A, N-terminal extension of AtSUM1 (uppercase letters) by tag3 to allow detection and enrichment of sumoylated protein substrates with minimal disruption of functionality. tag3 (single-letter code in lowercase letters) consists of a Strep tag (boldface), three HA tags (boldface, underlined), and an octa-His sequence (boldface). The K residue in position 10 of the SUM1 sequence (boldface) was previously identified as a site of SUMO attachment in SUMO chains. B, Alignment of C termini of mature SUMO proteins (single-letter code). HsSUM1, Human SUMO1; ScSMT3, SUMO of S. cerevisiae; AtSUM1, -2, -3, and -5, the four highly expressed SUMO isoforms of Arabidopsis. The Gln at position 4 from the C terminus (boldface; position 90 in AtSUM1) is conserved in animal and fungal SUMO proteins but replaced by a hydrophobic amino acid in AtSUM3 and AtSUM5. In AtSUM1 Q90A, this residue was changed to Ala in the sequence of Arabidopsis SUMO1.

SUMO isoforms with N-terminal extension are conjugated to protein substrates in vivo. Extracts from plants expressing different SUMO constructs were used for protein blotting and detection with antibody directed against the HA tag of the extension. SUMO1 (extended by tag1 at the N terminus as indicated in the text; lane 3) was compared with extract from plants expressing SUMO1 without the two C-terminal residues (SUMO1 ΔGG; lane 2), indicating dependence of the conjugation reaction on an intact C terminus. Lane 1 shows an extract from nontransgenic plants. SUMO isoforms SUMO3 and -5 (lanes 5 and 6, respectively), as well as a variant of SUMO1 with change Q90A (lane 7), N-terminally extended by tag3 (compare with Fig. 1), can also form conjugates in vivo. The conjugate patterns of SUMO1 and SUMO1 Q90A are similar. Lanes 8 and 9 show examples of SUMO expression by an inducible promoter. Extracts from cells transformed with an inducible tag3-SUMO5 construct contain conjugates to this protein only after induction (lane 9 versus lane 8). Bars at right indicate the position of unconjugated tag-SUMO, and the dotted line indicates the positions of SUMO conjugates. Molecular mass markers in kD are indicated at left.

Qualitative assessment of in vitro desumoylation of a substrate conjugated to SUMO1 versus SUMO1 Q90A. NAF protein with Flag tag was purified from E. coli and subjected to in vitro sumoylation, using either SUMO1 or SUMO1 Q90A. After reisolation with anti-Flag resin, the material was incubated with SUMO protease ESD4 and harvested at the indicated times. Reaction products were detected on western blots using anti-Flag antibody. SUMO1 Q90A-NAF (A, bottom panel) was gradually desumoylated, while SUMO1-NAF (A, top panel) was deconjugated quantitatively within 15 min. At lower ESD4 concentration (B), 70% of SUMO1-NAF was desumoylated in a 30-min incubation, while SUMO1 Q90A-NAF was almost unchanged. Band intensities in B were quantified with secondary antibody coupled to infrared dye and detection of light emission. Unmodified NAF protein was used as an internal standard.

Enrichment of sumoylated proteins. A, An enriched protein fraction from tag-SUMO-expressing plants was used for antibody detection using either rat monoclonal anti-HA antibody (lane 1) or polyclonal anti-SUMO antiserum (lane 2). The polyclonal serum detects additional bands. B, Crude plant extract (lane 3) was compared with an enriched fraction (lane 4) using gel blot and anti-HA detection. The intensity difference of the tag-SUMO band (bar at right) suggests an approximately 50-fold enrichment of SUMO and sumoylated proteins. C, Side-by-side comparison of a Coomassie Brilliant Blue-stained preparative gel of an enriched protein fraction (lane 5) with a western blot of the same material (lane 6). The blot demonstrates enrichment of sumoylated proteins, but the bands detected by the anti-HA antibody do not comigrate with prominent bands of the Coomassie Brilliant Blue-stained gel. D, Material as shown in C was further purified using anti-HA affinity chromatography. The Coomassie Brilliant Blue-stained gel (lane 7) has prominent bands at the position of the most intense bands of the western blot (lane 8), indicating that preparations as shown in C do contain significant amounts of SUMO conjugates. E, Coomassie Brilliant Blue-stained gel loaded with protein extract enriched by one step (Ni²⁺ affinity; compare with C), as used for mass spectrometric protein identification. Lane 9, extract from tag-SUMO transgenic plants (Prep); lane 10, material from nontransgenic plants (background control). In A and D, prominent bands visualized by two distinct detection methods are linked by horizontal lines. Dots show the major contaminant Rubisco large subunit; bars at right show the position of tag-SUMO. Molecular mass markers in kD are indicated at left.

In vitro sumoylation of protein tags. UBC27-Flag, a ubiquitin-conjugating enzyme with a Flag tag extension, or GST encoded by vector pET42c (GST-S) can serve as a negative control. However, extension of GST-S by a Flag tag converts GST into a substrate (GST-S-Flag; dot at right indicates the position of the sumoylated form). Sumoylation is abolished by deletion of the S peptide from the linker region between the GST core and Flag peptide (GST-Flag). Control lanes (−) contain an inactive (Cys-94-to-Ser change) version of SCE; + indicates the presence of wild-type SCE. Proteins were detected by western blotting, using antibodies against Flag, of GST tags.

Test of sumoylation enzymes by formation of SUMO chains. SAEs SAE1a/SAE2 or SAE1b/SAE2 (labeling at top) were incubated with SCE (+ label) or without SCE (− label) together with tag3-SUMO1 (left panel) or tag3-SUMO1 Q90A (right panel) in the presence (+) or absence (−) of SUMO ligase SIZ1. Note that SUMO1 can form SUMO-SUMO linkages in a pattern that is identical to that of SUMO1 Q90A. Bars at right indicate the positions of di-, tri-, and tetra-SUMO (several bands, presumably due to conformation differences or to different SUMO attachment sites). Bands were detected with antibody against the HA epitope present on SUMO.

In vitro sumoylation of potential in vivo substrate proteins. Proteins identified as potential in vivo substrates were expressed in E. coli, purified, and incubated with sumoylation enzymes. NAF, At2g19480; RRM, At3g56860; RXT3, At5g08450; TAF7, At1g55300. Control lanes labeled SCE (−) contain an inactive (Cys-94-to-Ser change) version of SCE. SUMO1 and SUMO1 Q90A give identical patterns, while SUMO3 results in a single, considerably weaker sumoylation band of NAF or TAF7. Proteins NAF and RRM have a C-terminal Flag peptide that was used for antibody-based detection, whereas RXT3 and TAF7 have an N-terminal GST tag (without the S peptide) and were detected by anti-GST antibody.