The autophagic deterioration of misfolded and also ubiquitinated proteins is essential for cellular homeostasis. In this procedure, which is governed by cargo receptors, ubiquitinated proteins are condensed into bigger structures and consequently end up being targets for the autophagy machinery. Here we employ in vitro reconstitution and also cell biology to define the duties of the human cargo receptors p62/SQSTM1, NBR1 and TAX1BP1 in the selective autophagy of ubiquitinated substprices. We show that p62 is the significant driver of ubiquitin condensate development. NBR1 promotes condensate development by equipping the p62-NBR1 heterooligomeric complicated with a high-affinity UBA domain. In addition, NBR1 recruits TAX1BP1 to the ubiquitin condensates formed by p62. While all three receptors communicate through FIP200, TAX1BP1 is the major driver of FIP200 recruitment and therefore the autophagic deterioration of p62–ubiquitin condensates. In summary, our study defines the duties of all three receptors in the selective autophagy of ubiquitin condensates.

You are watching: Which of the following best defines validation?


Macroautophagy (hereafter autophagy) is a conserved intracellular process, which aids cellular homeostasis by the disposal of harmful structures including protein aggregateways, damaged organelles, and also intracellular pathogens1,2,3. Defects in autophagy have actually been connected to a plethora of illness consisting of cancer and neurodegeneration4. The deterioration of the harmful product, described as cargo, is mediated by its encapsulation within de novo developed double-membrane vesicles, termed autophagosomes, which fuse through lysosomes wherein the cargo is degraded. Autophagosome formation is mediated by the autophagy machinery5,6. In selective autophagy, during which certain cargo is targeted for elimination, this machinery is recruited by cargo receptors such as p62/SQSTM1, NBR1, NDP52, and optineurin7,8,9,10,11,12,13. Many of the various cargo receptors in mammalian cells, consisting of p62 and also NBR1, acknowledge the cargo by means of its ubiquitin tags14.

A significant attribute of p62 is the destruction of ubiquitinated, misfolded proteins by selective autophagy. In this procedure, it plays several roles15,16,17. First, it mediates the condensation of ubiquitinated proteins right into larger structures18,19,20. Subsequently, it contributes to the regional development of autophagosomes around these condensates by recruiting the FIP200 scaffold protein10. Finally, it web links the cargo to the nascent autophagosomal membrane through its interactivity with LC3 and GABARAP proteins, which decoprice the forming autophagosomal membrane21,22. p62 oligomerizes right into filaments via its N-terminal PB1 domain23,24,25. This oligomerization is forced for its capacity to develop condensates with ubiquitinated proteins however also to avidly bind the LC3/GABARAP decorated autophagosomal membrane through its LC3 connecting area (LIR) motif and also the ubiquitinated cargo using its C-terminal UBA domain (Supplementary Fig.1a)19,20,26.

In these processes, p62 is aided by the cargo receptor NBR1. Similar to p62, NBR1 binds LC3/GABARAP proteins by means of LIR motifs and ubiquitin via its UBA domain27. NBR1 and p62 straight interact via their N-terminal PB1 domains (Supplementary Fig.1a)20,24,25. In cells, NBR1 colocalizes through p62, and its depletion results in fewer p62 condensates27,28. In vitro, NBR1 straight enhances the formation of p62–ubiquitin condensates20. The certain mechanisms via which NBR1 promotes condensate formation and also thus cargo degradation remajor unclear.

A third cargo receptor, TAX1BP1 (Supplementary Fig.1a), was newly presented to colocalize with NBR113. TAX1BP1 is compelled for the clearance of protein aggregates29. It interacts via NBR1 to recruit FIP200 and to trigger its succeeding destruction in LC3 lipidation independent autophagy13.

Here we employ in vitro reconstitutions and cell organic ideologies to present that p62, NBR1, and TAX1BP1 cooperate throughout the formation and also deterioration of p62–ubiquitin condensates by selective autophagy. We uncovered that p62 is the significant driver of ubiquitin condensate formation. We further present that NBR1 promotes p62–ubiquitin condensate formation through its PB1 domain-mediated binding to p62. This interactivity equips the p62—NBR1 heterooligomeric facility via a high-affinity UBA domajor offered by NBR1, enabling for even more reliable cargo acknowledgment. In enhancement, NBR1 serves to recruit TAX1BP1 to the condensates. While all three cargo receptors are able to connect through the FIP200 scaffold protein to initiate autophagosome development, TAX1BP1 is the primary driver for its recruitment to the condensates. In this research we disentangle the individual contributions of the 3 mammalian cargo receptors p62, NBR1, and also TAX1BP1 in the clearance of ubiquitin condensates through selective autophagy.


Colocalization of p62, NBR1, and also TAX1BP1 in ubiquitin-containing condensates

To identify the mechanisms of activity of the p62, NBR1, and also TAX1BP1 cargo receptors and their cooperation in the formation and also deterioration of ubiquitin-containing condensates, we stupassed away their colocalization in HAP1 cells expressing endogenously tagged GFP-p62 and also mScarlet-AID-NBR1 (Fig.1a and Supplementary Fig.1 b–d). In enhancement to a fluorophore, NBR1 was likewise tagged through an auxin-inducible destruction (AID) tag to permit its acute depletion on a protein level (Supplementary Fig.1b)30. The tags did not interfere through the lysosomal shipment of p62 and also NBR1, as their levels were enhanced upon inhibition of lysosomal task through bafilomycin therapy (Fig.1b and also Supplementary Fig.1d)20. Even in resting HAP1 cells, i.e., in the lack of known activators of autophagy such as starvation, treatment via drugs or overexpression of aggregation-vulnerable proteins, p62 forms multiple dynamic condensates (Supplementary video 1). When we stained the HAP1 GFP-p62, mSc-AID-NBR1 cells via an anti-TAX1BP1 antibody (Fig.1a), we noticed that about 13% of TAX1BP1 puncta colocalized with p62 and also NBR1 (Fig.1b). A comparable percent of p62 puncta colocalized via NBR1 and also TAX1BP1 (Fig.1c). Both these numbers boosted upon bafilomycin therapy saying that the receptors are degraded in lysosomes (Fig.1b, c). Consistent through previous findings29, TAX1BP1 puncta colocalized via ubiquitin (Supplementary Fig.1e) and also with LC3B (Fig.1d).


*

a The cargo receptors NBR1 and p62 were endogenously tagged via mScarlet-AID and GFP tags respectively, using CRISPR (Supplementary Fig.1b). Cells were left untreated (DMSO) or treated through bafilomycin (400 nM for 2 h) and also solved. After fixation, NBR1 and also p62 were detected using their endogenous fluorescent tags, while TAX1BP1 was detected by immunofluorescent staining. Scale bar, 10 µm. Validation of endogenous protein tagging in the cell line provided for the experiment is displayed in Supplementary Fig.1c and also d. b, c Colocalization of TAX1BP1 through p62, NBR1 or both (b) and colocalization of p62 through TAX1BP1, NBR1 or both (c), based on the experiments in Fig.1a. Colocalization evaluation was performed via ImageJ. Average percent of colocalization and SEM for three independent experiments are plotted. An unpaired, two-tailed Student’s t test was supplied to estimate definition. P worths are shown in the number. d Colocalization of TAX1BP1 through LC3B in HAP1 WT cells mock-treated through DMSO or treated through bafilomycin (400 nM) for 2 h. LC3B and also TAX1BP1 were detected by immunofluorescence staining. Scale bar, 10 µm. For the colocalization analysis, average percenteras of colocalization ± SEM for n = 3 are plotted. An unpaired, two-tailed Student’s t test was provided to estimate significance. P values are suggested in the figure.


NBR1 modulates p62–ubiquitin condensate development in vitro

Next off, we went on to dissect the roles of the 3 cargo receptors in the formation and clearance of ubiquitin condensates. Due to the fact that the depletion of p62 results in a sevecount diminished number or also complete lack of ubiquitin condensates18,29, its functional interactivity through NBR1 and also TAX1BP1 in this process is tough to examine in cells. Therefore, we turned to a reconstituted system10,20. Consistent through our previous results, we oboffered that NBR1 alone did not induce condensation (Supplementary Fig.2a) but that it markedly amplified the condensation of p62 and also GST fsupplied to 4 M1-connected ubiquitin moieties (GST-4xUb), as judged from the number (left panel) and size (right panel) of the condensates (Fig.2a–c and also Supplementary Fig.2a, b)20. NBR1 was effectively recruited to p62-containing condensates (Fig.2b). In order to test if there was an optimal molar proportion of NBR1 to p62 with respect to its condensation promoting activity, we titrated NBR1 into condensate formation asclaims containing p62 and also GST-4xUb (Fig.2d and also Supplementary Fig.2c). We observed that the promoting activity of NBR1 gradually increased through increasing concentrations, but that it dropped at an equimolar proportion. This said that a sub-stoichiometric concentration of NBR1 loved one to p62 is ideal for the proactivity of condensate development.


*

Source data for the assays in this number are provided as a Source Data document. a Schematic depiction of the condensate development assay. b Condensate formation assay with GST-4xUb (5 µM), mCherry-p62 (2 µM) and also GFP-NBR1 (2 µM). Condensate formation over time in the mCherry and also GFP networks was complied with by spinning disk microscopy. Scale bar, 15 µm. c Quantification of the experiment in (b). An average of the number (left) and also the dimension (right) of the condensates developed in the mCherry channel were measured via ImageJ and also plotted through conventional deviations versus time for n = 3. The number of condensates created in the GFP channel and quantification of the control experiment are shown in Supplementary Fig.2a. A Coomassie stained SDS-Page gel of the proteins provided for the assay is presented in Supplementary Fig.2b. d Titration of GFP-NBR1 (concentration range: 0–2 µM) in a condensate development assay containing 5 µM GST-4xUb and 2 µM mCherry-p62. The average number of mCherry-p62 condensates per area of imaging for n = 3 and traditional deviations are plotted versus time. Coomassie stained SDS-Page gel via protein inputs is shown in Supplementary Fig.2c. e Quantification of a condensate development assay performed via 5 µM GST-4xUb or in vitro synthesized K48- and also K63-connected ubiquitin chains of assorted length (Supplementary Fig.2d), incubated with mCherry-p62 (2 µM), via or without GFP-NBR1 (2 µM). BSA was added as a crowding agent to a last concentration of 2%. The average number of mCherry-p62 condensates for n = 3 and also conventional deviations are plotted versus time. f Microscopy-based pull-dvery own showing the recruitment of GFP-NBR1 (WT or ∆PB1 – 2 µM) to mCherry-p62 coated RFP-trap beads. Average GFP signal intensities and also traditional deviations for n = 3 are displayed. An unpaired, two-tailed Student’s t test was provided to estimate significance. P values are suggested in the figure. Purified NBR1 variants provided for this experiment are displayed in Supplementary Fig.2e. g Microscopy-based pull-down mirroring the recruitment of GFP-NBR1 (WT or ∆UBA – 2 µM) to GST-4xUb coated glutathione beads. Typical GFP signal intensities and standard deviations for n = 3 are displayed. An unpaired, two-tailed Student’s t test was supplied to estimate significance. P values are shown in the figure. Purified NBR1 variants provided for this experiment are shown in Supplementary Fig.2e. h Condensate formation assay was percreated with NBR1 WT, ∆PB1, and ∆UBA variants (2 µM), mCherry-p62 (2 µM) and GST-4xUb (5 µM). The average number of mCherry-p62 condensates and also typical deviations for n = 3 are plotted against time. Representative imperiods of the GFP and mCherry networks at the 60 min time allude are shown in Supplementary Fig.2g. SDS-Page gels of purified recombinant GFP-NBR1 (WT and mutants) and the condensate formation assay protein inputs are shown in Supplementary Fig.2e and f. i Condensate formation assay was performed via mCherry-p62 (2 µM) and GST-4xUb (5 µM) with the enhancement of GFP-NBR1 (2 µM) and/or its isolated PB1 doprimary (2 µM). The average number of mCherry-p62 condensates and conventional deviations for n = 3 are plotted versus time. SDS-Page gel via the protein inputs for the condensates assay is presented in Supplementary Fig.2h.


Next off, we asked if NBR1 would certainly additionally promote the condensation of in vitro synthesized K48- and also K63-linked ubiquitin chains (Fig.2e and also Supplementary Fig.2d). NBR1 promoted the development of condensates of these chain forms also. There are distinctions in the level to which the condensation of the three substrates was promoted, however as a result of the different length of the chains (Supplementary Fig.2d), we cannot distinguish in between chain form and chain size. We conclude that NBR1 can boost the condensation of a broad spectrum of substrates by p62, which is consistent via a low level of ubiquitin affiliation specificity of its UBA domain31.

We went on to dissect which properties of NBR1 are forced to promote condensation. To this finish, we expressed and purified two deletion mutants of NBR1 (Supplementary Fig.2e). The initially mutant lacked the PB1 domain (NBR1∆PB1) and, as intended from previous studies24,25, verified a sevedepend diminished binding of NBR1 to p62 in our microscopy-based pull-down assay (Fig.2f). We additionally deleted the C-terminal UBA domajor (NBR1∆UBA), which was formerly presented to mediate binding to ubiquitin via a higher affinity than the UBA domajor of p62, at leastern in the context of monoubiquitin, and K48- and K63-connected di-ubiquitin27,31,32,33,34. In the conmessage of the recombinant protein, this deletion additionally aboliburned ubiquitin-binding totally (Fig.2g). When tested in the condensate development assay, the NBR1∆PB1 mutant presented a sevedepend decreased condensate cultivating activity (Fig.2h and Supplementary Fig.2f, g). The PB1 domajor alone did not promote condensation and also in reality showed a dominant-negative result on the reaction (Fig.2i and Supplementary Fig.2h). This suggests that the interactivity of the NBR1 PB1 doprimary via p62 is not adequate for the promovement of condensate development yet that this interaction mediates the recruitment of one more biochemical task of NBR1 to the p62 filaments. In line through this, we oboffered that the UBA domain deletion mutant of NBR1 also showed a sevecount lessened cultivating task in our assay (Fig.2h and also Supplementary Fig.2f, g). Both the PB1 and also the UBA deletion mutants were still recruited to the condensates (Supplementary Fig.2g). We, therefore, conclude that NBR1 aids in effective cargo clustering by p62, by bringing its high-affinity UBA domajor to the p62 filaments by means of its PB1 domajor. Consistently, once the p62 UBA domain was reput via the NBR1 UBA domain the resulting chimeric protein was even more efficient than wild-kind p62 in forming condensates. In enhancement, the condensate development activity of the chimera was not stimulated by the addition of NBR1 (Supplementary Fig.2i).

The PB1 and UBA domain names of NBR1 are forced to promote p62 condensate formation in cells

In order to compare our in vitro results concerning the proactivity of p62-positive condensate development by NBR1 to the normally occurring process in cells, we made usage of the AID tag attached to endogenous NBR1 in our cell line with endogenously tagged p62 and NBR1 (Supplementary Fig.1b). The fluorophores on p62 and also NBR1 permitted us, for the first time, to photo their dynamics at endogenous expression levels in live cells (Fig.3a). mScarlet retains some stcapacity under acidic problems and also we observed a band positive for mScarlet at ~30 kDa which we interpret as the cleaved create of the fluorophore after NBR1 has actually been degraded within the lysosome (Supplementary Fig.1d). Treatment via bafilomycin, which blocks the acidification of the lysosome, resulted in stabilization of complete size mScarlet-AID-NBR1 and a much less influential complimentary mScarlet band also (Supplementary Fig.1d). Live imaging of the circulation of GFP-p62 and also mScarlet-AID-NBR1 was likewise complicated by the pH resistant mScarlet signal emitted from the lysosomes (Fig.3a). We therefore excluded the mScarlet signal which overlapped through acidified vesicles, stained by LysoTracker Blue and also considered only the signal which was outside of these compartments. We quantified the complete number of mScarlet-NBR1 pshort articles external of lysosomes and also the variety of these pwrite-ups which colocalized through p62. A big populace of NBR1 puncta did not overlap via p62. Upon therapy via wortmannin, which blocks autophagosome development, we oboffered a greater level of colocalization of the 2 proteins, saying that the double-positive condensates are especially turned over by autophagy. Consistently, therapy with bafilomycin verified a boost in the population of NBR1 colocalizing with p62 (Fig.3a).


*

a HAP1 mSc-AID-NBR1, GFP-p62 cells were left untreated (DMSO) or treated through bafilomycin (400 nM) or wortmannin (1 µM) for 2h. Endogenously tagged proteins and LysoTracker Blue (LysoTB) stained lysosomes were visualized by live-cell imaging usinga Live Spinning Disk microscopic lense. Scale bar, 10 µm. The full variety of NBR1 pwrite-ups not overlapping via the LysoTB signal are plotted in addition to the subset of NBR1 pposts which colocalize with GFP-p62. Average percenteras of colocalization and also conventional deviations for n = 3 are shown. An unpaired, two-tailed Student’s t test was supplied to estimate meaning. P worths are indicated in the number. b HAP1 GFP-p62, mSc-AID-NBR1, TIR1 cell line was left untreated (DMSO) or treated with 1-NAA for 3 h. p62 puncta in the cells (GFP channel) were visualized by Live Spinning Disk microscopy. Scale bar, 10 µm. The average variety of GFP-p62 puncta/cell and also conventional deviations for n = 3 are presented. An unpaired, two-tailed Student’s t test was used to estimate definition. P worths are suggested in the number. The exact same experiment performed on the manage cell line not containing TIR1 is displayed in Supplementary Fig.3b. c HAP1 mSc-AID-NBR1, GFP-p62, TIR1 cells were left untreated (DMSO) or treated with 1-NAA for 3 h in combination through puromycin (5 µg/ml) or wortmannin (1 µM). GFP-p62 puncta in the cells were visualized by live-cell imaging. Mean p62 puncta number and also standard deviation for n = 3 are plotted. An unpaired, two-tailed Student’s t test was provided to estimate meaning. P worths are suggested in the number. d, e HAP1 mSc-AID-NBR1, GFP-p62, TIR1 stably expressing iRFP-NBR1 D50R (d) or iRFP-NBR1 F929A (e) were left untreated (DMSO) or treated via 500 µM 1-NAA via or without 50 ng/ml doxycyclin for 12 h. After therapy GFP-p62 and iRFP-NBR1 puncta were visualized by live-cell imaging. Scale bar, 10 µm. Expression levels of p62 and also NBR1 upon therapies are presented in Supplementary Fig.3c. The exact same experiment, performed through the GFP-p62, mSc-NBR1, TIR1 cell line stably transfected via iRFP-NBR1 WT, is presented in Supplementary Fig.3d. f Quantification of the experiments in (d) and (e). The average number of GFP-p62 puncta/cell and standard deviations for n = 4 are displayed (top graph for the iRFP-NBR1 D50R expressing cells, bottom graph for the NBR1F929A expressing cells). An unpaired, two-tailed Student’s t test was supplied to estimate meaning. P values are suggested in the figure.


Having establiburned the cell line and conditions for the live imaging of NBR1 and also p62, we stably presented TIR1, an E3 ligase which upon stimulation through 1-NAA (1-naphtaleneacetic acid) is able to acknowledge the AID tag fprovided to NBR1 and induce its proteasomal degradation30 (Supplementary Fig.1b). The NBR1 protein in the parental cell line in which no TIR1 was presented was not impacted by treatment through 1-NAA (Supplementary Fig.3a). The TIR1-containing cell line, however, was able to diminish the majority of the NBR1 protein properly within 3 h of therapy (Supplementary Fig.3a). In spite of being a prominent interactor of NBR1 in vivo, p62 was not collaterally degraded by the utilized degron mechanism upon the addition of 1-NAA (Supplementary Fig.3a).

Next off, we examined the results of acute depletion of NBR1 on the number of p62 condensates. We treated the cell line containing GFP-p62, mScarlet-AID-NBR1, and also TIR1 via 1-NAA for 3 h and subjected the cells to live-cell imaging. We oboffered that the variety of GFP-p62 puncta was considerably decreased (Fig.3b and also Supplementary Fig.3b). Treatment via wortmannin and puromycin supported greater numbers of full p62 puncta, but, still confirmed a lower amount of p62 puncta compared to when the 1-NAA treatment was omitted (Fig.3c). In conclusion, a rapid, targeted depletion of endogenous NBR1 from cells caused a significantly reduced number of p62 puncta, indicating a perhaps diminimelted capacity to cluster cargo for selective autophagy.

We went on to ask if the expression of NBR1 or its PB1 and UBA domain mutants might rescue the depletion of NBR1. To this end, we created secure cell lines expressing doxycycline-inducible 3xFLAG-iRFP-NBR1 in the HAP1 GFP-p62, mScarlet-AID-NBR1, TIR1 background (Supplementary Figs. 1b and also 3c). The NBR1 variants included into the cells were either wild-kind NBR1 (Supplementary Fig.3d), a D50R mutant (Fig.3d), which shows lessened binding to p6225,27 or an F929A mutant (Fig.3e), which is defective in ubiquitin binding31. Stable clones were schosen based upon comparable expression levels of iRFP-NBR1 variants after doxycycline treatment (Supplementary Fig.3c). Despite of not observing any kind of changes of the p62 levels in the cell lines in between the clones on a western blot level (Supplementary Fig.3c), the baseline levels of p62 puncta between the clones differed. In particular, the F929A cell line presented even more p62 puncta than the wild-type or D50R clones (Fig.3d–f and also Supplementary Fig.3d). Taking this right into consideration, we compared the number of p62 puncta within each generated cell line at relaxing state and also upon NBR1-depletion and also doxycycline re-expression. Overexpression of wild-form NBR1 rescued depletion of endogenous NBR1 by 1-NAA as we observed no substantial distinction in between untreated cells and cells rescued via wild-type NBR1 in regards to p62 puncta number (Supplementary Fig.3d). The location of the puncta, but, appeared to be bigger once NBR1 was overexpressed (Supplementary Fig.3d), consistent via a previous report28. Performing the exact same therapies for the PB1-mutated D50R cell line we oboffered a different pattern of NBR1 re-expression (Fig.3d). The signal for iRFP-NBR1 D50R was a lot even more diffusage and also the developed puncta were smaller and also much less prominent. Quantification confirmed that, in comparison to the wild-form protein, the overexpression of the D50R mutant of NBR1 was not able to rescue the number of p62-puncta compared to pre-depletion of endogenous NBR1 (Fig.3f—height graph, Supplementary Fig.3d). The UBA-mutant F929A cell line, equivalent to the PB1-mutant cell line, was not able to rescue the p62-puncta phenokind upon 1-NAA-mediated depletion of endogenous NBR1 and mutant re-expression. The overexpressed iRFP-NBR1 F929A protein proved a diffuse pattern of signal via incredibly few huge puncta (Fig.3e). The levels of p62 puncta were fewer than the pre-treatment levels and similar to when tright here was no doxycycline-mediated re-expression (Fig.3f—bottom graph).

We conclude that the PB1 and UBA domain names of NBR1 are required to promote optimal p62 condensate formation in vitro and in cells.

See more: Wh A Sheet Or Web Supported By Metal Frames And Used As A Springboard

NBR1 straight recruits TAX1BP1 to p62–ubiquitin condensates

Due to the fact that TAX1BP1 colocalizes through p62 and NBR1 in condensates in cells (Fig.1a) and also bereason TAX1BP was recently presented to connect with NBR113, we asked if it is recruited to p62–ubiquitin condensates and also what its duty in their development can be. We first tested for a straight interaction of TAX1BP1 with p62 and also NBR1. TAX1BP1 interacted with both proteins yet the binding to NBR1 was notably stronger (Fig.4a and also Supplementary Fig.4a, b). As the interaction of p62 with TAX1BP1 is weak and also provided that NBR1 might bind to both p62 and TAX1BP1, we asked if NBR1 can improve the interaction of p62 through TAX1BP1 by bridging the two molecules. To test this, we immobilized GST-TAX1BP1 on glutathione beads and also included mCherry-p62 (Fig.4b). Thirty minutes afterwards, we supplemented the reactivity through GFP-NBR1. Starting from a low level, the signal of mCherry-p62 at the GST-TAX1BP1 beads raised concomitantly through the signal of GFP-NBR1 (Fig.4b, c). The signal of GFP-NBR1, however not mCherry-p62 alone additionally increased over time and also this rise for GFP-NBR1 was also better in the absence of mCherry-p62 (Fig.4c), likely bereason the mCherry-p62 on the TAX1BP1 beads sterically excludes some GFP-NBR1. These outcomes suggest that NBR1 can bridge p62 and also TAX1BP1. We went on to dissect which domajor of NBR1 would be responsible for TAX1BP1 recruitment. To this finish we offered NBR1 fragments covering the majority of its sequence (Supplementary Fig.4c) and tested their interactivity via GST-TAX1BP1 immobilized on glutathione beads (Supplementary Fig.4d, e). The fragment of NBR1 extending the CC1 and also FW domain names (aa 257-498) verified durable recruitment to the beads and also the FW domain alone was enough for TAX1BP1 binding (Supplementary Fig.4d). The even more durable recruitment of the CC1-FW fragment, compared to the FW domain alone may be because of CC1-mediated oligomerization, which brings even more GFP moieties to the beads, although a direct contribution of the CC1 domajor to the binding cannot be excluded. However before, as a result of challenges in obtaining a recombinant isolated CC1 domain, this could not be tested.