the C-terminus of the domain that leads to the WD40 domain. Residue E80 that is thought to coordinate the metal ion with the MIDAS domain of Rea1 is shown as a stick representation. (C) Composite omit map showing representative electron Downloaded from density of the structure at 1.7 Å resolution between residues E86 and T88. The map was contoured at 1.7 σ.

Figure 2. Comparison of the UBL domains from Ytm1/WDR12 and Rsa4/Nle1 (A) http://www.jbc.org/ Alignment of the crystal structures of the UBL domains from S. cerevisiae Ytm1 (Purple) and Chaetomium thermophilum Rsa4 (Pink, PDB ID 4WJS). (B) Multiple sequence alignment from the UBL domains of Ytm1/WDR12 and Rsa4/Nle1 from different homologues of Homo sapiens (H.S.), Mus musculus (M.m.), Drosophila melanogaster (D.m.), Caenorhabditis elegans (C.e.),

Chaetomium thermophilum (C.t.), and Saccharomyces cerevisiae (S.c.). (C) Alignment of the by guest on April 26, 2017 crystal structure of the UBL domain from S. cerevisiae Ytm1 (Purple) with Ubiquitin (Yellow, PDB ID 1UBQ). Residues F57 and L58 of Ytm1 are shown as cyan stick representations. (D) Yeast growth assay of Ytm1 UBL mutants. Transformants were spotted in 10-fold serial dilutions onto YPD plates in the presence or absence of 10 µg/ml doxycycline to deplete the expression of endogenous Ytm1 and were grown at 20, 30 and 37 °C for 3 days.

Figure 3. Human Midasin binds WDR12 and Nle1 through their UBL domains. (A) Schematic diagram of the WDR12, Nle1, Midasin, Pes1, and Bop1 constructs used for GST pull- down analysis. Abbreviations used are as follows: H (His-Tag), S (One-Strep-Flag Tag), Ext (Midas Extension Region). (B) The UBL domain of WDR12 is required to bind Midasin. HEK293 cells were transfected with plasmids harboring GST-Midasin-MIDAS and OSF-WDR12 with and without the UBL domain. GST pull-downs were analyzed by Western Blot. (C) The UBL domain of WDR12 is not required to form the PeBoW complex. HEK293 cells were transfected with plasmids harboring GST-Bop1, His-Pes1, OSF-WDR12 with and without the UBL domain and OSF-Midasin. (D) The UBL domain of Nle1 is required for binding Midasin. HEK293 cells were transfected with plasmids harboring GST-Midasin MIDAS and OSF-Nle1 with and without the UBL domain.

Figure 4. The MIDAS Extension Region of Midasin is required to bind WDR12. (A) Multiple sequence alignment of the MIDAS domain and ~100 residues upstream of the MIDAS domain of Midasin from different homologues of Homo sapiens (H.S.), Mus musculus (M.m.), Drosophila melanogaster (D.m.), Caenorhabditis elegans (C.e.), and Saccharomyces cerevisiae (S.c.). The predicted secondary structure of the MIDAS domain based on secondary structure alignments in HHPRED is shown below the sequence, and the numbers on the bottom of the first

16 Interactions between mammalian WDR12 and Midasin row correspond to the Homo sapiens Midasin sequence. The metal ion consensus sequence in MIDAS domains is composed of the following: hhhhDxSxS, followed by a conserved threonine in approximately 70 residues and an hhhh[S,T]DG in approximately 30 residues, where h is any hydrophobic and x is any residue (23,37). MIDAS consensus sequence residues are indicated above the sequence. (B). Western blot analysis of GST pull-downs of Midasin MIDAS N- terminal truncations with WDR12. The constructs used contained the following residues of Midasin fused to an N-terminal GST tag: 5287 (5287-5596), 5300 (5300-5596), 5310 (5310- 5596), 5320 (5320-5596) and 5530 (5530-5596) (C) Western blot analysis of GST pull-downs of Midasin MIDAS truncations with Nle1.

Figure 5. Metal Ion is required for WDR12 and Midasin binding. (A). Western blot analysis of GST pull-downs with WDR12 UBL mutations. (B) Model of the interaction between Midasin (blue) and the UBL domain of WDR12 (purple). The homology model of Midasin was generated from a crystal structure of the αL-β2 integrin (PDB ID 1TOP). Residues important for coordinating the metal ion (red) are shown as stick representations. (C) GST pull-downs of Midasin and WDR12 with and without EDTA. Cells were lysed in the presence or absence of EDTA and the binding was assayed by GST pull-downs followed by SDS-PAGE analysis (Simply Blue Stain (Thermo)). Downloaded from

Figure 6. WDR12 requires active Pol I transcription for nucleolar localization. U2OS were fixed and immunostained with an anti-WDR12 antibody (Red) or anti-Midasin antibody (Red),

and DNA was visualized by staining with DAPI (blue). Cells were treated with DMSO or http://www.jbc.org/ actinomycin D for 2 hours prior to fixing. Scale is representative of all panels.

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17 Interactions between mammalian WDR12 and Midasin

Table I Data Collection and Refinement Statistics Parentheses list statistic for the high-resolution shell

UBL Native UBL SeMet Data Collection Beamline SER-CAT 22ID SET-CAT 22BM Wavelength (Å) 1.0000 0.9649 Resolution Range (Å) 50-1.70 (1.76-1.70) 50.00 – 1.91 (1.98-1.91) Space Group P1 P1 Cell Dimensions a, b, c (Å) 35.299, 37.742, 41.135 37.525, 35.067, 41.012 α, β, γ (°) 82.542, 64.309, 69.619 64.395, 82.480, 69.558 Total Reflections 47152 (4135) 40477 (3359) Unique Reflections 18667 (1654) 12086 (1244) Completeness (%) 95.3 (85.4) 87.5 (88.5) Mean / 8.2 (2.5) 14.2 (2.7) Ι σ Downloaded from Multiplicity 2.5 (2.0) 3.3 (2.7) Rmerge (%) 9.1 (34.4) 9.0 (35.4)

Refinement

Resolution range (Å) 50-1.70 http://www.jbc.org/ Rwork/Rfree (%) 17.5/23.2 r.m.s.d. bond lengths (Å) 0.0058 r.m.s.d. bond angles (Å) 0.93 Ramachandran plot

Most favored (%) 96.5 by guest on April 26, 2017 Additional allowed (%) 2.94 Disallowed (%) 0.6 Wilson B factor (Å2) 28.96 PDB entry 5DTC

18 Interactions between mammalian WDR12 and Midasin

Table II Yeast Strains Used in this Study

Strain Genotype Reference tetO7-Ytm1 URA::CMV-tTA Ytm1:: kanR-tetO7TATA MATa OpenBiosystems his3-1 leu2-0 met15-0 tetO7-Ytm1 + Ytm1- URA::CMV-tTA Ytm1:: kanR-tetO7TATA MATa This Study ycplac111 his3-1 leu2-0 met15-0; ycplac111-Ytm1 tetO7-Ytm1 + URA::CMV-tTA Ytm1:: kanR-tetO7TATA MATa This Study ycplac111 his3-1 leu2-0 met15-0; ycplac111 tetO7-Ytm1 + URA::CMV-tTA Ytm1:: kanR-tetO7TATA MATa This Study Ytm1E80A his3-1 leu2-0 met15-0; ycplac111-Ytm1E80A tetO7-Ytm1 + URA::CMV-tTA Ytm1:: kanR-tetO7TATA MATa This Study Ytm1E86A his3-1 leu2-0 met15-0; ycplac111-Ytm1E86A tetO7-Ytm1 + URA::CMV-tTA Ytm1:: kanR-tetO7TATA MATa This Study

Ytm1R65A his3-1 leu2-0 met15-0; ycplac111-Ytm1R65A Downloaded from tetO7-Ytm1 + URA::CMV-tTA Ytm1:: kanR-tetO7TATA MATa This Study Ytm1L58S his3-1 leu2-0 met15-0; ycplac111-Ytm1 L58S tetO7-Ytm1 + URA::CMV-tTA Ytm1:: kanR-tetO7TATA MATa This Study Ytm1F57S his3-1 leu2-0 met15-0; ycplac111-Ytm1 F57S tetO7-Ytm1 + URA::CMV-tTA Ytm1:: kanR-tetO7TATA MATa This Study http://www.jbc.org/ Ytm1F57S L58S his3-1 leu2-0 met15-0; ycplac111-Ytm1 F57S L58S

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19 Figure 1 Downloaded from

A B http://www.jbc.org/ E80 N Ytm1 92 460 UBL Domain by guest on April 26, 2017 WDR12 91 423

α2 β1 β2 β4 E86 C T88 α1

β3

Y87 Downloaded from Figure 2

A E80 B S.c. ------MTEDKSQVKIRFFT-REKDELLHVQDTPMYAPISLKRYGLSEIVNHhttp://www.jbc.org/ LLGSE----KPV 57 E114 C.t. ------MDAPMEDAPAPVAQVKVIFTTTEPDLELPES-KRQLLVPADIRRYGLSRILNSESMLD---TGSI 61 Ytm1/ C.e. ------MDLADLMEVENGDHSINQKHVQITFFS--KDEEIPQIPDAVFDVPTGAECDDMNLLLNKTIEANDGAWKER 69 D.m. ------MDVDNGEGQVQVHLKT-—KQ-EHYAVPDVPYAIDGTVTTVELNTFVNALLRQK-DGSSDT 57 WDR12 M.m. ------MAQLQARFYS---ENKKYAVDDVPFSIPAAAEVADLSNIINKLLETKNELHKHV 51 H.s. ------MAQLQTRFYT---DNKKYAVDDVPFSIPAASEIADLSNIINKLLKDKNEFHKHV 51 S.c. MSTLIPPPSKKQKKEAQLPREV-----AIIPKDLPNVSIKFQALDTG--DNVGGALRVPGAISEKQLEELLNQLNGTS---DDPV 78 C.t. MATLAPPPSKRQRREEIQRTQTQQDVTPLVATDLGSFKANFIDSDG--NQMTDVVEINFADATEKNISNLLNTLLGRD--REEFT 81

Rsa4/ C.e. ------MTESPQISVSFVSEDEN-EL-GGSGILVPVDISTNELQILCNQLL by guest on April 26, 2017 GSS---DDPV 53 D.m. ------MLAKKQKMQET-----DTEQEATPHTIQARLVSDTG-EE-AGPPIDLPAGITTQQLGLICNALLKN----EEAT 67 N Nle1 M.m. ------MAAAV-----VEEAAAGDVQRLLVQFQDEGG-QL-LGSPFDVPVDITPDKLQLVCNALLAQ----EEPL 62 H.s. ------MAAAV-----PDEAVARDVQRLLVQFQDEGG-QL-LGSPFDVPVDITPDRLQLVCNALLAQ----EDPL 62

L58 E80 WD40 Domain S.c. PFDFLIEG------ELLRTSLHDYLTKK-GLSSEASLNVEY------TRAILPPSYLNSFSNEDWVSSLDV---- 111 C.t. PFDFLING------SFLRSSLEDYLTSN-GLSLETTLTLQY------VRSLIPPVYEASFEHDDWVSAVDVLSAT 123 Ytm1/ C.e. RFEFLVGET------FLRTSLAEFIEEY-EVETETILKVEC------VIGIEAPKPPLHDIQAPDWVSSVQI---- 127 D.m. DFDFLVFD------EYLRGRLCDHLREK-AISFEDAIEIEY------VERFPAPEPPQDCLLHDDWVSAVKA---- 114 WDR12 M.m EFDFLIKG------QFLRVPLVKHMELE-NISSEEVVELEY------VEKYTAPQPPEQCMFHDDWISSIEG---- 109 H.s. EFDFLIKG------QFLRMPLDKHMEME-NISSEEVVEIEY------VEKYTAPQPPEQCMFHDDWISSIKG---- 109 S.c. PYTFSCTIQGKKASDPVKTIDITDNLYSSLIKPGYNSTEDQITLLYTPRAVFKVKPVTRSSSAIAGHGSTILCSAFAPHTSSRMV 160 C.t. PYRFRIHIPGKDLIID---QYPNDLLSLLQKHGVTNPFETTITLSAEPQAIFKVHAVSRLAHRIPGHGQPILSCQFSPVSSSRLA 163 Rsa4/ C.e. PISFFTTEG------AEIVDSIRKSLE---EIDFETTLKLVYQPQAVFRVRPVTRCSASIPGHGEPVISAQFSPDG-RGLA 121 D.m. PYLFFVGE------DEIKKSLEDTLDLA-SVDTENVIDIVYQPQAVFKVRPVTRCTSSMPGHAEAVVSLNFSPDG–AHLA 135 Nle1 M.m. PLAFYVHD------AEIVSSLGKTLESQ-SVETEKIVDIIYQPQAVFRVRAVTRCTSSLEGHSEAVISVAFSPTG–KYLA 130 H.s. PLAFFVHD------AEIVSSLGKTLESQ-AVETEKVLDIIYQPQAIFRVRAVTRCTSSLEGHSEAVISVAFSPTG-KYLA 130 !

C D YPD YPD+Dox YPD YPD+Dox YPD YPD+Dox WT Empty Vector E80A E86A L58 R65A F57 F57S L58S F57S L58S 20 ° C 30 ° C 37 ° C Figure 3 Downloaded from

1 423 A WDR12 S UBL WD40 84 423 WDR12ΔUBL S WD40 http://www.jbc.org/ 1 485 Nle1 S UBL WD40 100 485 Nle1ΔUBL S WD40 5287 5380 5596 by guest on April 26, 2017 G-Midasin GST Ext MIDAS 5287 5380 5596 S-Midasin S Ext MIDAS 1 583 Pes1 H Pes1 1 746 Bop1 GST Bop1 B C D H-Pes1, G-Bop1 - + + + +! S-WDR12 - + - + -! S-WDR12 + + -! S-WDR12ΔUBL - - + - +! S-WDR12ΔUBL - - +! S-Nle1 + + -! G-Midasin - + + ! S-Midasin + - - + +! S-Nle1ΔUBL - - +! 75 150 α-GST G-Midasin - + + ! α-GST 100 (G-Bop1) 50 75 S-WDR12 75 50 α-GST S-WDR12ΔUBL α-Strep 50 37 S-Midasin 50 α-Strep 75 GST Resin GST 37 Resin GST 75 α-His 50 α-Strep

(H-Pes1) Resin GST 75 50 75 50 α-Strep 50 S-WDR12 S-WDR12ΔUBL α-Strep 50 α-Strep 37 37 S-Midasin WCL WCL

50 WCL WCL WCL 50 α-Actin 50 α-Actin 37 37 α-Actin 37 Figure 4 A MIDAS Extension Region Downloaded from ! S.c. IEDNPPDASASLTPERSLE------ESRELWHKSEISTADLVSRLGEQLRLILEPTLATKLKGDYKTGKRLNMKRIIPYIASQFRKDKIWLRRTKPSK 4700! C.e. ------ADEQWAAMSRTVGMLAAELAENLRLILEPQRANKMQGDYRSGKRLNMRRLIPYIASEYRKDRIWMRRTKRAQ 4160 D.m. LSSAEQIELRQQYQKQLTTFRVAQPEHEDYETWQGISNRMTQNARELCEQLRLILEPTKCTRLKGDYRTGRRINMKKIIPYIASQFRKDKIWLRRTKPAQ 5318 M.m. ISELRQEMERQLETWQAHDYGNAEEEKAAAEMWQNYLVLTASLSQQLCEQLRLLLEPTQAAKLRGDYRTGKRLNMRKIIPYIASQFRKDRIWLRRTKPSK 5366 H.s. VNELRQELERQLEMWQPRESGNPEEEKVAAEMWQSYLILTAPLSQRLCEELRLILEPTQAAKLKGDYRTGKRLNIRKVIPYIASQFRKDKIWLRRTKPSK 5380 5290 5300 5310 5320 5330 5340 5350 5360 5370! http://www.jbc.org/ hhhhDxSxS T S.c. RQYQIMIALDDSKSMSESKCVKLAFDSLCLVSKTLTQLEAGGLSIVKFGENIKEVHSFDQQFSNESGARAFQWFGFQETKTDVKKLVAESTKIFERAR-- 4798 C.e. REYQILIAVDDSESMNENGIHQNTCESVCIVEDALRRCDAGRVSVCSFGADVNTIIPFGEASAS-SSIEMLKQMTFSQKKTDLLLLLKTAKQQLDEMR-- 4257 D.m. RDYKITIAIDDSKSMHHNNSKTLTLEAISLVSQALTLLESGRLSIVSFGEAPQIILNHTEQFD---GPRLVNALNFAQDKTKIAGLLNFIRTANAEESGT 5415 M.m. RQYQICLAIDDSSSMVDNHTKQLAFESLAVIGNALTLLEVGQIAVCSFGESVKLLHPFHEQFNDSSGTHILRLCTFQQRKTKIAQFLETVAKMFAAAQKL 5466 H.s. RQYQICLAIDDSSSMVDNHTKQLAFESLAVIGNALTLLEVGQIAVCSFGESVKLLHPFHEQFSDYSGSQILRLCKFQQKKTKIAQFLESVANMFAAAQQL 5480

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hhhhSDG S.c. -AMVHNDQWQLEIVISDG---ICEDHETIQKLVRRARENKIMLVFVIIDGITSNESILDMSQVNYIPDQYGNPQLKITKYLDTFPFEFYVVVHDISELPE 4894 C.e. ----TGTSEQMLIVISDGRGALSQGADKVRALYSALQG--VTVLFIVLD--SGKKSIEDHTVASFKDNK-----VVLTPYLALFPFPFYALVKSVQQLPS 4344 D.m. GGDNG-LFENLLLILSDGRNIFSEGAQNVKNAIKLARLQRIFLVYIIIDNPDNKNSILDIQHVAVNADG----SVNINSYLDSFPFPYYVIVRDLNQLPL 5511 M.m. SQNVSPETAQLLLIVSDGRGLFLEGKDRVLAAVQAAQNANIFVIFVVLDNPNSRDSILDIKVPIFKGPGE---MPEIRSYMEEFPFPFYIILRDVNALPE 5563 H.s. SQNISSETAQLLLVVSDGRGLFLEGKERVLAAVQAARNANIFVIFVVLDNPSSRDSILDIKVPIFKGPGE---MPEIRSYMEEFPFPYYIILRDVNALPE 5577

S.c. MLSLILRQYFTDLASS--- 4910 C.e. VIAESIRQWFEMTTTHHS- 4362 D.m. VLSEAMRQWFELVNSE--- 5526 M.m. TLSDALRQWFELVTASDHS 5582 H.s. TLSDALRQWFELVTASDHP 5596

!

B G-Midasin C G-Midasin Truncation! Truncation! 5287 5300 5310 5320 5330 5287 5300 5310 5320 5330 S-WDR12 + + + + +! S-Nle1 + + + + +! 75 75 α-GST α-GST 50 50 75 75

α-Strep 50 α-Strep GST Resin GST GST Resin GST 50

75 75 -Strep 50 α-Strep 50 α WCL WCL WCL WCL 50 50 α-Actin α-Actin 37 37 Downloaded from Figure 5 http://www.jbc.org/

A S-WDR12 B

Mutation! WT S76L S76L S76E E78A E78A Δ 47-50 G-Midasin + + + + +!

75 by guest on April 26, 2017 α-GST 50 75 50 α-Strep GST Resin GST 75 50 α-Strep

WCL WCL 50 α-Actin 37 S76 S E78 C D T S

MW - EDTA + EDTA D

GST-Midasin WDR12 Figure 6 Downloaded from

Anti- Anti- WDR12 DAPI Merged Midasin DAPI Merged http://www.jbc.org/

Untreated by guest on April 26, 2017

DMSO

Actinomycin D The Crystal Structure of the Ubiquitin-Like Domain of Ribosome Assembly Factor Ytm1 and Characterization of its Interaction with the AAA-ATPase Midasin Erin M. Romes, Mack Sobhany and Robin Evans Stanley J. Biol. Chem. published online November 24, 2015

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