SCCRO interacts with Cullins, CAND1, and Ubc12. A, Coomassie-stained SDS-PAGE gel showing GST (lane 1) and GST-SCCRO (lane 2) pulldown products from HeLa extracts. Unique bands from GST-SCCRO pulldown assays were excised and subjected to tandem mass spectroscopy analysis (MALDI-ReTOF) revealing the 120-kDa band as CAND1 and the 80–90-kDa bands as cullins 1–4. B, Western blot on the pulldown products of GST-SCCRO and GST-SCCRO mutants from HeLa extracts probed with indicated antibodies showing SCCRO binds to CAND1, cullins 1–5, ROC1, and Ubc12 (lane 2). The C-terminal (SCCROΔ210–259; lane 4) but not the N-terminal deletion of SCCRO (SCCROΔ1–33; lane 3) lost interaction with the Cullin-ROC1, CAND1, and Ubc12 (lane 4). SCCRO-D241N lost interaction with the Cullin-ROC1 and CAND1 while retaining binding to Ubc12 (lane 6). The level of the various GST-tagged proteins used in the pulldown experiment was confirmed by probing a Western blot with anti-GST antibody (bottom panel). C, Western blot showing products from an HA immunoprecipitation (IP) of lysates from HeLa cells transfected pCMV-HA-SCCRO or selected SCCRO deletions/mutants probed with indicated antibodies showing SCCRO maintains binding to neddylation components in vivo (lane 2). SCCROΔ210–259 (lane 4) and SCCRO-D241N (lane 6) lose binding to cullins, ROC1, and CAND1. D, Western blot on immunoprecipitates from HeLa extract prepared with anti-SCCRO (left panel; lane 2) anti-ROC1 (right panel; lane 2) or preimmune serum (PIS; lane 1) probed with indicated antibodies showing in vivo interaction between native SCCRO and CAND1, Cul1, and ROC1 but not SKP2 (left panel), whereas ROC1 binds to CAND1, Cul1, SCCRO, and SKP2 (right panel). E, summary of results from pulldown assays using GST-SCCRO or SCCRO deletions/mutations showing binding of CAND1, Cul-ROC1, and Ubc12 requires C-terminal 50 amino acids. SCCRO-D241N loses binding to CAND1 and Cul1-ROC1 but retains binding to Ubc12.

SCCRO augments Cullin neddylation in vitro. A and B, plot showing levels of Cul1 (A) and Cul3 (B) (mean ± S.E.) neddylation quantified by densitometry of Western blots from three independent in vitro neddylation reactions containing HeLa extract (as a source of cullin substrate), E1, E2, ATP, NEDD8, and a concentration gradient of SCCRO. The fraction of neddylated Cul1 (A) and Cul3 (B) increased with increasing SCCRO concentration. Representative Western blots from in vitro neddylation reactions are shown as insets. C, a plot showing the fraction of neddylated Cul3 (means ± S.E.) against time in minutes from three independent neddylation assays. SCCRO but not SCCRO-D241N enhances neddylation efficiency. D, representative Western blot from C showing Cul3 neddylation in presence of SCCRO or SCCRO-D241N. E, results from in vitro neddylation reaction supplemented with SCCRO (lanes 2 and 3) or SCCRO deletions showing SCCROΔ1–33 (lanes 4 and 5) but not the C-terminal deletion SCCROΔ210–259 (lanes 6 and 7) retained neddylation activity. F, Western blot for Cul1 from a time course in vitro reaction containing purified, bacterially expressed Cul1-ROC1 showing SCCRO enhances efficiency but is not required for neddylation.

SCCRO augments cullin neddylation in vivo. A, Western blot on HeLa lysates showing elevated SCCRO protein levels in SCCRO transfected (lane 3) relative to untransfected (lane 1) or vector-transfected (lane 2) cells. B, Western blot showing a higher level of neddylated cullins in lysates from SCCRO transfected (lane 2) relative to vector-transfected cells (lane 1). Western blot on HeLa lysates from B after addition of neddylation components (E1, E2, NEDD8, and ATP) showing increased neddylated Cul1 levels in SCCRO-transfected (lane 4) relative to empty vector (lane 3)-transfected cells. C, Western blot on lysates from SCC15 cells showing a decrease in SCCRO protein levels in cells transfected with specific RNAi against SCCRO (lane 3) relative to untransfected (lane 1) or scrambled RNAi-transfected cells (lane 2). D, in vitro neddylation reaction of the same lysates showing decreased Cul1 neddylation in SCCRO-RNAi transfected (lane 3) compared with untransfected (lane 1) or scrambled RNAi (lane 2)-transfected cells. The addition of recombinant SCCRO to the lysate from SCCRO RNAi-transfected SCC15 cells (lane 6) recovers Cul1 neddylation to levels observed in controls (lanes 4 and 5). E, Western blot showing the absence of detectable SCCRO protein in testis lysates from SCCRO^–/– mice (left panel, lane 2) in contrast to a SCCRO^+/+ (left panel, lane 1) litter mate control. The same lysates were subjected to neddylation assays (right panel) showing a significant decrease in neddylated Cul3 levels in SCCRO^–/– mice (lane 2).

SCCRO interacts with the unique N terminus of Ubc12∼NEDD8. A, Western blot for Ubc12 after a thioester reaction showing generation of Ubc12∼NEDD8 thioester (top panel, lane 1, upper band). GST (lane 2) and GST-SCCRO (lane 3) pulldown on the same reaction products showing preferential interaction of SCCRO with Ubc12∼NEDD8 despite a large excess of free Ubc12 (top panel, lane 3). Autoradiograph of products from a thioester reaction containing E1, E2, ATP, and ³²P-labeled PK-NEDD8 showing Ubc12∼NEDD8 (bottom panel, lane 1, upper band) and free NEDD8 (bottom panel, lane 1, lower band). GST (lane 2) or GST-SCCRO (lane 3) pulldown assays on thioester reaction components showing SCCRO binds to Ubc12∼NEDD8 but not to free NEDD8 (bottom panel, lane 3) B, Western blot showing levels of Ubc12∼NEDD8 and Ubc12 from a thioester reaction supplemented with varying concentrations of free Ubc12 after quenching with EDTA (see “Experimental Procedures”; top panel). The same reactions were subjected to pulldown assays using limiting amounts of GST-SCCRO (70 pm) showing preferential binding to Ubc12∼NEDD8 even in the presence of 20-fold excess of free Ubc12 (bottom panel). C, Western blots for NEDD8 (top panel) and Ubc12 (middle panel) on thioester reactions with and without ATP in lysates from HeLa cells transfected with pCMV or pCMV-SCCRO showing lower levels of Ubc12∼NEDD8 in reactions not containing ATP (compare first and third lanes with the second and fourth lanes). HA immunoprecipitation of the same reaction mix showing preferential binding of SCCRO (in limiting concentrations) to Ubc12∼NEDD8 (bottom panel, third and fourth lanes), even in the presence of a large excess of free Ubc12 (bottom panel, third lane). D, Western blot for HA after thioester reactions on HeLa cell lysates transfected with HA-Ubc12 or HA-Ubc12Δ26 showing equal amounts of NEDD8 thioester formation after 10 and 30 min of incubation, respectively. HA immunoprecipitation on the same reaction products followed by Western blot with the indicated antibody showing SCCRO interacts with full-length Ubc12∼NEDD8 but not with Ubc12Δ26∼NEDD8, whereas ROC1 interacts with both (bottom right).

SCCRO does not overcome the inhibitory effects of CAND1 on neddylation. A, Western blot on GST-SCCRO pulldown products after incubation with purified, bacterially expressed Cul1-ROC1 and CAND1 showing CAND1 is pulled down by SCCRO only when it is complexed with Cul1-ROC1 (lanes 1 and 3). A CAND1 deletion (CAND1Δ604–1230) that loses binding to cullins is not pulled down by SCCRO (lane 4 and 5). B, Western blot from reciprocal GST-CAND1 pulldown assay showing interaction of CAND1 with SCCRO requires Cul1-ROC1 (lanes 2–4). C, Western blot for Cul1 following in vitro neddylation reaction using purified recombinant components showing complete inhibition of neddylation in the presence of 2-fold or higher molar excess of CAND1. D, Western blot on products from an in vitro neddylation reaction confirming increasing Cul1 neddylation with increasing amounts of SCCRO (lanes 2–4). The addition of CAND1 inhibits Cul1 neddylation (lane 5), which is not rescued even by 5-fold molar excess of SCCRO (lane 6; same blot, excess lanes removed). E, Western blot showing GST-CAND1 pulldown products after incubation with purified, bacterially expressed Cul1-ROC1, Ubc12∼NEDD8, and SCCRO. CAND1 interacts with Ubc12∼NEDD8 only when Cul1-ROC1 is present (lanes 1 and 2), and this interaction is enhanced by the presence of SCCRO (lane 3). F, Western blot on lysates from HA-SCCRO-transfected HeLa cells subjected to neddylation reaction showing an increase in cullin neddylation with the addition of ATP (lanes 1 and 2). HA immunoprecipitation (IP) of the same reaction products showed SCCRO preferentially interacts with unneddylated cullins (lanes 3 and 4). G, Western blot on products from an in vitro neddylation reaction using purified recombinant components showing an increase in the fraction of neddylated Cul1 with increasing reaction time (top panel). Western blot on the same reaction after GST-SCCRO (middle panel) and GST-CAND1 (bottom panel) pulldown assays showing that SCCRO and CAND1 interact with both free and neddylated cullins (same blot, excess lanes removed). H, Western blot on products from pulldown assays after GST-CAND1 complexed with either NEDD8-Cul1-ROC1 or free Cul1-ROC1 was incubated with increasing amounts of testis lysate from SCCRO^+/+ mice showing release of NEDD8-Cul1-ROC1 but not unneddylated Cul1-ROC1. I, Western blot on products from pulldown assays after GST-CAND1 complexed with either NEDD8-Cul1-ROC1 or free Cul1-ROC1 was incubated with increasing amounts of testis lysate from SCCRO^–/– mice showing release of Cul1-ROC1-NEDD8 only with addition of SCCRO and not SCCRO-D241N (same blot, excess lanes removed).