Uch37 associates with both proteasome and hINO80. (A) Affinity-purified 2XHA-Uch37 and its co-purified proteins from cytoplasm (S100) or the nuclei (NE) of HEK293 cells were analyzed by SDS-PAGE and silver staining. Bovine 26S proteasome (0.5 μg) () and hINO80 affinity-purified through Flag-CCDC95 were included for comparison. To facilitate recovery of 26S proteasomes, 3 mM ATPγS was included in buffers during isolation of Uch37-containing complexes in lanes 2 and 3. (B) The samples shown in (A) were analyzed by mass spectrometry. Numbers are NSAFs (Normalized Spectral Count Abundance Factors) () multiplied by 1000. Proteins listed were not detected in mock purifications from parental HEK293 cells.

NFRKB recruits Uch37 to hINO80. (A) NFRKB interacts with Uch37 in yeast two-hybrid assay. Full-length (FL) Uch37 or N-terminal UCH domain (UCH) was fused to Gal4 activation domain (AD); full-length or fragments of NFRKB were fused to Gal4 DNA binding domain (BD). (B) [³⁵S]-methionine-labeled NFRKB fragments made by in vitro translation were incubated with purified GST or GST-Uch37 at 37 °C for 2 h. Inputs (15%) and proteins bound to glutathione-agarose were analyzed by SDS-PAGE and detected using a phosphorimager. Panels show similar exposures. (C) 2 μg purified recombinant N101 or N465 was incubated with 2 μg GST, GST-UCH, or GST-Uch37 at 30 °C for 1 h and incubated with glutathione-agarose. After four washes with PBS, bound proteins and inputs (20%) were analyzed by immunoblotting with anti-GST and anti-NFRKB antibodies. (D) Anti-Flag immunoprecipitates from extracts of HEK293 cells stably expressing N-terminally Flag-tagged full-length NFRKB, NFRKB lacking the N-terminal 101 amino acids (NFRKB Δ101), or CCDC95 analyzed by SDS-PAGE and blotted with anti-Flag or anti-Uch37 antibodies. Endogenous Uch37 runs as a doublet (). Degradation products are marked by asterisk. (E) hRpn13 and NFRKB compete for binding to Uch37. GST-Uch37 (1 μM) was incubated with equimolar N101 and 0, 0.5, 1, 2, 3, 4, or 5 equivalents of hRpn13 (lanes 1–7) or equimolar hRpn13 and increasing N101 (lanes 9–15). GST-Uch37 and bound proteins were pulled-down with glutathione-agarose and detected by anti-His immunoblotting. GST-Uch37 contains a 6XHis-tag whereas N101 and hRpn13 contain 10XHis tags. (F) Model for Uch37 partitioning between proteasome and hINO80 complexes in cells. Solid black and dashed gray arrows indicate strong and weak interactions, respectively; hRpn2, the 19S RP subunit that binds hRpn13.

Regulation of Uch37 activity by interaction with NFRKB or incorporation into hINO80. (A) GST-Uch37 alone or in complex with Flag-tagged hRpn13, NFRKB, or NFRKB fragments expressed in insect cells, affinity purified using glutathione-agarose (lanes 1 and 2) or anti-Flag agarose (lanes 3–10), incubated with or without 2 μM UbVS at 37 °C for 1 h, and analyzed by anti-GST immunoblotting. For a coomassie-stained gel of the proteins, see . (B) 0.1 μM GST-Uch37 or GST-UCH were pre-incubated for 1 h with or without 0.5 μM hRpn13, N101, or N465 at 21°C; 0.25 μM UbVS was then added. After 30 min, reaction products were analyzed by anti-GST immunoblotting. The asterisk denotes a degradation product. (C) UbAMC hydrolysis monitored by AMC fluorescence. As indicated, reactions contained 2.5 nM full-length GST-Uch37, 2.5 nM GST-UCH, 12.5 nM hRpn13, 12.5 nM N101, or 6.25 nM N465. A coomassie-stained gel of recombinant proteins is shown in . (D) hINO80-associated Uch37 did not react with UbVS. Affinity-purified HA-Uch37 from cytoplasmic (lanes 1 and 2) or nuclear (lanes 3 and 4) fractions of HEK293 cells was incubated with or without 2 μM UbVS at 37 °C for 1 h. Lanes 5–10, hINO80 affinity-purified from stable cell lines expressing HA-tagged hINO80 subunit Amida, HA-tagged CCDC95, or Flag-tagged CCDC95 was incubated with or without UbVS. Reaction products were detected by anti-Uch37 immunoblotting.

hRpn13 or proteasomes activate hINO80-associated Uch37. (A) hINO80 (~50 nM) containing either wild-type or C88A mutant Uch37 was isolated by sequential immunoprecipitation of Flag-CCDC95 and HA-Uch37. UbAMC hydrolysis was assayed with or without 50 nM hRpn13. (B) Affinity purified hINO80 (~0.25 μM) was incubated with 1 μM UbVS with or without 1 μM recombinant hRpn13 at 37 °C; an additional 1 μM UbVS was added every 2 h. Reactions were stopped at the indicated times and analyzed by immunoblotting with anti-HA antibody. (C) Exogenous hRpn13 does not displace Uch37 from hINO80. hINO80 (~35 pmol) was immobilized on anti-Flag agarose (lanes 3–6), and 100 pmol of hRpn13 was added (lanes 1, 2, 5, 6). After 2 h rotation at 4 °C, unbound proteins (U) were recovered, the agarose beads were washed, and bound proteins (B) were eluted with Flag peptide. These fractions were immunoblotted for hRpn13, Uch37, and NFRKB. Equivalent amounts of anti-Flag agarose without hINO80 (lanes 1 and 2) served as a control for non-specific binding. Aliquots of bound fractions shown in lanes 4 and 6 were further incubated for 1 h with 1 μM UbVS, and Uch37 was detected by immunoblotting with anti-HA antibody (lanes 7 and 8). hRpn13 was added back to the bound fraction shown in lane 6 and incubated with UbVS for 1 h (lane 9). (D) Purified recombinant His-Myc-hRpn13 was immobilized on anti-Myc agarose (lanes 1, 2, 5, 6); equivalent amounts of beads without hRpn13 served as controls (lanes 3, 4, 7, 8). Purified hINO80 (lanes 1–4) or GST-Uch37 (lanes 5–8) were added to beads and rotated at 4 °C for 3 h. Unbound (U) and bound (B) proteins were recovered as described in C except that elution was with Myc peptide. As a positive control, beads, bead-bound His-Myc-hRpn13, or soluble hRpn13 were incubated with hINO80 and UbVS at 37 °C for 30 min with rotation (lanes 9–11). Asterisk, immunoglobulin heavy chain. (E) Relative abundance of Uch37 in PA700 from bovine erythrocytes, HeLa cell proteasomes and hINO80. Proteasomes were affinity-purified from HeLa cells through a Flag-tagged hRpt6 subunit, and hINO80 was affinity-purified from HEK293 cells through HA-tagged Arp8 or Flag-tagged CCDC95 subunits. PA700, the 19S RP from bovine erythrocytes, contains 0.5–1 Uch37 polypeptides per complex (; ) and was included as a standard. Purified complexes were normalized by SYPRO-Ruby staining after SDS-PAGE and measuring fluorescence intensities of similar sized bands using a Typhoon 8600 Imager (lanes 1–9). Circles mark the tagged subunits in each complex. To estimate relative molar concentrations of Uch37 in each complex, various amounts of each complex were analyzed by immunoblotting using antibodies against Uch37, hRpn13, hRpn2, and Ies2 (lanes 10–20). Uch37 appears as a single band in lanes 19 and 20 because the doublet was not resolved on the 4–15% gradient gel used in this experiment. (F) Proteasomes can activate hINO80-associated Uch37. hINO80 (~5 pmol) that had been affinity purified from a cell line stably expressing both Flag-tagged CCDC95 and HA-tagged Uch37 was incubated with 1 μM UbVS at 37 °C for 2 h. Reactions were supplemented with 5 pmol recombinant hRpn13 (lane 3), proteasomes immunoaffinity purified from S100 fraction in 150 mM NaCl (lane 4) or 300 mM NaCl (lane 5), or proteasomes from nuclear extract purified in 300 mM NaCl (lane 6).