Proximity mapping of human separase by the BioID approach

Biochemical and Biophysical Research Communications xxx (2016) 1e7

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Proximity mapping of human separase by the BioID approach Fikret Gurkan Agircan, Shoji Hata, Carmen Nussbaum-Krammer, Enrico Atorino, Elmar Schiebel* €t Heidelberg, DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany Zentrum für Molekulare Biologie der Universita

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 July 2016 Received in revised form 26 July 2016 Accepted 1 August 2016 Available online xxx

Separase is a caspase-like cysteine protease that is best known for its essential role during the metaphase-to-anaphase transition when it cleaves the cohesin ring complex that keeps the sister chromatids together. Another important function of separase is to regulate the process of centriole separation, known as centriole disengagement, at the end of mitosis. We used proximity-dependent biotin identification (BioID) to expand our knowledge on the identity of separase's proximity interactors. We show that separase BioID labeled two domains at the mother centriole: an area underneath the centriolar appendages and another at the proximal end of the mother centriole. BioID analysis € m Syndrome Protein 1 identified more than 200 proximity interactors of separase, one being the Alstro (ALMS1) at the base of centrioles. Other proximity interactors are the histone chaperons NAP1L1 and NAP1L4, which localize to the spindle poles during mitosis and the spindle assembly checkpoint proteins BUBR1, SKA1 and SKA3 that reside at kinetochores in early mitosis. Finally, we show that depletion of BUBR1 homolog from Caenorhabditis elegans delayed the recruitment of separase to mitotic chromosomes, and eventually anaphase onset. © 2016 Elsevier Inc. All rights reserved.

Keywords: Separase BioID Kinetochore Centrosome

1. Introduction Cells replicate their genome in S-phase. The duplicated sister chromatids are kept together by an encircling cohesion complex until anaphase onset [1]. At the metaphase-to-anaphase transition, activation of separase (ESPL1), a cysteine caspase like protease, leads to cleavage of the cohesin ring complex subunit RAD21SCC1, to liberate the two sister chromatids. This release is necessary to enable the successful segregation of the newly separated sister chromatids into the two daughter cells. Besides the established function of separase at chromosomes, separase's caspase activity has also been linked to centriole disjunction in mitosis [2,3]. Both the canonical substrate, RAD21SCC1, and a centrosomal protein, known as pericentrin, have been implicated as linker molecules that maintain centriole cohesion. Cleavage of SCC1 and pericentrin by separase have been proposed to be the molecular mechanism by which the disjunction of the two centrioles is promoted [4]. While separase's function is well studied at the kinetochore and reasonably well described at centrosomes, we do not know much about separase's habitat at the centrosome and kinetochore.

* Corresponding author. E-mail address: [email protected] (E. Schiebel).

Affinity purification coupled to mass spectrometry (AP-MS) is the most common method to identify protein complexes. It is also known that AP-MS works well when the purified complex is soluble and of high affinity [5]. However, AP-MS is not very applicable to identify protein complexes at centrosome and kinetochore as it is well established that the integrity of centrosomes and kinetochores is preserved under low salt concentration. High salt conditions dissolve these structures but also affect the protein-protein interactions [6]. Proximity-dependent biotin identification (BioID) is an alternative method to AP-MS [7]. For BioID the protein of interest is fused to the hyperactive biotin ligase BirAR118G (BirA*). Neighboring proteins that lie within a 20 nm radius are biotinylated by the BirA*. The biotinylated proteins can then be purified using the high affinity binder streptavidin followed by their identification by mass spectrometry. Another limitation for AP-MS at centrosomes and kinetochores is the lack of accessibility to such densely packed protein complexes. In contrast, BioID is much better at identifying weak and transient interactions [5]. Here we used BioID to identify proximity interactors of separase in human cells. Separase was tagged at its either N- or C-terminus with BirA*. Both N- and C-BioID for separase identified overlapping proximity partners with enrichment at centrosomes and kinetochores. The combination of the BioID approach with proteasome

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Please cite this article in press as: F.G. Agircan, et al., Proximity mapping of human separase by the BioID approach, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.002

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inhibition to stabilize proteins, or mitotic enrichment with inhibition of the kinesin-5 motor protein Eg5, identified a novel set of separase proximity interactors related to the kinetochores and the centrosomes. 2. Materials and methods 2.1. Antibodies The following antibodies were used for in this study: ALMS1 (1:300 [8]), CEP164 (1:1000 [9]), Centrin 2 (1:100, Millipore 20H5), HA (IF 1:100; IB: 1:1000, Roche 3F10), Myc (IF 1:100; IB 1:1000, Santa-Cruz 9E10), pericentrin (1:2000, Abcam, #ab4448), pericentrin (1:800, guinea pig, this study), SAS6 (1:50, Santa-Cruz sc81431), Streptavidivin-Alexa647 (1:1000, Invitrogen, #s32357), and g-tubulin (1:1000, Abcam, #ab27074), Streptavidin-HRP (1:2000, Pierce, #21130).

partner if the protein was enriched in the separase BioID at least 4 fold over the level seen in the Only-BioID control in two independent experiments. 2.6. Time-lapse imaging of C. elegans embryos and eGFP-Separase transfected HeLa cells Time-lapse images were acquired using Leica DMi8 Spinning Disk confocal microscope, equipped with an Hamamatsu Orca Flash 4.0 LT (C11440-42U) camera. SEP-1::GFP localization in C. elegans embryos was monitored using an HC PL APO 63/1.40e0.60 oil objective, by obtaining 4  0.3-mm z-stacks every 30 s, with 500-ms exposure times for GFP at 30% 488 nm laser power. eGFP-Separase localization in transiently transfected HeLa cells were monitored using an HC PL APO 40/1.3 oil objective, by obtaining 13  1-mm zstacks every 20 min, with 100-ms exposure times for GFP at 15% 488 nm laser power. Images were analyzed and time-lapse movies were made using FiJi.

2.2. Cell culture conditions 3. Results Stable U2OS FRT TReX cell lines were created and cultured as described [10]. Cells were induced with 1 mg/ml of doxycycline for 24 h, and incubated in 50 mM of biotin plus 1 mg/ml doxycycline for another 24 h. For MG132 treatment, cells were treated with 1.25 mM for 24 h at the same time with doxycycline and biotin. HeLa cells were seeded on Ibidi 8-well mSlide (Ibidi, # 80826) with ~40% confluency. Next day, they were transfected with Lipofectamine 2000 according to the manufacturer's protocol. 2.3. Caenorhabditis elegans strains, RNA interference of embryos Nematodes were grown using standard methods [11]. The strain WH416 ojIs58 [pie-1p::sep-1::GFP þ unc-119(þ)]III was obtained from the Caenorhabditis Gene Center (CGC) [12]. The bub-1 RNAi clone was obtained from the Ahringer RNAi feeding library, the empty vector L4440 (Addgene) was used as control [13]. L4 animals were transferred onto fresh RNAi plates and fed at 20  C for 48 h prior to imaging. For isolation of one-cell embryos, hermaphrodite adult worms were dissected in 2 ml nanosphere size standards solution (100 nm polystyrene beads, ThermoScientific) on an 18  18 nm coverslip, which was subsequently placed onto an 2% agarose pad on a microscope slide. 2.4. Immunofluoresence microscopy Immunofluoresence staining and imaging were performed as previously described [14]. For the subcellular localization of NAP1L1/4, z-stack images were acquired using Zeiss LSM780 confocal microscopy with standard equipment. Acquired images were z-projected using FiJi. 2.5. Immunoprecipitation and mass spectrometry analysis 10  15-cm plates were used for growing the cells for total cell lysate, MG132 treatment and CSK extraction. Sample preparation and mass spectrometry analysis were performed as previously described with the exception that label-free quantification (LFQ) was performed using default MaxQuant settings with a minimum ratio count of 2 and the “Stabilize large LFQ ratios”-option enabled [15e17]. For data analysis, we used values obtained from the formula of LFQNET ¼ log2(LFQsample)  log2(LFQcontrol). LFQ (label free quantification) is based on quantification with MaxQuant software [15]. We only considered proteins with LFQNET values > 2 in duplicates. This means that we only identified a molecule as being a proximity

3.1. Separase proximity interactors localize to centrosomes and chromosomes We used the AP-MS approach to identify novel interactors of separase at centrosomes and kinetochores. N-terminally eGFP tagged separase, which localized to both the centrosomes and chromosomes (Fig. S1A, Supplementary Movie 1), was transiently expressed in HEK293T cells. eGFP-Separase and its interactors were pulled down using GFP binder and analyzed by mass spectrometry (Fig. S1B). Although we could identify 101 distinct proteins as candidates of separase interactors in comparison with control (Table S1), none of the known centrosome or kinetochore components were detected (Table S1, Fig. S1C). Given the limitations of APMS to detect transient protein-protein interactions, we changed our strategy to a recently developed technique called BioID to identify proximity interactors of separase [7]. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.bbrc.2016.08.002. We first constructed stable U2-OS cell lines expressing either inducible BirA*-Separase, Separase-BirA* or BirA* only constructs (Fig. 1A). Both BirA*-Separase and Separase-BirA* fusions were subject to auto-cleavage, indicating that separase-BioID fusion proteins had proteolytic activity (Fig. 1B) [18]. The subcellular localization of the Separase-BirA* and the signals generated by separase's proximity interactions was investigated by indirect immunofluorescence. Initially, we confirmed that the biotin signal was detectable on metaphase chromosomes (Fig. 1C), illustrating the correct localization of separase to the chromosomes during mitosis. Moreover, the Separase-BirA* colocalized with the centrosome marker pericentrin (Fig. 1D). This centrosomal signal became readily apparent upon extraction of soluble cytoplasmic proteins. In control cells expressing only BirA*, the biotin signal was detected at structures resembling mitochondria (Fig. 1D). This staining pattern was expected since some of mitochondrial proteins are naturally biotinylated [7]. In case of Separase-BirA*, the streptavidin signal localized in the cytoplasm, at the centrosomes and weakly in the nucleus (Fig. 1D). In order to gain insights into the localization of separase binding partners at centrosomes, we analyzed the sub-localization of the centrosomal biotin signal in greater detail. N- and C-BioID detected two spatially distinct pools of biotin at the mother centriole (Fig. 2A). The biotinylation signals partially overlapped with CEP135 at the proximal end of the mother centriole. At the distal end, it was found below the CEP164 ring overlapping with the sub-distal

Please cite this article in press as: F.G. Agircan, et al., Proximity mapping of human separase by the BioID approach, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.002

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Fig. 1. BirA*-fused Separase and proximity interactors localize to the centrosome and chromosomes. (A) Separase was either N- or C-terminally tagged with a hyperactive biotin ligase (BirA*). A non-fused biotin ligase expressing construct was used as negative control. (B) U2OS Flp-In T-REx cell lines were used to construct stably expressing BirA* constructs. Protein levels of BirA-fused separase's were confirmed by immunoblotting with either myc or HA antibody, respectively. Both constructs were subject to self-cleavage. (C) From both control BirA* and Separase-BirA* expressing cells, metaphase spread chromosomes were prepared and stained with 647 nm Alexa-tagged Streptavidin. Scale bar: 10 mm. (D) The localization of the BirA*-fused separase and proximity interactors were determined with HA antibody and Alexa 647-labeled streptavidin, respectively. In the presence of biotin, biotinylated proteins were located at centrosomes and cytoplasm. Centrosomal localization of BirA*-fused separase became readily visible upon extraction of soluble cytoplasmic proteins. Scale bar: 10 mm.

appendage protein ODF2 (Fig. 2A). In contrast, the newly assembled daughter centriole was not labeled. This labeling pattern was similar to the localization of eGFP tagged separase (Fig. 2B). Thus, these data suggest that separase interacts with proximal and distal domains of the mother centriole but does not have affinity to the daughter centriole (Fig. 2C).

kinetochores or the mitotic spindle [19]. These hits were named as mitosis-related proteins. 28 mitosis-related proteins were found overlapping in both BioID datasets. 6 and 13 proximity interactors were specific to N-BioID and C-BioID, respectively (Fig. 3A). The centrosomal proteins ALMS1, CEP131, PCM1, CEP170 and the PP6 phosphatase subunits ANKRD17 and ANKRD52 were identified with high confidence in both BioIDs.

3.2. The separase proximity interactome identifies proximity interactors at centrosome and kinetochore We next performed mass spectrometric analysis of biotinylated proteins enriched via streptavidin affinity purification from whole cell lysate of BirA*-Separase or Separase-BirA* expressing cells in order to identify proximity interactors of separase. Silver staining and immunoblotting detected biotinylated proteins after streptavidin purification of proteins from cell extracts (Fig. S2), indicating the separase BioID approach was working. N-BioID (BirA*-Separase) and C-BioID (Separase-BirA*) identified 111 and 123 proximity interactors, respectively. LFQNET intensity (See Materials and Methods) comparison showed that most of the proteins were identified with nearly equal frequency in the N-BioID and C-BioID (Fig. 3A). SecurinPTTG1, a well known interactor of separase, was found with high confidence in the CBioID, suggesting BioID approach worked well in this study (Table S2). Out of 145 individual proteins detected in the N- and CBioID, 47 were annotated as localizing to centrosomes,

3.3. Combination of BioID with proteasome inhibition is necessary for detection of cell cycle regulated proximity interactors In order to determine unstable separase proximity interactors, we repeated the separase BioID in the presence of the proteasome inhibitor MG132 (Fig. 3B and Table S2). Following proteasome inhibition, 55 novel separase BioID interactors were detected (Table S2). Importantly, novel kinetochore proteins such as BUB1B (BUBR1) and SKA3 were detected only in the MG132 BioID. BUB1B functions in the spindle assembly checkpoint [20]. Protein level of BUB1B fluctuates during the cell cycle, suggesting that its MG132dependent BioID detection reflects an increase in protein level per se [21]. In addition, BUB1B and separase concentrate at kinetochores during metaphase [22,23]. As this particular cell cycle phase is enriched by proteasome inhibition, it is plausible that BUB1B and separase remain in close proximity for longer at the kinetochores of MG132 treated cells.

Please cite this article in press as: F.G. Agircan, et al., Proximity mapping of human separase by the BioID approach, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.002

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Fig. 2. Separase proximal interactors localize to the mother centriole as two pools. (A) Biotinylated proteins in separase BioID localized to two sites of the mother centriole. Proximal pool partially overlapped with CEP135 and distal pool overlapped with the sub-distal appendage component ODF2. (B) eGFP tagged separase also localized to the mother centriole, giving similar staining pattern as separase-BioID. Scale bar: 1 mm. (C) Schematic illustration of separase and its proximal interactors at the mother centriole.

3.4. Enrichment of cells in mitosis identifies novel proximity interactors of separase Cyclin B1 that interacts with separase during mitosis was absent from the streptavidin-enriched proteins of either N- or CBioID [24]. The low frequency of mitotic cells may underlie this failure in detection. To close this gap, we enriched cells in mitosis through the use of an Eg5 inhibitor STLC to trigger a mitotic checkpoint arrest. Upon mitotic enrichment, we could detect Cyclin B1CCNB1 and Cdk1 in N- and C-BioID (Fig. 3C and Table S2). Interestingly, N-BioID and C-BioID from mitotic cells showed 47 common proteins, 30 of which were found at centrosomes, kinetochores or the mitotic spindle (Fig. 3C). The PP6 phosphatase subunits ANKRD17, ANKRD52, PPP6C, PPP6R1, and PPP6R3, and the ATPases RUVBL1 and RUVBL2 were among the mitotic separase proximity interactors together with NAP1L1 and NAP1L4 complex that belong to the nucleosome assembly protein (NAP) family. (Table S2, Fig. 3D). Next, we checked where the centrosomal protein ALMS1 and NAP1L1/4 complex might interact with separase. ALMS1 localized to the proximal end of mother centriole, with a significant overlap with the proximal pool of separase BioID signal (Fig. 4A),

suggesting that at least a fraction of the proximal biotin marker on the mother centriole represents the ALMS1-separase interaction. NAP1L1 localized to the microtubules in interphase and spindle pole in mitosis (Fig. 4B, Fig. S3A). NAP1L4, moreover, localized to the cytoplasm in interphase and spindle poles in mitosis (Fig. S3B). 3.5. BUB1B depletion delays recruitment of separase to chromosomes in C. elegans embryos We identified several kinetochore and centrosome components as the proximity interactors of separase. Among the several kinetochore localizing proteins, we focused on BUB1B to show the impact of this BioID study on separase regulation. BUB1B is a core component of the spindle assembly checkpoint (SAC) [25]. We detected BUB1B as a proximity interactor of separase in MG132 treated cells (Table S2). The BioID interaction with BUB1B may indicate that separase is part of this recruitment cascade. Because separase is readily detected at the holocentric kinetochores of C. elegans, we used C. elegans embryos as a model to study localization dependencies between BUB1B and separase. The BUB1B homolog (BUB1) was depleted from C. elegans embryos expressing

Please cite this article in press as: F.G. Agircan, et al., Proximity mapping of human separase by the BioID approach, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.002

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Fig. 3. Mass Spectrometry Analysis of Separase Proximity Interactors. The LFQNET values of detected proteins were compared in BirA*-Separase and Separase-BirA* from (A) total cell lysates, (B) MG132 treated samples, and (C) STLC treated (mitotic enriched) samples. Known interaction partners of separase are shown in the graph. Venn diagram was created to show the distribution of proteins in comparison to mitosis related proteins, which is determined using the database MicroKits 4.0. (D) Summary of the proximity interactors of separase at kinetochores (blue) and centrosomes (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

GFP tagged separase. In agreement with published data, separase localized to centrosomes and chromosomes of C. elegans embryos during mitosis (Fig. 4C) [26]. BUB1 depletion by RNAi decreased separase levels at chromosomes in prometaphase/early metaphase without having an impact on separase at centrosomes (Fig. 4D and Fig. S4). Interestingly, separase association with chromosomes increased just before anaphase onset and therefore mitotic progression was only marginally delayed (Fig. 4C). 4. Discussion BioID offers a good approach to reveal the identity of separase partners, especially if they are present in low copy number or stably bound to chromatin or the centrosomes [5]. First, we performed the BioID experiments using both N- and C-terminal hyperactive BirA* tagged separase fusion proteins. Moreover, given the arduous regulation of separase, we combined the BioID screens with either proteasome inhibition or mitotic enrichment and showed that combination of BioID with proteasome-inhibited or synchronized cells is essential to uncover the full range of the interaction map. Later, we identified that centrosomal signals generated by eGFPseparase and separase-BioIDs were restricted to the mother centriole. Presently, it is unclear why separase preferentially localizes to mother centrioles. The association with the proximal end of the centriole can be attributed to separase's function in centriole disengagement. The distal BioID mark on the mother centriole, however, could arise as a consequence of the relocalization of separase proximity interactors to the sub-distal appendages. Alternatively, separase might be targeted to the centrosome through sub-distal appendage as suggested for some centrosomal proteins [27].

Next, upon analysis of BioID results of the combined experiments and based on the previous studies, we suggest that separase interact with NAP1L1, NAP1L4, ALMS1, CEP131, CEP170, PCM1 and the PP6 complex at centrosomes/spindle poles; SKA1, SKA3, SPAG5, and BUB1B at kinetochores [28e30]. CEP131 and PCM1 are centrosomal satellites proteins and they appear as core components of the centrosomal protein BioID as centrosomal proteins are commonly shuttled between the centrosome and satellites [17,31]. Currently the functional link between PP6, NAP1L1/4 and separase is not known. Nonetheless, PP6 has been suggested to regulate microtubule dynamics at the spindle pole and spindle pole localization of NAP1L1 and NAP1L4 suggests that these proteins might interact with separase at spindle poles [32]. Furthermore, SKA1 and SKA3 localize to the plus-end of microtubules and have been shown to recruit APC/C and PP1 to the kinetochore [33,34]. Moreover, BUB1B is important for the recruitment of kinetochore proteins including Mad2 and CENP-E [23]. Therefore, separase might be targeted to the kinetochore in similar mechanism involving SKA1, SKA3 and BUB1B. Finally, we depleted BUB1 mRNA in C. elegans and showed that the localization of separase to chromosomes was delayed until anaphase onset. Consistent with our result, it was shown that depletion of BUB1 in C. elegans slightly delays anaphase onset by regulating the separase activity [35]. Thus, we propose that BUB1B promotes recruitment of separase to sister chromatids in early mitosis, probably by directly interacting with separase. Thus, our study has identified separase proximity interactors at kinetochores and centrosomes that may function as regulators or docking sites for separase. Our proximity interaction maps is an invaluable resource for further studies on the regulation of separase.

Please cite this article in press as: F.G. Agircan, et al., Proximity mapping of human separase by the BioID approach, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.002

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Fig. 4. Analysis of Novel Separase Proximity Interactors. (A) The sub-localization of the separase BioID signal was determined with respect to the ALMS1 and CEP164. ALMS1 colocalize with separase BioID signal at the centrosome. Scale bars: 1 mm. (B) The sub-localization of NAP1L1 was determined in U2-OS cells upon transient transfection and subsequent immunofluoresence confocal imaging. The boxed areas on the right are the enlargements of the depicted areas in the figure. NAP1L1 localizes to microtubules and spindle poles in interphase and mitosis, respectively. Scale bars: 1 mm. (C) Maximum intensity projected z-stack confocal images from time-lapse series of one-cell embryos expressing GFP::SEP-1. In control treated embryos, GFP::SEP-1 was highly accumulated at the chromosomes starting from early metaphase (t ¼ 30s). During the late metaphase and anaphase (t ¼ 90s  150s), GFP::SEP-1 localized to chromosomes (red arrowhead) and centrosomes (white arrowhead). In BUB1 RNAi treated embryos, significantly less GFP:SEP-1 signal was detectable in early metaphase (t ¼ 30s); however, in late metaphase GFP::SEP-1 was recruited back to the chromosomes (t ¼ 150s). The signal on the chromosomes during anaphase was comparable to control (t ¼ 180s). Scale Bar: 5 mm, time in seconds. (D) Quantification of percentage of the animals, which exhibited high GFP::SEP-1 accumulation at the chromosomes in early- and mid-metaphase during the first cell division. Bar graph: SD, n > 30 (from 4 independent experiments). Two-tailed t-test was performed, p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Acknowledgements

References

We are grateful to Drs. L. Pelletier (University of Toronto, Can€t Basel, Switzerland) ada) for helpful discussions; E. Nigg (Universita for U2-OS FRT TReX cell line; T. Hearn for ALMS1 antibody (Swansea University, UK); K. Goto (Yamagata University, Japan) for NAP1L1 and NAP1L4 constructs; ZMBH mass spectrometry/proteomics core facility for mass spectrometry and proteomics analysis. C. elegans strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The work in E.S.'s group is funded by Deutsche Forschungsgemeinschaft DFG SCHI 295/6-1. Work in C. N.-K.’s group is supported by the Deutsche Forschungsgemeinschaft (SFB1036, TP20).

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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.08.002. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.08.002.

Please cite this article in press as: F.G. Agircan, et al., Proximity mapping of human separase by the BioID approach, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.002

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Please cite this article in press as: F.G. Agircan, et al., Proximity mapping of human separase by the BioID approach, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.002