Rae1 interaction with NuMA is required for bipolar spindle formation

Richard W. Wong, Gu¨ nter Blobel*, and Elias Coutavas*

Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021

Contributed by Gu¨nter Blobel, November 1, 2006 (sent for review October 8, 2006) In eukaryotic cells, the faithful segregation of daughter chromo- (13), is one of Ϸ30 different proteins (14) (nucleoporins or nups) somes during depends on formation of a microtubule found in the nuclear pore complex. Rae1 has been shown to bind (MT)-based bipolar . The Nuclear Mitotic Appa- to the nucleoporin Nup98 (15) and the mitotic checkpoint kinase ratus protein (NuMA) is recruited from interphase nuclei to spindle Bub1 (16) through their so-called GLEBS (Gle2-binding site) MTs during . The carboxy terminal domain of NuMA binds domains (17) and to function with Nup98 in securin degradation MTs, allowing a NuMA dimer to function as a ‘‘divalent’’ crosslinker (18). The vesicular stomatitis virus M protein blocks host cell that bundles MTs. The messenger RNA export factor, Rae1, also mRNA export by binding to Rae1 (19). Although Rae1 has been binds to MTs. Lowering Rae1 or increasing NuMA levels in cells reported to bind to MTs (20, 21), these binding sites have not results in spindle abnormalities. We have identified a mitotic- been mapped. Interestingly, several nucleoporins uniquely lo- specific interaction between Rae1 and NuMA and have explored calize to the spindle (22), including the Nup107–160 complex the relationship between Rae1 and NuMA in spindle formation. We recently shown to be required for spindle assembly (23), but the have mapped a specific binding site for Rae1 on NuMA that would mechanistic aspects and functional relevance of these mitotic convert a NuMA dimer to a ‘‘tetravalent’’ crosslinker of MTs. In redistributions are largely unknown. mitosis, reducing Rae1 or increasing NuMA concentration would be Aberrant expression of either Rae1 or NuMA has been linked expected to alter the valency of NuMA toward MTs; the ‘‘density’’ to formation of multipolar spindles. In the case of NuMA, of NuMA-MT crosslinks in these conditions would be diminished, multipolarity is linked to overexpression, whereas in the case of even though a threshold number of crosslinks sufficient to stabilize Rae1, multipolarity is linked to its depletion (21, 24). Here we CELL BIOLOGY aberrant multipolar spindles may form. Consistent with this inter- identify a mitotic interaction between Rae1 and NuMA, map this pretation, we found that coupling NuMA overexpression to Rae1 interaction to a specific domain of NuMA, and demonstrate that overexpression or coupling Rae1 depletion to NuMA depletion a balance of these two proteins is required for bipolar spindle prevented the formation of aberrant spindles. Likewise, we found formation. We propose a model wherein Rae1 modulates the that overexpression of the specific Rae1-binding domain of NuMA MT crosslinking valency of NuMA in mitotic spindles to prevent in HeLa cells led to aberrant spindle formation. These data point to segregation defects that are commonly found in the Rae1–NuMA interaction as a critical element for normal spindle cells. formation in mitosis. Results ͉ mitotic spindle nucleoporin Rae1 and NuMA Form a Transient Complex During Mitosis. To elu- cidate in greater depth the specific role of mitotic Rae1, we n eukaryotic cells upon entry into mitosis, interphase micro- analyzed the composition of purified Rae1 complexes in mitotic Itubules (MTs) are reorganized into the spindle apparatus, a HeLa cells. The major Rae1-associated proteins from mitotic IP complex and dynamic macromolecular machine composed of were subjected to MALDI mass spectrometry after trypsin polymerized tubulin and many interacting proteins (1). MTs are digestion, leading to the identification of NuMA (data not polymers of ␣-␤-tubulin dimers with distinct plus and minus shown). By immunoblotting of anti-Rae1 immunoprecipitates, ends. The typical spindle apparatus contains two we detected coprecipitating NuMA along with Nup98 and poles at with ␥-tubulin at the minus ends of MTs. dynein (Fig. 1A). Conversely, using anti-NuMA antibodies, we Bipolar spindle assembly requires organization of MTs and their immunoprecipitated Rae1 and dynein but not Nup98 (Fig. 1B). selective local stabilization. Chromatin and kinetochores stabi- These data suggested that Rae1 and NuMA interact. To further lize the plus ends of MTs and become aligned in the center of define the specificity for this mitotic Rae1–NuMA interaction, the spindle awaiting successful biorientation of all sister chro- we prepared extracts from HeLa cells synchronized using a matids before . In some settings, notably plant cells and double thymidine block followed by release into and out of the oocytes, spindles form in the absence of centrosomes by MT MT destabilizer nocodazole. HeLa cells were released from an nucleation on chromatin followed by bundling at the minus S phase double thymidine block into nocodazole for 12 h and ends (2–4). arrested in mitosis. At this time, mitotic cells were collected by The Nuclear Mitotic Apparatus protein (NuMA) is a 237-kDa Ϸ shake-off and released out of nocodazole for 4 h. These exper- protein with an 1,500-aa discontinuous coiled-coil spacer iments revealed a transient association of Rae1 and NuMA between N- and C-terminal globular domains (5, 6) that can form during mitosis (Fig. 1C). Consistent with the IP data, we found parallel coiled-coil dimers Ϸ200 nm in length (6). The C- terminal domain of NuMA contains a site for multimerization (6), a nuclear localization sequence that interacts with karyo- Author contributions: R.W.W. and E.C. designed research; R.W.W. and E.C. performed pherin ␣ (7), a MT-binding site that overlaps with a binding site research; R.W.W., G.B., and E.C. analyzed data; and R.W.W., G.B., and E.C. wrote the paper. for LGN (8) (a leucine-glycine-asparagine-repeat containing The authors declare no conflict of interest. protein) and a site for binding the polyADP-ribose polymerase, Abbreviations: MT, microtubule; IP, immunoprecipitation. tankyrase (9). The N-terminal domain of NuMA is believed to *To whom correspondence may be addressed. E-mail: [email protected] or contain a binding site for dynactin that acts as an adaptor for [email protected]. dynein, a minus end-directed motor known to target NuMA to This article contains supporting information online at www.pnas.org/cgi/content/full/ the spindle pole (10). The WD (tryptophan-aspartic acid) repeat 0609582104/DC1. ␤ propeller protein Rae1 (11), also known as gle2 (12) or mrnp41 © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0609582104 PNAS ͉ December 26, 2006 ͉ vol. 103 ͉ no. 52 ͉ 19783–19787 Downloaded by guest on September 26, 2021 Fig. 2. Simultaneous depletion of Rae1 and NuMA rescues bipolarity. (A) HeLa cells were transfected with either siRNA duplexes against Rae1 (Left)or Rae1 and NuMA together (Right). After 72 h, cells were stained with ␣-tubulin antibody (red) and analyzed by confocal laser microscopy. Chromatin was stained with DAPI (blue). [Scale bars, 25 ␮m(Upper); 5 ␮m(Lower).] (B and C) Representative figures of cells treated with Rae1 siRNA, fixed, and stained with anti-pericentrin and either ␥-tubulin (B)or␣-tubulin (C) antibodies. DNA is counterstained with DAPI.

dle poles (data not shown) and stained positive for the centro- Fig. 1. Rae1 and NuMA form a transient complex during mitosis. (A and B) ␥ IP from mitotic HeLa extracts with ␣-Rae1 and ␣-NuMA or control antibodies somal markers pericentrin and -tubulin (Fig. 2 B and C). Given (IgG), followed by immunoblotting with ␣-NuMA, ␣-Nup98, ␣-dynein, and our observation of a mitotic Rae1–NuMA interaction, we were ␣-Rae1. In lanes marked ‘‘2% input,’’ 5 ␮lof250␮l extract used per IP was interested in exploring the effect of NuMA down-regulation on analyzed directly. (C) Synchronized HeLa cells were collected at the indicated the multipolar spindle phenotype of Rae1-depleted cells. NuMA time points, and extracts were analyzed by immunoblotting directly (Input overexpression is also linked to multipolar spindle formation ␣ ␣ 2%) or after IP with -Rae1. Anti-phospho-Histone H3 and -tubulin were that may be rescued by reduction of NuMA levels (24). Indeed, used as mitotic index and loading controls. (D) Asynchronous HeLa cells costained with ␣-Rae1 (green) and ␣-NuMA (red); chromatin was visualized when NuMA and Rae1 levels were reduced simultaneously by using DAPI (blue). The large yellow arrow points to metaphase cell, small siRNA, the incidence of multipolar spindles was greatly reduced white arrowpoints to interphase, and the large white arrowpoints to late (Fig. 2A and Table 1). telophase. (Scale bar, 25 ␮m.) Simultaneous Overexpression of NuMA and Rae1 Rescues Bipolarity. To further test our hypothesis that mitotic Rae1 can bind to that Rae1 and NuMA colocalized transiently on HeLa cell NuMA and influence spindle formation, we explored the effect spindle poles from to anaphase (Fig. 1D and SI Fig. 6). of overexpressing Rae1 in cells overexpressing NuMA and

Simultaneous Depletion of Rae1 and NuMA Rescues Bipolarity. Be- cause NuMA or Rae1 are known to individually impact spindle Table 1. RNAi and protein overexpression spindle phenotypes formation, we altered their balance in vivo by modulating their Mitotic Percent Percent Percent concentrations using RNAi and overexpression strategies and cells bipolar monopolar multipolar assayed the effect on spindle polarity. Consistent with previous observations (21), reduction of Rae1 by RNAi (SI Fig. 7) led to Control 300 98.5 Ϯ 3 0 1.5 Ϯ 1 the formation of multipolar spindles (Fig. 2A). We quantified the Rae1 siRNA 300 67 Ϯ 70 33Ϯ 3 mitotic defects at 72 h after transfection with siRNA duplexes (Rae1 ϩ NuMA) siRNA 300 91 Ϯ 30 9Ϯ 6 targeting Rae1 and found a high proportion (Ͼ30%) of cells Control 200 98 Ϯ 20 2Ϯ 1 displayed strikingly altered spindle morphology compared with GFP-NuMA 200 59 Ϯ 512Ϯ 329Ϯ 4 controls treated with buffer alone (transfection efficiency GFP-NuMA ϩ HA-Rae1 200 83 Ϯ 24Ϯ 213Ϯ 4 Ϯ Ϯ Ͼ90%; see Fig. 2A and Table 1). The extra spindle poles GFP 200 98 30 22 Ϯ Ϯ Ϯ appeared to pull away from the main spindle, GFP-NuMA325–829 200 65 2133222 contributing to serious chromosome-alignment defects. The Quantitation of spindle defects represented in Figs. 2 and 3. n ϭ three Rae1 siRNA-treated cells displayed NuMA localization to spin- independent experiments.

19784 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0609582104 Wong et al. Downloaded by guest on September 26, 2021 Fig. 3. Simultaneous overexpression of Rae1 and NuMA rescues bipolarity. Representative figures of HeLa cells transfected with plasmids overexpressing either GFP-NuMA or GFP-NuMA and Rae1-HA together. After 24 h, cells were fixed, stained with ␣-tubulin antibody (red in overlay; GFP is green), and analyzed by confocal laser microscopy. Chromatin was stained with DAPI (blue). (Scale bar, 5 ␮m.)

displaying the multipolar spindle phenotype. First we transfected GFP-NuMA into HeLa cells and 72 h later observed the cells using confocal microscopy. A variety of phenotypes was ob- served: in 29% of the cells (n ϭ 300), additional poles were CELL BIOLOGY observed and in 12% of the cells monopolar spindles formed (Fig. 3 and SI Fig. 8). Interestingly, if we cotransfected NuMA with Rae1, we found that additional poles were observed in only 13% of the cells (n ϭ 300); 4% of the cells remained monopolar; and most of the cells appeared normal during prometaphase/ metaphase (83%; Table 1, Fig. 3 and SI Fig. 8). These data therefore further suggest that normal spindle pole formation requires balanced concentrations of NuMA and Rae1 during mitosis.

Mapping the Rae1 Interaction Domain on NuMA. To investigate the basis of the Rae1 NuMA interaction at mitosis, we generated a series of NuMA deletion mutants and tested them for their Fig. 4. Mapping the Rae1 interaction domain on NuMA. (A) Schematic of NuMA and five FLAG-tagged fragments of NuMA. Numbers on the left refer ability to interact with Rae1. We coexpressed Rae1 and various to amino acids (aa); all fragments are continuous (e.g., NuMA2 ends at amino fragments of NuMA (see Fig. 4A) in a cell-free reticulocyte acid 829 and NuMA3 starts at amino acid 829). (B) Autoradiograph of [35S]me- translation system. Only the fragment NuMA325–829 interacted thionine-labeled Rae1 and NuMA-FLAG fragments coexpressed in vitro, af- directly with Rae1 (Fig. 4B). This fragment of NuMA contains finity-purified, and separated by SDS/PAGE. Rae1 is untagged. Asterisks indi- a potential GLEBS-like motif (SI Fig. 9). The Nup43 or Seh1 cate the five FLAG-tag NuMA fragments expressed in varying amounts using this system. Numbers indicate molecular weight markers in kilodaltons. (C) WD-repeat ␤ propellers did not interact with NuMA325–829 (data Immunoblotting of ␣-GFP IPs from either GFP- or GFP-NuMA325–829-expressing not shown). We therefore conclude that a fragment of NuMA ␣ ␣ spanning amino acids 325–829, at the N-terminal end of the HeLa cells. IPs are blotted with -Rae1 or -NuMA [using BD Biosciences clone 22 monoclonal that recognizes an epitope (amino acids 658–691) within coiled-coil domain, interacts specifically with Rae1. NuMA325–829]. (D) HeLa cells overexpressing GFP-NuMA325–829 (green) To test whether NuMA325–829 interacts with Rae1 in vivo,we costained with tubulin (red) and DAPI (blue). expressed NuMA325–829-GFP in HeLa cells. We then used anti-GFP antibodies for IPs from extracts of cells expressing ϭ either NuMA325–829-GFP or GFP alone and tested for the cells (n 200; Fig. 4D and Table 1). Although we did not observe presence of Rae1. As seen in Fig. 4C, Rae1 was specifically any gross mislocalization of Rae1 in these cells (not shown), a coimmunoprecipitated with NuMA325–829-GFP but not GFP possible interpretation of these results is that NuMA325–829 binds alone (asterisks). We could also detect a small amount of to and sequesters some Rae1, making it unavailable to interact full-length NuMA (arrow) in the IPs, suggesting that full-length productively with full length NuMA. This would then be anal- NuMA associates with the NuMA325–829 fragment (arrowhead). ogous to the multipolar spindle phenotype observed after re- This observation implies that at least some dimerization deter- duction of Rae1 by RNAi. The NuMA325–829 fragment could also minants reside between amino acids 325 and 829 at the beginning dimerize with full-length NuMA, and the resulting hybrid of the predicted coiled-coil region of NuMA. NuMA-NuMA325–829 heterodimers would lack one C-terminal Because the NuMA325–829 fragment seems to bind Rae1 in domain and would potentially have reduced ability to link MTs. cells, we examined the effect of expressing this domain in HeLa cells. NuMA325–829-GFP localized in part to spindles (upper row Discussion of Fig. 4D) and colocalized with tubulin (lower row of Fig. 4D). Spindle assembly requires the temporal and spatial coordination We found that additional poles were observed in 30% of these of multiple overlapping pathways involving MT nucleation and

Wong et al. PNAS ͉ December 26, 2006 ͉ vol. 103 ͉ no. 52 ͉ 19785 Downloaded by guest on September 26, 2021 established, the resulting supernumerary poles may be suffi- ciently stable to persist, because none of the spindle poles have accumulated enough of the tetravalent crosslinks to compete with each other for stability. In this scenario, the minus ends of MTs are capped with ␥-tubulin and surrounded by pericentrin. In contrast, if NuMA overexpression is accompanied by Rae1 overexpression, the formation of a high density of tetravalent crosslinks may kinetically favor the formation of a bipolar spindle destabilizing any supernumerary poles that lack a critical density of tetravalent crosslinks. A spindle with a higher density of tetravalent crosslinks could successfully compete for NuMA and Rae1 against spindles with low-density crosslinks that are less stable. Our results with the overexpression of a Rae1-binding fragment of NuMA (NuMA325–829) would be entirely consistent with our speculations regarding the valency and density of NuMA-mediated minus-end MT bundling. The kinetics of spindle formation are influenced by many other components, including MT-based motors (26), MT dy- namics (27), gradients of Ran and kinases (28), and polyADP- ribosylation (29), that will likely modify the effects we reported here for Rae1 and NuMA. In any case, our results should provide a useful framework for further testing the dynamics of MT bundling in mitosis and elucidating the role of a Rae1–NuMA imbalance in chromosome segregation defects leading to Fig. 5. A ‘‘valency’’ model of MTs interacting with NuMA and Rae1. NuMA . is assumed to be a dimer (6) with the C-terminal (C) indicated to directly interact with MTs (7). A region (residues 325–829) at the N-terminal end of the Materials and Methods coiled coil of NuMA interacts with Rae1 (data in this paper) and therefore Plasmids. The plasmid-encoding full length human Rae1 (Image converts NuMA from a divalent to a tetravalent MT ‘‘crosslinker.’’ ID LIFESEQ95168410; Open Biosystems, Huntsville, AL) was subcloned into pcDNA3 with HA tag and pET28a. The NuMA domains were subcloned by PCR from pCDNA3-GFP-NuMA stabilization, pole formation and attachment and alignment of into pET28a with a C-terminal simian virus 40 T antigen nuclear chromosomes (3, 25). MTs are dynamically unstable structures localization sequence and a FLAG tag. All constructs were that are stabilized by a variety of MT-associated proteins. In confirmed by DNA sequencing. addition, ‘‘crosslinking’’ among MTs is required to bundle them at their minus end at the spindle pole. The C-terminal domain Cell Culture, Transfections, and Synchronization. HeLa cells were of NuMA has been shown to bundle MTs. If NuMA is a dimer transfected with Rae1 and NuMA siRNAs using Oligofetamine and (ref. 6 and Fig. 5), it could act as a divalent crosslinker of MTs. with GFP-NuMA and HA-Rae1 plasmids using Lipofectamine In addition, any NuMA-associated protein that also binds MTs 2000 following the manufacturer’s protocol (Invitrogen, Carlsbad, could function in the MT crosslinking. Rae1 has previously been CA). Cells were synchronized in S phase by double thymidine block shown to bind to MTs (21). Here we show that Rae1 also (30) using 2 mM thymidine with the following modifications. In interacts with NuMA, and we have mapped this interaction to experiments involving siRNA oligos [Fig. 2 and supporting infor- the N-terminal end of the coiled-coil domain. To our knowledge, mation (SI) Fig. 7], the cells were transfected 24 h before the this is a previously undescribed biochemical mapping of a initiation of the first thymidine block and collected or imaged after specific interaction between a nucleoporin and any component 72 h. In experiments involving plasmid-mediated protein overex- of the mitotic spindle. We suggest that by interacting with NuMA pression (Figs. 2 and 3 and SI Fig. 8), the transfection was initiated and MTs, Rae1 could increase the MT crosslinking valency of before the second thymidine block for 4 h. For experiments NuMA (Fig. 5) and further stabilize MTs at their minus ends. We represented in Fig. 1C, cells were released into 30 ng/ml Nocoda- propose this interaction is critically required for normal bipolar zole after the second thymidine block, harvested for analysis at spindle formation. We observed the presence of centrosomal hourly intervals for 12 h during Nocodazole incubation, and then markers in the aberrant spindle poles of Rae1-depleted cells, and collected by mitotic shakeoff, replated in fresh medium, and the same has been reported in the case of NuMA overexpression harvested for analysis at hourly intervals for 4 h. For the RNAi in cancer cell lines (24); therefore, we cannot preclude the experiments, siRNA duplexes targeting Rae1 [5Ј-GCAGUAAC- possibility that Rae1 and NuMA are involved in CAAGCGAUACA-3Ј] (21) or NuMA [5Ј-GGCGUGGCAG- duplication or stabilization. GAGAAGUUC-3Ј] (31) were purchased from Integrated DNA Our data suggest that a balance between NuMA and Rae1 is Technologies (Coralville, IA). Mock transfection was with buffer critical for bipolar spindle formation. Specifically, in the case of alone (control). Transfection efficiency was monitored with overexpression of NuMA coupled with concomitant overexpres- Block-iT (Invitrogen). sion of Rae1 and, conversely, in the case of depletion of Rae1 coupled with concomitant depletion of NuMA, formation of Antibodies and Immunofluorescence. In initial experiments, anti- supernumerary spindle poles is suppressed (Figs. 3 and 4D). We Rae1 antibodies from K. Weis (University of California, Berke- speculate that the additional crosslinking valency of NuMA, by ley, CA) (21) and J. van Deursen (Mayo Clinic, Rochester, MN) virtue of its interaction with MT-bound Rae1, increases the (32) were used (Fig. 1 A and B). For all subsequent experiments, ‘‘density’’ of crosslinks and therefore enhances the bundling of peptides based on human Rae1 residues 313–327 (FYNPQKK- MTs at their minus ends. In the case of NuMA overexpression NYIFLRNAAEE) (21), with N-terminal acetylation and C- or Rae1 depletion, many of the crosslinks at the minus end of terminal amidation, were injected in rabbits (Cocalico Biologi- MTs would be divalent rather than tetravalent (Fig. 5). Never- cals, Reamstown, PA). Antibodies were affinity-purified before theless, if a critical number of these divalent crosslinks has been use. Anti-NuMA polyclonal antibody used in Fig. 1 A and B was

19786 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0609582104 Wong et al. Downloaded by guest on September 26, 2021 from D. Compton (Dartmouth Medical School, Hanover, NH) In Vitro Binding Assays. Proteins were expressed by using the (33); anti-NuMA monoclonal antibody (clone 22) from BD Promega (Madison, WI) TNT coupled transcription/translation Biosciences (Franklin Lakes, NJ) was used in all other experi- system according to the manufacturer’s protocol. Five microli- ments. DM1A monoclonal ␣-tubulin antibody and ␥-tubulin ters of Flag beads (ANTI-FLAG M1 Agarose Affinity Gel from antibody were from Sigma–Aldrich (St. Louis, MO). The dynein Sigma–Aldrich) were washed three times with binding buffer (20 monoclonal antibody (clone 74.1) was from Chemicon (Te- mM Hepes, pH 7.5/100 mM KCl/5 mM MgCl2/0.1% Tween mecula, CA), Phospho-Histone H3 antibody was from Upstate 20/20% glycerol/0.01% BSA/1 mM DTT/1 mM PMSF/1ϫ com- Biotechnology (Lake Placid, NY). ␣-HA, ␣-GFP, and ␣-peri- plete protease inhibitor mixture), preblocked for 10 min with 10 centrin antibodies were from Abcam (Cambridge, U.K.). Sec- ␮l of nonspecific rabbit serum, washed with binding buffer, and ondary antibodies were from Molecular Probes (Eugene, OR). resuspended in 60 ␮l of binding buffer. Then, 10 ␮lofin vitro For immunofluorescence, synchronized HeLa cells were transcribed and translated [35S]methionine labeled Rae1 and washed in PBS and fixed for 10 min in methanol at Ϫ20°C. Cells NuMA-Flag mutants were added to the beads, and the mixture were then permeabilized with 0.2% Triton X-100 in PBS for 10 was incubated at 4°C for 1 h. Beads were washed six times with min at room temperature. Samples were examined on a Zeiss binding buffer and boiled in 15 ␮l of SDS/PAGE sample buffer. (Oberkochen, Germany) LSM510 MEGA confocal microscope, Samples were analyzed by SDS/PAGE (4–20% Tris-glycine gels; ϫ and all images were acquired by using a plan-Apochromat 100 Invitrogen), followed by autoradiography. 1.4-N.A. objective. We thank M. Blower and K. Weis (University of California, Berkeley, 7 Immunoprecipitations (IPs). For IPs, Ϸ10 cells were seeded and CA) and J. van Deursen (Mayo Clinic, Rochester, MN) for the initial synchronized as described above. Mitotic HeLa cells were col- supply of human Rae1 antibodies, D. Compton (Dartmouth Medical lected, washed with PBS, spun at 400 ϫ g for 10 min, and lysed School, Hanover, NH) for NuMA antibodies, A. Merdes (Centre Na- in 1 ml of cold Lysis buffer (50 mM Tris⅐HCl, pH 7.2/250 mM tional de la Recherche Scientifique) for the GFP–NuMA construct, and NaCl/0.1% Nonidet P-40/2 mM EDTA/10% glycerol) containing M. Kastan (St. Jude Children’s Research Hospital, Memphis, TN) for the 1ϫ protease inhibitor mixture (Roche, Indianapolis, IN) and 1 pcDNA3-HA tag vector. The FLAG tag pET28a plasmid was a kind gift mM PMSF. Lysates were centrifuged for 30 min at 4°C at of K. Yoshida (the Blobel laboratory). We thank K. Hsia (the Blobel ␤ 14,000 ϫ g. The resulting lysate supernatants were precleared laboratory) for various propeller constructs. We also thank Haiteng with 50 ␮l of Protein A/G bead slurry (Santa Cruz Biotechnol- Deng and Joseph Fernandez for the mass spectrometry analysis, Henry Zebroski for the Rae1 peptide synthesis, and A. North for support with ␮ CELL BIOLOGY ogy, Santa Cruz, CA), mixed with 5–10 l of various antibodies confocal microscopy at The Rockefeller University Bioimaging facility. as specified, and incubated for1hat4°Cwithrocking. The beads ␮ We thank members of the Blobel laboratory for helpful discussions and were then washed five times with 500 l of Lysis buffer. After the Megan King, Patrick Lusk, Joe Glavy, and Hang Shi for critical reading last wash, 50 ␮lof1ϫ SDS/PAGE blue loading buffer (New of the manuscript. This work was supported in part by the National England Biolabs, Ipswich, MA) was added to the bead pellet Institutes of Health (to E.C.) and by a grant from the Leukemia and before loading. Lymphoma Society (to G.B).

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Wong et al. PNAS ͉ December 26, 2006 ͉ vol. 103 ͉ no. 52 ͉ 19787 Downloaded by guest on September 26, 2021