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).