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Supplementary Figure 1. Similarity relationship among human SUMOs, yeast SMT3,
Drosophila SUMO, Arabidopsis SUMO1, and Chlamydomonas SUMO homologs.
Supplementary Data Figure 1
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Supplementary Data Figure 2. Purification of endogenous free CrSUMO96 by
immunoprecipitation followed by immunoblot detection (left panel) and silver stain
detection (right panel). N, non-boiled cell lysate; H, heated cell lysate; IP input, extract
prior to immunoprecipitation; IP output, protein immunoprecipitated with CrSUMO96
antisera.
Supplementary Data Figure 2
2
Supplementary Data Figure 3. Confirmation of the identity of CrSUMO96 in boiled cell
lysates using mass spectrometry–based amino acid sequence analysis.
Supplementary Data Figure 3
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A.
C.r. E2 ID
CrUBCE2_1
CrUBCE2_2
CrUBCE2_3
CrUBCE2_4
UBC9 ID
CrUBC9B_1
CrUBC9B_2
CrUBC9C_1
CrUBC9C_2
JGI
Protein
ID
57440
135149
152525
160028
GenBank ID
XP_001694849.1
XP_001703521.1
XP_001699308.1
XP_001701577.1
HMM
No.
IEP score
residues
159
163
148
155
8.81
5.9
6.8
7.73
339.9
247.1
335.5
223.8
AtUBC9_NP_849462
148
8.07 384.5
AtUBC9_NP_567791
178
7.5
HsE2I_NP_919237
158
8.9 448.4
157
9.65 430.9
ScUbc9p
NP_010219.1
Annotation
393
UBC9 (UBIQUITIN
CONJUGATING
ENZYME 9)
UBC9 (UBIQUITIN
CONJUGATING
ENZYME 9)
ubiquitin-conjugating
enzyme E2I variant
SUMO-conjugating
enzyme Ubc9p
B.
Supplementary data Figure 4. A. Potential CrUbcE2 molecules. B. Similarity relationship
among human, yeast, Arabidopsis and CrUbcE2 enzymes.
Supplementary data Figure 4
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Supplementary Data Figure 5. Amino acid sequence of CrUbcE2_1 [SUMO-conjugating
enzyme (E2)] from Chlamydomonas reinhardtii.
Supplementary Data Figure 5
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Supplementary Data Figure 6. Comparison of SUMO and SUMOylated proteins extracted
from control and heat shocked C. reinhardtii cells and analyzed by two-dimensional PAGE.
SUMO and SUMOylated proteins blotted to nitrocellulose membranes following 2-D PAGE
were detected using anti-CrSUMO96 antibodies (top figures) or pre-immune serum
(bottom figures). Putative noncongugated SUMO species decreasing after heat shock
treatment are noted in thick-walled red boxes. Other SUMOylated proteins decreasing in
amounts following heat shock treatment are noted in thin-walled red boxes. SUMOylated
proteins markedly increasing following heat shock treatment are enclosed within green
boxes. Equal quantities of proteins (400 µg) were loaded on each 2-D gel. Migration and
sizes (in kDa) of protein size markers during SDS-PAGE are noted to the left of the
images.
Supplementary Data Figure 6
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Supplementary Data Figure 7 Multiple alignment of C. reinhardtii ubiquitins and related
proteins XP_001694608 and Cr1794 including two homologs of A. thaliana Rub1 protein.
Five of the proteins shown contain tandem duplicates of the ubiquitin domain starting with
an [IL][FYK]VKTL motif starting at the third column of both panels of the alignment. Here
square brackets indicate possible amino acid substitutions. A variant of the SUMO-binding
ΨKXE motif, [ILV]KXK[IV][DEQ][DE] (dark blue) is present in five of the seven proteins,
including two copies in Cr140045 and Cr1794. Alternatives to this are six LKCMIDEL and
one IKERVEEK motifs. Double-glycine cleavage site motifs are well conserved at the Cterminal positions of the duplicate ubiquitin domains (black boxes). Note that arginines
precede all domain-terminal double-glycine motifs. For brevity, the N-terminal segments of
XP_001684320 and XP_001702404, and an insertion from Cr195594 close to the Cterminus of the sequence have been omitted.
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