TOPORS, a dual E3 ubiquitin and Sumo1 ligase, interacts with 26 S protease regulatory subunit 4, encoded by the PSMC1 gene

RESEARCH ARTICLE TOPORS, a Dual E3 Ubiquitin and Sumo1 Ligase, Interacts with 26 S Protease Regulatory Subunit 4, Encoded by the PSMC1 Gene Barbara Czub 1‡ *, Amna Z. Shah 1‡ *, Giovanna Alfano 1 , Przemysław M. Kruczek 1 , Christina F. Chakarova 1 , Shomi S. Bhattacharya 1,2 1Institute of Ophthalmology, University College London (UCL), London EC1V 9EL, United Kingdom, 2Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Seville-41092, Spain ‡These authors are joint first authors on this work. *[email protected].uk (BC); [email protected] (AZS) Abstract The significance of the ubiquitin-proteasome system (UPS) for protein degradation has been highlighted in the context of neurodegenerative diseases, including retinal dystrophies. TOPORS, a dual E3 ubiquitin and SUMO1 ligase, forms a component of the UPS and selected substrates for its enzymatic activities, such as DJ-1/PARK7 and APOBEC2, are important for neuronal as well as retinal homeostasis, respectively. TOPORS is ubiquitously expressed, yet its mutations are only known to result in autosomal dominant retinitis pigmentosa. We performed a yeast two-hybrid (Y2H) screen of a human retinal cDNA library in order to identify interacting protein partners of TOPORS from the retina, and thus begin delineating the putative disease mechanism(s) associated with the retina-specific phenotype resulting from mutations in TOPORS. The screen led to isolation of the 26 S protease regulatory subunit 4 (P26s4/ PSMC1), an ATPase indispensable for correct functioning of UPS-mediated proteostasis. The interaction between endogenous TOPORS and P26s4 proteins was validated by co-immuno-precipitation from mammalian cell extracts and further characterised by immunofluorescent co-localisation studies in cell lines and retinal sections. Findings from hTERT-RPE1 and 661W cells demonstrated that TOPORS and P26s4 co-localise at the centrosome in cultured cells. Immunofluorescent staining of mouse retinae revealed a strong P26s4 reactivity at the interface between retinal pigmented epithelium (RPE) layer and the photoreceptors outer segments (OS). This finding leads us to speculate that P26s4, along with TOPORS, may have a role(s) in RPE phagocytosis, in addition to contributing to the overall photoreceptor and retinal homeostasis via the UPS. Introduction The significance of the ubiquitin-proteasome system (UPS) for protein degradation has been greatly emphasised in neurodegenerative diseases [1–3]. In particular, its importance has been established in context of retinal dystrophies, highlighting the insufficient capacity of the 26 S proteasomes to degrade excess misfolded proteins as a major factor in the aetiology of photoreceptor degeneration [4–6]. TOPORS (MIM 609507), a topoisomerase I-binding, arginine/ PLOS ONE | DOI:10.1371/journal.pone.0148678 February 12, 2016 1/20 OPEN ACCESS Citation: Czub B, Shah AZ, Alfano G, Kruczek PM, Chakarova CF, Bhattacharya SS (2016) TOPORS, a Dual E3 Ubiquitin and Sumo1 Ligase, Interacts with 26 S Protease Regulatory Subunit 4, Encoded by the PSMC1 Gene. PLoS ONE 11(2): e0148678. doi:10.1371/journal.pone.0148678 Editor: Olaf Strauß, Eye Hospital, Charité, GERMANY Received: September 5, 2015 Accepted: January 20, 2016 Published: February 12, 2016 Copyright: © 2016 Czub et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The work was funded by the following: 1. BRITISH EYE RESEARCH FOUNDATION (Fight for Sight; charity number 1111438): www.fightforsight. org.uk (PhD Studentship 1816 grant to BC and SSB); 2. THE TERESA ROSENBAUM GOLDEN CHARITABLE TRUST (Rosetrees Trust; charity number 298582): www.rosetreestrust.co.uk (Grant number M233-CD1 to BC AZS GA SSB); 3. THE SPECIAL TRUSTEES OF MOORFIELDS EYE HOSPITAL GENERAL FUND (Charity number serine-rich protein, which was initially identified as a binding partner of human topoisomerase I (hTop1) and p53 in two independent yeast two-hybrid (Y2H) screens [7,8], is a component of the UPS. It acts as a dual E3 ubiquitin and SUMO1 ligase [9–11], these two ligase activities being mutually exclusive and dependent on its phosphorylation status [12,13]. Since cross-talk between the ubiquitination and SUMOylation pathways has been repeatedly demonstrated [14–17], it could be speculated that both TOPORS ubiquitination as well as SUMOylation activities affect UPS function. E3 enzymes are a vast group of proteins mediating the final step of ubiquitination (reviewed in [18]). They convey specificity in the protein degradation processes by identifying substrates for the UPS and usually recognize only specific E2 enzyme-substrate combinations. The ubiquitin ligase activity of TOPORS is conveyed by its RING domain [10]. Interestingly, TOPORS has the capacity to recognise a greater variety of E2 ubiquitin-conjugating enzymes than most other RING E3 ligases [10,19], suggesting that it may have a more prominent regulatory role in the UPS than E3 ligases with a narrower spectrum of specificity. Several substrates for both its activities have been identified for TOPORS. Remarkably, DJ-1 (MIM 602533), implicated in earlyonset Parkinson’s disease (PARK7, MIM #606324), in the aetiology of which the UPS is involved [20,21], as well as Apolipoprotein B mRNA-editing Enzyme, Catalytic Polypeptide 2 (APOBEC2) required for zebrafish retina regeneration are among the SUMOylation substrates of TOPORS [22,23], further supporting the role for the UPS in neuronal and retinal homeostasis. Our laboratory was the first to link mutations in TOPORS with inherited autosomal dominant retinitis pigmentosa (adRP, locus RP31, MIM #609923) [24], later shown to cause approximately 1% of all adRP [25,26]. A mutation in TOPORS leading to an autosomal dominant pericentral retinal dystrophy (adPRD, MIM 180210) was additionally identified in a large multi-generational Scandinavian pedigree [27]. These phenotypes are of special interest since TOPORS is a ubiquitously expressed gene, yet its mutations have been known to result only in retinal degeneration with no other symptoms. Hence, our goal was to identify protein interacting partners of TOPORS from the human retina by performing a yeast two-hybrid (Y2H) screen. We identified the 26 S protease regulatory subunit 4 (P26s4, UniProt #P62191), encoded by the PSMC1 gene (MIM 602706). The 26 S proteasome comprises a barrel-shaped core catalytic particle (CP) and one or two flanking regulatory particles. The latter consist of at least twenty protein subunits constituting a base and a cap. The base includes a ring of six ATPases [28], of which P26s4 is the only one, whose ATPase activity is essential for the peptidase activity of the CP [29]. The regulatory particles confer dependence on ATP and substrate specificity. Here we present evidence that the dual E3 ubiquitin and SUMO1 ligase, TOPORS, interacts with the 26 S protease regulatory subunit 4 (P26s4) in yeast and in mammalian cells. Materials and Methods Yeast-Two Hybrid cDNA Library Construction The human retinal cDNA library was created in yeast using the Make Your Own “Mate & Plate™”Library System (Clontech, CA, USA) according to the manufacturer’s instructions in the pGADT7-Rec vector (Clontech, CA, USA). The library was constructed using an oligo-dT primer (OdT). The resulting preys were fused with GAL4 AD at their amino-terminus. Yeast Two-Hybrid Vectors The Y2H bait construct (pBD-TOPORS: GAL4 DNA binding domain (BD) fused with fulllength TOPORS) was generated using the Gateway Cloning System (Life Technologies, CA, USA) according to the manufacturer’s instructions, using vectors, which had been modified from the original Stratagene (CA, USA) Y2H vectors to contain the att sequences, allowing for TOPORS Interacts with Regulatory Subunit 4 of the 26 S Proteasome PLOS ONE | DOI:10.1371/journal.pone.0148678 February 12, 2016 2/20 228064): www.moorfields.nhs.uk (Personal award to SSB for infrastructures); 4. MOORFIELDS EYE CHARITY (Moorfields Eye Hospital NHS Foundation Trust; charity number 1140679): www.moorfields.nhs. uk (Personal award to SSB for infrastructures); and 5. National Institute for Health Research: http://www. nihr.ac.uk (Personal award to SSB for infrastructures). Competing Interests: The authors have declared that no competing interests exist. compatibility with the Gateway Cloning System. The candidate interacting partner of TOPORS, P26s4 encoded by the PSMC1 gene, was cloned in frame with the GAL4 activation domain (AD) using the Gateway Cloning System and the modified Stratagene vectors (pAD-PSMC1). In-Fusion1Advantage PCR Cloning Kit (Clontech, CA, USA) was used to generate TOPORS deletion constructs, specifically pGBKT7-N-TOPORS (residues 1–380), pGBKT7-M-TOPORS (residues 373–781)and pGBKT7-C-TOPORS (residues 705–1045), for interaction characterisation in yeast. The compatibility of the modified Stratagene and Clontech plasmid constructs was thoroughly validated prior to the Y2H screens. Library Screening Matchmaker™Yeast Two-Hybrid (Y2H) System (Clontech, CA, USA) was used following the manufacturer’s instructions. The Saccharomyces cerevisiae Y2H Gold strain was transformed with pBD-TOPORS and the GAL4 DNA-BD control plasmids. The S.cerevisiae Y187 strain was transformed with the human retinal cDNA library fragments and linearised pGADT7-Rec vector and the GAL4 AD control plasmids according to the manufacturer’s instructions (Yeastmaker™Yeast Transformation System 2, Clontech, CA, USA). The Y2H screen was performed by mating the Y2H Gold strain, expressing pBD-TOPORS, with the Y187 strain, expressing the cDNA library clones. A two-tier protein-protein interaction (PPI) selection process was used. Initially, two reporter genes, Mel1 and Aur1-C,wereusedto detect interactions. Mel1 encodes X-α-galactosidase, which allows for utilisation of X-α-galactose from the medium as a nutrient source. Mel1 expression is triggered by a PPI, which is indicated by colonies excreting a blue pigment, a product of X-α-galactose utilisation. Aur1-C is the dominant mutant version of the Aur1 gene, encoding an enzyme, which breaks down the highly toxic anti-fungal Aureobasidin A (AbA) drug present in the growth medium. Medium selecting for X- α-galactose utilisation and AbA resistance is referred to in the results sections as ‘Medium D.’ Positive clones were picked and re-patched on higher stringency media, referred to in the results sections as ‘Medium Q’, selecting for activation of auxotrophic markers His3 and Ade2 in addition to the initial Mel1 and Aur1-C reporter genes (the higher stringency medium lacked histidine and adenine in addition to containing X-α-galactose and AbA). After the second selection library plasmids were isolated from the positive clones, using the Easy Yeast Plasmid Isolation Kit (Clontech, CA, USA) and sequenced, using the The BigDye TM Terminator v3.1 Cycle Sequencing Kit and the ABI PRISM13730 DNA Analyser (Applied Biosystems, UK). The UCSC BLAT search engine was employed to identify the inserts. The Human Genome Assembly Dec. 2013 (GRCh38/hg38) was searched, using the 'BLAT's guess' query type and the output sorting option of 'query, score'. The BLAT Search Result with the highest score was selected. In order to confirm and characterise the interactions pAD-PSMC1 was re-transformed into yeast. PPIs between full-length TOPORS (pBD-TOPORS) or its fragments/deletion constructs (pGBKT7-N-TOPORS, pGBKT7-M-TOPORS and pGBKT7-C-TOPORS), and full-length P26s4 (pAD-PSMC1) were tested in separate experiments according to the two-tier selection processes described above. Positive (pAD-SV40 T Ag x pBD-p53) and negative (pAD-SV40 T Ag x pBD-Lamin C) interaction controlswere used throughout. In Silico Identification of Ciliary-Targeting Sequences Several ciliary-targeting sequences (CTS) were previously described, including VxPx, RVxP, KVHPSST, AxEGG and Ax(S/A)xQ [30,31]. The PRALINE multiple sequence alignment tool TOPORS Interacts with Regulatory Subunit 4 of the 26 S Proteasome PLOS ONE | DOI:10.1371/journal.pone.0148678 February 12, 2016 3/20 (The Centre for Integrative Bioinformatics VU (IBIVU), University of Amsterdam, Netherlands) [32] was used to determine the conservation of P26s4 and TOPORS peptide sequences. Once the alignment files had been generated, the results were saved as.pdf files and the sequence was annotated manually. In silico identification of CTS in TOPORS (empirically confirmed to localise to centrosomes [33]) was performed as a control. Antibodies Mouse monoclonal anti-TOPORS antibody (H00010210-M01) was obtained from Abnova (Taiwan); used at 1:100 for immunofluorescence (IF) and at 1:250 for Western blotting (WB). Rabbit polyclonal anti-PSMC1 (P26s4) antibody (HPA000872) was purchased from Sigma- Aldrich (MO, USA), used at 1:250 for IF and at 1:1000 for WB. Mouse (F3165) anti-FLAG antibody was purchased from Sigma-Aldrich (MO, USA) and used at 1:500. Goat polyclonal antibody against peri-centriolar material 1 (PCM1) protein (sc-50164, used at 1:250 for IF) and goat anti-V5 antibody (sc-83849, used at 1:2500) were obtained from Santa Cruz Biotechnology (TX, USA). Goat polyclonal anti-polo-like kinase 4 (PLK4) antibody (Ab2642, used at 1:150 for IF), rabbit polyclonal anti-pericentrin antibody (Ab4448, used at 1:1000) and mouse monoclonal alpha-tubulin antibody (Ab7291, used at 1:5000) were purchased from Abcam (UK). Alexa Fluor 1488-conjugated goat anti-mouse (A11001, used at 1:400) and anti-rabbit (A11008, used at 1:400) antibodies, Alexa Fluor 1594-conjugated goat anti-mouse (A11005, used at 1:400) and anti-rabbit (A11034, used at 1:400) antibodies, and donkey anti-goat antibodies conjugated with Alexa Fluor 1488 (A11055, used at 1:400) and with Alexa Fluor 1 A594 (A11058, used at 1:400), were purchased from Life Technologies (CA, USA). FITC-con- jugated donkey anti-mouse, anti-rabbit and anti-goat IgG (1:300) and Cy ™3-conjugated donkey anti-mouse, anti-rabbit and anti-goat IgG (1:300); and horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG (1:10000) were purchased from Jackson Immuno Research Laboratories, Inc. (PA, USA). Mammalian Cell Culture The murine 661W cone photoreceptor cell line was maintained in in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, CA, USA) supplemented with 10% FCS and penicillin–streptomycin (1000 μg/ml). Human (h)TERT-RPE1 retinal pigment epithelial and SK-N-SH neuroblastoma cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM)/F-12+GlutaMAX (Life Technologies, CA, USA) supplemented with 10% FCS and penicillin–streptomycin (1000 μg/ml). All cells were grown in 6-well plates at 1.2 x10 6 cells per well at 37°C in an atmosphere of 5% CO 2 . Co-Immuno-Precipitation and Western Blot Analysis Cellular lysates were prepared from human (h)TERT-RPE1 cells and from murine 661W cells in RIPA buffer. Lysates from cellular fractions of hTERT-RPE1 cells were prepared using the ProteoExtract1Subcellular Proteome Extraction Kit (Merck Group, Germany). Prior to immuno-precipitation and SDS-PAGE/WB analysis protein concentrations were determined using the BCA Protein Assay Kit (Merck Group, Germany) and/or the Non-Interfering Protein Assay™Kit (Merck Group, Germany). Prior to immunoprecipitation (IP) studies SureBeads Protein G-conjugated magnetic beads (BioRad, CA, USA) were washed thoroughly and used for co-immuno-precipitation (coIP) according to the manufacturer’s instructions. Four micrograms of the mouse monoclonal anti- TOPORS antibody (Abnova) were incubated with 100 μl of beads for ten min at room temperature with rotation. The antibody-bound beads were then washed and, subsequently, incubated TOPORS Interacts with Regulatory Subunit 4 of the 26 S Proteasome PLOS ONE | DOI:10.1371/journal.pone.0148678 February 12, 2016 4/20 with 100 μl of cellular lysate (containing 0.8–1.2 mg/ml of protein) for one hour at room temperature with rotation. All concentrations and volumes are given per coIP sample. The precipitated complexes were then denatured in 1X Laemmli Sample Buffer at 75°C for ten min and loaded onto polyacrylamide gels for SDS-PAGE analysis (40 μg of cellular lysate were used per each input lane, followed by Western blotting (WB) with rabbit anti-PSMC1 (P26s4) antibody. Gradient gels (4%–20%) and PVDF membranes were used (BioRad, CA, USA). Membranes were blocked in 5% non-fat dried milk with 0.1% Tween20 in TBS. Primary and secondary antibodies were diluted as described above. ECL solution (BioRad, CA, USA) was used for visualization. Mammalian Expression Vectors For characterisation in human cell lines the Gateway Cloning System (Life Technologies, CA, USA) was used to generate V5-TOPORS deletion constructs, specifically p-nV5-N-TOPORS (residues 1–380), p-nV5-M-TOPORS (residues 373–781)and p-nV5-C-TOPORS (residues 705– 1045) tagged at the amino terminus. Primers, designed to generate N-, M- and C-TOPORS with att tails, compatible with the Gateway Cloning System, were ordered from Sigma-Aldrich (MO, USA). Full-length TOPORS, cloned into pCATCH vector tagged at the amino-terminus with FLAG, was a gift from Professor Stefan Weger (Free University of Berlin, Germany). Immunocytochemistry Prior to immunofluorescence (IF) studies cells were seeded in 24-well plates on glass coverslips at 5.0 x 10 4 cells per well and incubated for 24 h. The cells were then subjected to fixation for 20 min in 4% PFA in PBS at room temperature. They were subsequently incubated in the permeabilisation solution (4% Triton-X100 with 0.3% BSA in PBS) for five min at room temperature. The cells were washed twice in immunocytochemistry (ICC) blocking buffer (0.5% bovine serum albumin (BSA) and 20 mM glycine in PBS), followed by incubation in the blocking buffer for 15 min before staining. Cells on cover slips were incubated with the primary antibodies at room temperature overnight. Cells were washed in ICC blocking solution and incubated with secondary antibodies for 45 min at room temperature in the dark. Cells were again washed in ICC blocking solution and mounted using DAKO Fluorescence Mounting Medium (Agilent Technologies, CA, USA). Primary and secondary antibodies were diluted in concentrations described above. Nuclei were stained using 4’,6-diamidino-2-phenylindole (DAPI). Transfections were performed in confluent hTERT-RPE1 cells using the Lipfectamine12000 Transfection Reagent (Thermo Fisher Scientific, MA, USA), according to the manufacturer’s instructions. The cells were fixed 24 h post transfection prior to IF staining, as described above. Immunohistochemistry Eyes of C57 BLACK 6 mice were obtained from the UCL Institute of Ophthalmology Biological Resource(s) Unit (BRU) in accordance with the regulations of the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. The work was approved by the UCL Institute of Ophthalmology Institutional Animal Care and Use Committee (IACUC # 70/2710). Adult mice (6 months of age, used for all retinal studies described herein) were kept under standard housing conditions (12 hour light/dark cycle). The animals were euthanised under light conditions two to three hours after the onset of light by exposure to an increasing concentration of carbon dioxide (CO 2 ) gas. Death was ensured by subsequent dislocation of the neck. The eyes were enucleated and fixed in 4% PFA in PBS for 24 h at 4°C. Sucrose gradient infiltration was performed using: 20% sucrose for 5 h at 4°C, followed by 30% sucrose at 4°C TOPORS Interacts with Regulatory Subunit 4 of the 26 S Proteasome PLOS ONE | DOI:10.1371/journal.pone.0148678 February 12, 2016 5/20 overnight, and a solution of 15% sucrose and 50% Optical Cutting Temperature (OCT) (VWR International Ltd, UK) medium for 2 h at room temperature. The eyes were then embedded in OCT and cryo-sectioned at 10 μm. For immuno-staining retinal sections were permeabilised (0.3% Triton X-100 in PBS for ten min at room temperature) and then pre-treated with immunohistochemistry (IHC) blocking solution (5% normal goat serum (NGS) and 0.1% Triton X- 100 in PBS) for 1 hour at room temperature, followed by incubation with primary antibodies. The sections were washed in 0.1% Triton X-100 in PBS and treated with appropriate secondary antibodies. Cell nuclei were stained with DAPI. Filamentous actin (F-actin) was stained with A594 conjugated-phalloidin, used at 1:400 (A12381; Life Technologies, CA, USA). Primary and secondary antibodies were diluted in concentrations described above. Fluorescent Microscopy Imaging Immunostained cells and tissue cryo-sections were imaged using a ZEISS Axiovert S100 fluorescent inverted microscope and ZEISS LSM 700 laser scanning confocal microscope (Carl Zeiss, Germany). Images were processed using ZEN (blue edition) Image Browser and Adobe Photoshop CS5 (Adobe Systems, WA, USA). Deconvolution of indicated cell photomicrographs was achieved by imaging the cell in the focal plane of the centrosomal/centriolar marker first and imaging the P26s4 signal in the same plane, using the ZEISS Axiovert S100 fluorescent inverted microscope. The nucleus was then imaged in its focal plane and the three individual channels were combined manually using Adobe Photoshop CS5. This resulted is 'losing' the additional staining throughout the cell. Results TOPORS and P26s4 Interact in Yeast and Mammalian Cell Lines The goal of this study was to identify candidate interacting partners of TOPORS from human retina in order to delineate the mechanism of retinal degeneration associated with mutations in this ubiquitously expressed gene. Over 10 7 clones were screened (Table 1) in a two-tier selection process, utilising the Aur1-C PPI reporter gene at both stringency levels. Its expression results in inhibition of Aureobasidin A (AbA), which is toxic to yeast, thus reducing background growth. Firstly, the yeast culture was spread on medium supplemented with AbA and X-alpha-galactose (reporter genes Aur1-Cand Mel1, respectively), resulting in growth of 24 colonies representing candidate interacting partners of TOPORS. Each observed colony was patched on more stringent media, which led to a successful isolation of 21 positive clones (PPIs indicated by expression of four reporter genes: Aur1-Cand Mel1 as well as auxotrophic His3 and Ade2). DNA was extracted from the resulting yeast patches and primers, specific to the cDNA library-vector (pGADT7-Rec), were used for amplification of cDNA inserts encoding the Table 1. A two-tier selection process led to identification of 21 putative interacting partners of TOPORS after the second selection level. Number of cDNA library clones Total clones screened 3.29 x 10 7 First selection level: AUR1-C and MEL1 1 24 Second selection level: AUR1-C,MEL1,HIS3 and ADE2 1 21 1 Reporter genes expressed as a result of protein-protein interactions in the Y2H screen: AUR1-C, selects for resistance to AbA; MEL1,α-galactosidase synthesis (blue-white marker); HIS3, histidine synthesis (auxotrophic marker); ADE2, adenine synthesis (auxotrophic marker). doi:10.1371/journal.pone.0148678.t001 TOPORS Interacts with Regulatory Subunit 4 of the 26 S Proteasome PLOS ONE | DOI:10.1371/journal.pone.0148678 February 12, 2016 6/20 putative interacting proteins. The resulting PCR products were purified, sequenced and the UCSC BLAT search engine was employed to identify the inserts. One of the inserts (clone 2) was confirmed as exons 1–6ofPSMC1 encoding the amino-terminal half of the 26 S protease regulatory subunit 4 (P26s4) (Fig 1A). These exons encode a fragment of the protein comprising an N-myristoylation site as well as an NADH-binding site, but they do not encode regions involved in ATP binding and hydrolysis. The TOPORS-P26s4 interaction was subsequently validated by co-immuno-precipitation (coIP) of both endogenous proteins from human retinal pigment epithelial cells (hTERT-RPE1) and from murine 661W cone photoreceptor cell line (Fig 1B and S1 Fig). Cell extracts were precipitated using anti-TOPORS antibody, followed by Western blotting for P26s4, which generated a band of approximately 60 kDa in both cell types. In the hTERT-RPE1 cell line TOPORS also interacted with a larger P26s4 species migrating at approximately 75 kDa, which could reflect a covalent modification of P26s4, such as SUMOylation. Immunoblot analyses, performed on protein extracts from specific cellular fractions revealed that the 75 kDa is present in all cellular fractions, yet it appears most abundant in the cytosol and the nucleus. Another band unique to the fraction of membranes and organelles was additionally observed migrating approximately half-way between the 60 kDa and 75 kDa band. Fig 1. P26s4 interacts with TOPORS in yeast and mammalian cells. A) APSMC1 fragment from the human retinal library encoding P26s4 was identified as a novel interacting partner of TOPORS in the Y2H screen. The sequence alignment was generated using the UCSC Genome Browser (http://genome. ucsc.edu) on the Human Feb. 2009 (GRCh37/hg19) Assembly [34,35]. B) P26s4 is detected in endogenous complexes precipitated from human TERT-RPE1 and murine 661W cell lysates with an anti-TOPORS antibody. A band migrating at approximately 60 kDa was detected in total lysates and positive coIP lanes in both cell lines, indicating positive coIP of TOPORS and P26s4. An additional band migrating at approximately 75 kDa was observedin total cell lysates in both cell lines and in the coIP lane in the hTERT-RPE1 cell line. Western blotting on lysates extracted from specific cellular fractions: cytosol, membranes and organelles (Memb./Organ.), nucleus and cytoskeleton of hTERT-RPE1 cells demonstrated that the larger band is present in all tested fractions, yet is most prominent in the cytosol and the nucleus. Stripping of both membranes and re-probing for TOPORS demonstrated the proteinis present in complexes precipitated from both cell lines. Re-probing to detect TOPORS in cellular fractions indicated species of different sizes are present reflecting the results from the coIP blot. doi:10.1371/journal.pone.0148678.g001 TOPORS Interacts with Regulatory Subunit 4 of the 26 S Proteasome PLOS ONE | DOI:10.1371/journal.pone.0148678 February 12, 2016 7/20 P26s4 Localises to the Centrosome The close relationship between the 26S proteasome and the centrosome has been demonstrated in dividing HEK293 and HeLa cells [36], in granular neurons [37], and in ciliated HEK293 cells [38], as well as in the fast-growing HEK293-FT cells, in zebrafish embryos and in ciliarich tissues, such as murine testis, kidney and retina [39]. In fact, we identified two ciliary-tar- geting sequences (CTS) within the peptide sequence of P26s4 (Table 2). The rhodopsin-type CTS motif (VxPx) [31] of P26s4 is located proximally to its amino-terminus. The second CTS of this protein resembles the CTS motifs found in G-protein-coupled receptors [30]; it is located approximately in the middle of the P26s4 peptide sequence. The whole protein, including its both CTS motifs, is strongly conserved among vertebrates, according to conservation scoring performed using PRALINE (The Centre for Integrative Bioinformatics VU (IBIVU), University of Amsterdam, Netherlands) [32] (data not shown). TOPORS, which is associated with the centrosome throughout the cell cycle and localises to basal body of primary cilium [33], possess two CTS motifs of the VxPx type itself, of which one (172-VTPD-175) is conserved among apes and monkeys, and the other one among vertebrates overall (484-VKPL- 487), according to conservation scoring performed using PRALINE IBIVU, University of Amsterdam, Netherlands) [32] (data not shown). Immunofluorescence staining of hTERT-RPE1 cells (Fig 2) demonstrated diffuse, granular P26s4 signal throughout the cell, which is in agreement with the house-keeping functions of the 26 S proteasome, of which P26s4 forms a crucial component. Co-localisation with TOPORS signal occurs at several P26s4-reactive granules, which could be the centrosomes (indicated with arrows in Fig 2). Deconvoluted images showing co-staining with centrosomal markers confirmed the centrosomal-targeting of P26s4. Interestingly, it also showed that in the hTERT-RPE1 cell line this protein localises to only one centriole of the centrosome. P26s4 was not observed at the ciliary basal body in the hTERT-RPE1 cells (S2 Fig). The localisation of P26s4 was then investigated in the murine cone photoreceptor 661W cell line. The experiments revealed that P26s4 co-localised with both: TOPORS and centriolar markers in dividing (Fig 3), but not in ciliated cells (S2 Fig). P26s4 Associates with the C-Terminal Region of TOPORS TOPORS is a multifunctional protein, its roles being conveyed by distinct domains located within different regions of its peptide sequence. In an attempt to separate the domains, and hence major functions, full-length TOPORS protein was divided into three portions (N-, M- and C-TOPORS; Fig 2A) to investigate, which of these domains could be responsible for mediating the interactions between TOPORS and P26s4. N-TOPORS comprised the RING finger domain conveying the E3 ubiquitin ligase activity and phosphoserine 98 involved in up-regulation of TOPORS ubiquitination activity [12]. M-TOPORS contained an SR/RS dipeptide region, which includes a region required for Table 2. Ciliary-targeting sequences identified in P26s4 in silico. CTS subtype CTS region in P26s4 Other proteins with this CTS sub-type 2 VxPx 52-VTPH-54 (VxPx) Rhodopsin (P08100), polycystin-1 (P98161), polycystin-2 (Q13563) Ax(S/A)xQ 238-AVANQ-242 G-protein-coupled receptors: Sstr3 (P32745), Htr6 (P50406), and Mchr1 (Q99705) 2 Reviewed in reference 31. doi:10.1371/journal.pone.0148678.t002 TOPORS Interacts with Regulatory Subunit 4 of the 26 S Proteasome PLOS ONE | DOI:10.1371/journal.pone.0148678 February 12, 2016 8/20 interaction with SUMO1 as well as a SUMO1 acceptor site at lysine 560 [40]. Serine 718 was also included; its phosphorylation leads to up-regulation of the ubiquitin ligase activity whilst down-regulating the SUMO1 ligase activity of TOPORS [13]. C-TOPORS included the RP31 mutational hotspot region [24–26] and fragments required for interaction with SUMO1 as well as its E2 conjugating enzyme, UBC9. Phosphoserine 718 was also included within C-TOPORS [40]. The deletion constructs (Fig 2A) were tested directly in yeast for interactions with fulllength P26s4. The findings demonstrated that P26s4 readily interacted with constructs M and C (Table in Fig 4B), which are associated with the E3 SUMO1 ligase activity of TOPORS. The interaction between M-TOPORS and P26s4 resulted in activation of two reporter genes in five out of six experiments at the lower stringency, and all four reporter genes were activated in five out of six experiments at the higher stringency. C-TOPORS interacted with P26s4 in four out Fig 2. P26s4 co-localises with TOPORS and centrosomal markers in hTERT-RPE1 cells, but only at one centriole of the centrosome. P26s4 localised throughout the cell in a diffuse speckled pattern; the signal included distinct points co-localising with TOPORS staining (arrows). P26s4 co-localised with both PCM1 and PLK4 at one of the two centrioles; P26s4 signal is also observed at the linker molecules holding the two centrioles together. Scale bar: 10 μm; insets show a magnification of signals enclosed in the dashed squares. Fluorescent microscope images were taken using Zeiss Axiovert S100 inverted microscope. Images in the middle and bottom panel were deconvoluted, as described in Materials and Methods. Secondary antibody control images were collected using the same settings and generated no signals in the red and green channels. doi:10.1371/journal.pone.0148678.g002 TOPORS Interacts with Regulatory Subunit 4 of the 26 S Proteasome PLOS ONE | DOI:10.1371/journal.pone.0148678 February 12, 2016 9/20 Supporting Information S1 Fig. P26s4 interacts with TOPORS in mammalian cells. Full-sized images of blots shown in Fig 1 are presented. P26s4 is detected in endogenous complexes precipitated from human TERT-RPE1 and murine 661W cell lysates with an anti-TOPORS antibody. A band migrating at approximately 60 kDa was detected in total lysates and positive coIP lanes in both cell lines, indicating positive coIP of TOPORS and P26s4. An additional band migrating at approximately 75 kDa was observed in total cell lysates in both cell lines and in the coIP lane in the hTERT-RPE1 cell line. Western blotting on lysates extracted from specific cellular fractions: cytosol, membranes and organelles (Memb./Organ.), nucleus and cytoskeleton of hTERT-RPE1 cells demonstrated that the larger band is present in all tested fractions, but is most prominent in the cytosol and the nucleus. Stripping of both membranes and re-probing for TOPORS demonstrated the protein is present in complexes precipitated from both cell lines. Re-probing to detect TOPORS in cellular fractions indicated species of different sizes are present reflecting the results from the coIP blot. (TIF) S2 Fig. P26s4 did not co-localise with primary cilia (marked with α-tubulin, inset) in ciliated hTERT-RPE1 cells; however, it co-localised with α-tubulin at a punctate localization, where no primary cilium was observed (indicated by arrow, upper panel), likely representing the centrosome. Scale bar: 10 μm. Deconvoluted images from ZEISS Axiovert S100 fluorescent inverted microscope. (TIF) S3 Fig. N-TOPORS localises to the nucleus and centrioles. Human (h)TERT-RPE1 cells were either co-transfected with V5-tagged N-TOPORS and FLAG-tagged full-length TOPORS (top panel), or with V5-tagged N-TOPORS only (middle and bottom panels). The V5-tagged artificial N-TOPORS fragment co-localised with FLAG-tagged full-length TOPORS (top panel) and endogenous TOPORS (middle panel) at nuclei of hTERT-RPE1 cells and it colocalised with pericentrin at the centrioles. (TIF) S4 Fig. M-TOPORS localises to the nucleus. Human (h)TERT-RPE1 cells were either cotransfected with V5-tagged M-TOPORS and FLAG-tagged full-length TOPORS (top panel), or with V5-tagged M-TOPORS only (middle and bottom panels). The V5-tagged artificial M-TOPORS fragment co-localised with FLAG-tagged full-length TOPORS (top panel) and endogenous TOPORS (middle panel) at nuclei of hTERT-RPE1 cells, but it did not co-localise with pericentrin. (TIF) S5 Fig. C-TOPORS localises to the nucleus and cytoplasm. Human (h)TERT-RPE1 cells were either co-transfected with V5-tagged C-TOPORS and FLAG-tagged full-length TOPORS (top panel), or with V5-tagged C-TOPORS only (middle and bottom panels). The V5-tagged artificial C-TOPORS fragment co-localised with FLAG-tagged full-length TOPORS (top panel) at nuclei of hTERT-RPE1 cells and it co-localised with endogenous TOPORS at the nucleus (middle panel), however it did not co-localise with pericentrin. (TIF) S6 Fig. P26s4 localises to distal photoreceptor outer segments (OS) and retinal pigment epithelium (RPE) in murine retina sections. P26s4 localises diffusely throughout the photoreceptor OS with distinct P26s4 signal being observed at the RPE-OS interface and the RPE (both panels). Phalloidin staining for filamentous actin was used as a marker delineating OS TOPORS Interacts with Regulatory Subunit 4 of the 26 S Proteasome PLOS ONE | DOI:10.1371/journal.pone.0148678 February 12, 2016 16 / 20 structure (bottom panel). RPE, retinal pigment epithelium; OS, rod photoreceptor outer segment; IS, rod photoreceptor inner segment. Nuclei stained using DAPI. Scale bar 20 μm. Confocal microscope images were taken using the Zeiss LSM 700. Secondary antibody control images were collected using the same settings and generated no signals in the red and green channels. (TIF) S7 Fig. TOPORS localises throughout the retina with the strongest signal observed at the photoreceptor connecting cilium, and also a distinct signal at the distal region of the outer segments (OS). OS, photoreceptor outer segment; CC, connecting cilium; IS, photoreceptor inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer. Nuclei stained using DAPI. Scale bar 20 μm. Confocal microscope images were taken using the Zeiss LSM 700. Secondary antibody control images were collected using the same settings and generated no signals in the red and green channels. (TIF) Acknowledgments We thank Professor Stefan Weger for the pCATCH-TOPORS plasmid, Beverley Scott for her support with DNA sequencing and DeQuincy Prescott for his technical assistance and laboratory management. Author Contributions Conceived and designed the experiments: AZS BC SSB. Performed the experiments: AZS BC PMK. 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