Molecular Cell, Volume 27
E1-L2 Activates Both Ubiquitin and FAT10
Yu-Hsin Chiu, Qinmiao Sun, and Zhijian J. Chen
Figure S1. Identification of E1-L2 by ubiquitin affinity chromatography. HeLa S100
was incubated with Ub-Sepharose in the presence of ATP at 25oC for 1 hour, and then the
slurry was poured into a column. The column was eluted sequentially with a buffer
containing 1M KCl (high salt wash), 10 mM DTT, a pH 9.0 buffer (high pH elution) and
finally a pH 9.0 buffer containing 1M KCl (high salt / high pH elution). The collection
tubes for the DTT and high pH elutions contain BSA as a carrier protein. Aliquots of the
eluted fractions were analyzed by SDS-PAGE followed by Coomassie blue staining.
IsoT: isopeptidase T.
Figure S2. Evolutionary conservation of E1-L2. (A) Alignment of E1-L2 protein
sequences from human (H. sapiens), mouse (M. musculus), chicken (G. gallus),
pufferfish (T. rubripes) and sea urchin (S. purpuratus). (B) Sequence identity scores of
E1-L2 proteins from different species.
Figure S3. Subcelluar localization of E1-L2. (A) Crude lysates from HEK293 cells
were fractionated into nuclei, heavy membranes, light membranes, and cytosol by
differential centrifugation as described in Experimental Procedures. Proteins in these
fractions were analyzed by immunoblotting with an antibody against E1-L2. (B) The
specificity of the E1-L2 antibody was verified by RNAi using siRNA oligos against E1-
L2 or GFP (control).
Figure S4. GST-FAT10 is not activated by E1-L2. (A) Coomassie blue staining of
recombinant FAT10 proteins. Lane 1: GST-FAT10; lane 2: GST was removed by Tev
protease; lane 3: His6-FAT10. (B &C) His6-E1-L2 was incubated with the recombinant
FAT10 proteins shown in (A) in the presence of ATP for 1 minute at 37oC, and then
aliquots of the reaction mixtures were analyzed by non-reducing (B) or reducing (C) gel
electrophoresis followed by immunoblotting with an antibody against His6. β-ME: β-
Figure S5. Competition of ubiquitin and FAT10 for E1-L2 in vitro. His6-E1-L2 (65
nM) was incubated with ubiquitin and FAT10 at the indicated concentrations for 15
minutes at 37oC. The resulting thioesters were analyzed by non-reducing gel
electrophoresis followed by immunoblotting with an antibody against His6.
Figure S6. E1, but not E1-L2, transfers ubiquitin to Ubc3 and E2-25K. GST-Ubc3
and GST-E2-25K were cleaved with thrombin to remove the GST tag, and the E2s were
incubated with ubiquitin and E1 or E1-L2 in the presence or absence of ATP. The
reaction mixtures were resolved by non-reducing SDS-PAGE and the gels were stained
with Coomassie blue.
Figure S7. E1-L2 transfers ubiquitin, but not FAT10, to Ubc5 and Ubc13. E1-L2 was
incubated with ubiquitin or FAT10 in the presence of Ubc5 or His6-Ubc13/Uev1A as
indicated. After incubation at 37oC for 15 minutes, the thioesters were analyzed by non-
reducing SDS-PAGE followed by detection with Coomassie blue (upper panel) or
immunoblotting with an antibody against Ubc5 or His6 (lower panels).