SUPPLEMENTARY INFORMATION Contents: Supplementary Figure 1. Additional structural and binding data for designed tuim peptides. Supplementary Figure 2. Subcellular localization patterns of designed tuim fluorescent fusion proteins expressed in cultured human cells. Supplementary Figure 3. HA-Vx3K0-EGFP is recruited to mitochondria in the presence of Parkin whereas HA-Vx3NB-EGFP is not. Supplementary Figure 4. Vx3-fusions inhibit polyub recognition and cleavage. Supplementary Figure 5. Control experiments for using fluorescent fusions of polyubsensor peptides as linkage-specific inhibitors inside cells. Supplementary Figure 6. Calculating the IC50 of TNF-a inhibition by expressed Rx3(A7)- megfp. Supplementary Table 1. Cloning details for all constructs used in this study. Supplementary Table 2. Sequences of all tuim peptides used in this study.
a Supplementary Figure 1 UIM linker UIM linker UIM b c Supplementary Figure 1. Additional structural and binding data for designed tuim peptides. (a) Predicted helical content of two linker modules used in this study. Shown is the predicted helicity of the weakly helical linkers used in the original Vx3 peptide and the all-alanine A7 linker, both in the context of the Vps27-derived 3-UIM peptide. Predictions of linker region helicity have been previously validated for several tuim peptides using circular dichroism (Sims & Cohen, 2009). (b) The interaction between Vx3(A7) and Lys63-Ub 4 was too tight for us to measure accurately by fluorescence anisotropy. Fluorescein-labeled Vx3(A7) peptide at 1 nm was titrated with Lys63-Ub 4 chains, and the fluorescence anisotropy was monitored; 1 nm represented the minimum concentration of fluorescent peptide that gave sufficient anisotropy signal in the experiment. Typically, the labeled species being titrated must be present at concentrations < 0.1-times the dissociation equilibrium constant (K d ) of the interaction to accurately measure the K d, thus setting a lower limit of the K d measurable in the assay. The K d for Vx3(A7) interacting with Lys63-Ub 4 is clearly below 1 nm, and so cannot be accurately fit. The best-fit parameters for the interaction (K d = ~60 pm) should thus be regarded as imperfect estimates for this fit. Based on this experiment, we estimate that an upper bound of the Vx3(A7) K d is 200 pm. (c) The equilibrium dissociation constants (K d ) for the interactions of Rx3(A6) and Rx3(A8) with K63 Ub 3 (2,900 nm and 160 nm, respectively) were determined by competition with fluorescein-labeled Vx3 peptide as described in the METHODS.
Supplementary Figure 2 a b wt K63R Vx3NB EGFP c LC3 LC3 Vx3K0-EGFP Vx3NB-EGFP merge merge
Supplementary Figure 2 Supplementary Figure 2. Subcellular localization patterns of designed tuim fluorescent fusion proteins expressed in cultured human cells. (a) Vx3K0- megfp and Vx3NB-mEGFP were transiently transfected into HeLa cells and imaged. The images were subsequently segmented into cytosolic and nuclear compartments as described in the METHODS, and the nuclear and cytoplasmic EGFP intensities were tabulated for thousands of cells. Nuclear localization of Vx3NB-mEGFP steadily increased with total amount of expressed protein, consistent with the proposed cryptic nuclear localization sequence of EGFP. In contrast, the polyub-binding Vx3K0-mEGFP protein was largely excluded from the nucleus at low expression levels. (b) The negative control construct Vx3NB-EGFP was expressed in singleubiquitin-wt and single-ubiquitin-k63r yeast strains. As expected, in the absence of a Ub-binding capacity, the tuim construct did not localize to the large, cytosolic aggregates seen when Vx3K0-EGFP was expressed in wild-type yeast. (c) Many but not all Vx3K0 foci appear to co-localize with LC3, consistent with recent reports that Lys63-polyUb is involved in selective cargo uptake for some autophagy pathways (Kraft, Peter, & Hofmann, 2010). Co-localization is not observed with the non-ubbinding control fusion protein, Vx3NB-EGFP.
HA Vx3K0 EGFP mcherry Parkin WT HA Vx3E EGFP mcherry Parkin WT Supplementary Figure 3 Merge Parkin GFP Tom2 0 HA Vx3K0 EGFP Parkin R275W 3h 10μM CCCP HA Vx3K0 EGFP (no Parkin) Supplementary Figure 3. HA-Vx3K0-EGFP is recruited to mitochondria in the presence of Parkin whereas HA-Vx3NB-EGFP is not. HeLa cells were transfected with the indicated polyub sensor constructs with or without co-transfection of mcherry-parkin. Cells were treated with 10 µ M CCCP for 3 h and then fixed and stained for the mitochondrial marker Tom20.
Supplementary Figure 4 Supplementary Figure 4. Vx3-fusions inhibit polyub recognition and cleavage. Recombinant MBP-His-Vx3 protein was incubated with mixed Lys63-linked Ub 4 and Ub 5 chains and recombinant BRISC, a Lys-63-specific deubiquitinating enzyme (Cooper et al., 2009), at various ratios of substrate to inhibitor. At an equimolar ratio, MBP-His-Vx3 partially inhibited the deubiquitination of the chains. With MBP-Vx3 in excess, cleavage inhibition was complete.
a b Supplementary Figure 5 c d e Supplementary Figure 5. Control experiments for using fluorescent fusions of polyubsensor peptides as linkage-specific inhibitors inside cells. For all experiments, U2OS cells were transfected and treated with ligand to activate NF-κB as described in the METHODS. (a) The non-ub-binding control Vx3NB-EGFP was equivalent to HA-EGFP in the NF-κB activation assay. Note that in these plots the nuclear:cytoplasmic p65-staining ratio is not scaled to a maximum of 1. (b) Vx3-EGFP and Vx3K0-EGFP acted equivalently to inhibit NF-κB activation in U2OS cells. (c-e) Vx3K0-mEGFP also distinguished the linkage-type polyub dependences of IL-1β from TNF-α. U2OS cells transiently transfected with a non-ub binding control, Vx3NB-mEGFP (c), or Vx3K0-mEGFP (d) were treated with IL-1β or TNF-α, fixed, and imaged as described in the text. As with Rx3(A7), Vx3K0- strongly inhibited signaling through IL-1R at all but the lowest doses of the inhibitor, whereas TNFR signaling was fully active at low doses of the inhibitor and was fully inhibited only at high doses. A zone of low Vx3K0-mEGFP concentrations exhibits high specificity for inhibition of the K63- polyub species responsible for IL-1β signaling (gray shaded region). (e) Cells falling into this high-specificity zone were analyzed from three independent experiments, and the average NF-κB activation scores are plotted with error bars representing the s.e.m. of the activation Nature value. Methods: Each doi:10.1038/nmeth.1888 point in c and d represents the average of 200 cells.
Supplementary Figure 6 a b c regfp lysate lysate value d TNF alpha uns-mulated IC50
Supplementary Figure 6 Supplementary Figure 6. Calculating the IC50 of TNF-α inhibition by expressed Rx3(A7)-mEGFP. We set out to determine the IC50 for TNF-α inhibition by Rx3(A7)-mEGFP. Evidence suggests that non-k63-polyub linkages, including K11-linked chains, play key signaling roles in this pathway; thus, the inhibition we observed may give a reasonable estimate of the inhibitor concentrations above which K63-linkage-specific action is unlikely. (a) First, recombinant EGFP (regfp) was purified from bacteria and examined by SDS- PAGE and Coomassie staining. Next, two coverslips (22 x 22 mm) were seeded with an identical number of HeLa cells, transfected identically using Lipofectamine 2000 (Invitrogen), and stimulated with 10 ng/ml TNF-α for 30 min as in Fig. 5c. After ligand stimulation, one coverslip was immediately lysed and compared to the regfp standard by quantitative western blot (qwb) (b). A standard curve representative of the four independent qwbs is shown in (c). A sample of the lysate was found to contain 98 nm EGFP. The second coverslip was immediately fixed and stained for p65, then imaged and scored for p65 translocation as in Fig. 5. 85% of the stained coverslip was imaged at 10x magnification. Each point in (d) represents the binned average p65 translocation score for 1,000 HeLa cells; 130,000 EGFP-positive cells were detected in the analysis, so approximately 150,000 EGFPpositive cells were on the coverslip. The average amount of EGFP per cell is thus 1.33 x 10-16 mol. Assuming that the volume of a HeLa cell is 1540 µm 3 (http:// bionumbers.hms.harvard.edu), the average cellular EGFP concentration was 87 µm. The mean cellular EGFP fluorescence intensity value for the imaged cells was 60 intensity units (I.U.); thus, 60 I.U. corresponds to 87 µm. As shown in (d), the inhibitor construct intensity that corresponded to 50% inhibition of signaling (IC50) was determined to be 11 I.U. Thus, the cellular inhibitor concentration corresponding to the IC50 for TNF-alpha inhibition is 16 µm. This is in terms of total inhibitor in the cell, whereas we know from imaging (see Fig. 2) that some fraction of the inhibitor is tied up in large aggregates. Thus, a reasonable estimate of the effective (or free) inhibitor concentration at 50% TNF-α signaling inhibition is probably 1-10 µm. This range is consistent with the measured equilibrium dissociation constant (K d ) for Rx3(A7) with non-k63-linked polyub chains, including K11 polyub (10 µm), and thus sets a reasonable threshold below which linkage-specific action can be expected from these reagents.
Supplementary Table 1. Cloning details for all constructs used in this study. Used in (figure) Insert name Vector backbone Cloning sites 1d Rx3(A7) pet28a Nde1/BamH1 1e Vx3 pet28a Nde1/ BamH1 2a; 4 Vx3K0-mEGFP pqcxip Not1/EcoR1 2b; 4 Vx3NB-mEGFP pqcxip Not1/EcoR1 2c NLS-Vx3-mEGFP pqcxip Not1/EcoR1 2d; 4 Rx3(A6)-mEGFP pqcxip Not1/EcoR1 2e; 4 Rx3(A7)-mEGFP pqcxip Not1/EcoR1 2f; 4 Rx3(A8)-mEGFP pqcxip Not1/EcoR1 2g i Vx3K0-EGFP prs425-adh 3a c MBP-Vx3 prs415-adh and prs425-adh 3a c NLS-MBP-Vx3 prs415-adh and prs425-adh 3d,e Vx3NB-EGFP pegfp-n1 Nhe1/ BamH1 3d f NLS-Vx3-EGFP pegfp-n1 Nhe1/ BamH1 3f mcherry-rap80 poz-n Xho1/ Not1 Table 1 Vx3K0 pet28a Nde1/BamH1 Table 1 Vx3(A7) pet28a Nde1/BamH1
Supplementary Table 2. Sequences of all tuim peptides used in this study. Insert name Vx3 Vx3K0 Vx3NB NLS- Vx3 Rx3(A6) Rx3(A7) Rx3(A8) Vx3(A7) Sequence MEEEEDPDLKAAIQESLREARAEEKVKEDEEELIRKAIELSLKESREVNAQEE DEEELIRKAIELSLKECRNSA MEEEEDPDLRAAIQESLREARAEERVREDEEELIRRAIELSLRESREVNAQEE DEEELIRRAIELSLRECRNSA MEEEEDPDEKAEIQEELREARAEEKVKEDEEELERKEIELELKESREVNAQE EDEEELERKEIELELKECRNSA MSPKKKRKVEASKMTASPKKKRKVEASKMTAMEEEEDPDLKAAIQESLREA RAEEKV KEDEEELIRKAIELSLKESREVNAQEEDEEELIRKAIELSLKESRNSA MTEEEQFALALKMSEQEAAAAAAAEEEEEQFALALKMSEQEAAAAAAAEEE EELLRKAIAESLNSCRPS MTEEEQFALALKMSEQEAAAAAAAAEEEEEQFALALKMSEQEAAAAAAAAE EEEELLRKAIAESLNSCRPS MTEEEQFALALKMSEQEAAAAAAAAAEEEEEQFALALKMSEQEAAAAAAAA AEEEEELLRKAIAESLNSCRPS MEEEEDPDLKAAIQESLREAAAAAAAAEDEELIRKAIELSLKESAAAAAAAED EEELIRKAIELSLKECRNSA
SUPPLEMENTARY REFERENCES Sims, J. J. & Cohen, R. E. Linkage-specific avidity defines the lysine 63-linked polyubiquitin-binding preference of Rap80. Mol. Cell 33, 775-783 (2009). Cooper, E. M. et al. K63-specific deubiquitination by two JAMM/MPN+ complexes: BRISCassociated Brcc36 and proteasomal Poh1. EMBO J. 6, 621-631 (2009). Kraft C., Peter M., & Hofmann K. Selective autophagy: ubiquitin-mediated recognition and beyond. Nat Cell Biol. 12, 836-841 (2010).