Evolution of a signalling system that incorporates both redundancy and diversity: Arabidopsis SUMOylation

Abstract

The reversible post-translational modifier, SUMO (small ubiquitin-related modifier), modulates the activity of a diverse set of target proteins, resulting in important consequences to the cellular machinery. Conjugation machinery charges the processed SUMO so that it can be linked via an isopeptide bond to a target protein. The removal of SUMO moieties from conjugated proteins by isopeptidases regenerates pools of processed SUMOs and unmodified target proteins. The evolutionarily conserved SUMO-conjugating proteins, E1 and E2, recognize a diverse set of Arabidopsis SUMO proteins using them to modify protein substrates. In contrast, the deSUMOylating enzymes differentially recognize the Arabidopsis SUMO proteins, resulting in specificity of the deconjugating machinery. The specificity of the Arabidopsis deSUMOylating enzymes is further diversified by the addition of regulatory domains. Therefore the SUMO proteins, in this signalling system, have evolved to contain information that allows not only redundancy with the conjugation system but also diversity with the deconjugating enzymes.

Figure 1. SUMO family members can be utilized by SUMO conjugation machinery in vitro

(A) Sequence alignment of amino acids of yeast Smt3, T-SUMO, AtSUMO-1, AtSUMO-2, AtSUMO-3, and AtSUMO-5. Numbering is shown with respect to yeast Smt3. Black circles above the alignment denote residues in yeast Smt3 that are in direct contact with yeast ΔUlp1 as described by Mossessova and Lima [23]. The arrow indicates where SUMO substrates are cleaved by ULP1s. (B) [³⁵S]Mammalian RanGAP was in vitro translated in an RRL. In the RRL, some of the RanGAP is SUMOylated by endogenous SUMOylation machinery (lane 1). The in vitro translated product was then used in an in vitro SUMOylation assay using purified recombinant GST–AtSUMO-1, GST–AtSUMO-2, GST–AtSUMO-3, GST–AtSUMO-5, GST–T-SUMO, GST–Smt3, GST–M-SUMO-1, GST–M-SUMO-2 and GST–M-SUMO-4 to produce GST–SUMO-modified RanGAP. A volume of 5 μl of the SUMO-modified RanGAP from each reaction was then added to 5× SDS sample buffer [250 mM Tris, pH 6.8, 50% glycerol, 5% (w/v) SDS, 5% 2-mercaptoethanol and Bromophenol Blue dye] and the samples were resolved on SDS/8% polyacrylamide gels and visualized by autoradiography.

Figure 2. Sequence alignments of the ULP1 family members

(A) Sequence alignment of amino acids in the catalytic core of yeast Ulp1, AtULP1A, AtULP1C, AtULP1D and AtESD4. Numbering is shown with respect to yeast Ulp1. Asterisks above the alignment denote the catalytic histidine, aspartic acid and cysteine residues. Black circles above the alignment denote residues in yeast Ulp1 that are in direct contact with yeast Smt3 as described by Mossessova and Lima [23]. The catalytic domains of all Arabidopsis ULP1s are extended to their native C-terminus. (B) Phylogenic tree representation of amino acid sequence distance among AtULP1s used in the present study. The tree was calculated using ClustalW (v1.4) algorithm in the MacVector program.

Figure 3. In vitro peptidase activity of AtULP1 family members

(A) Schematic diagram of in vitro SUMO peptidase assay. (B) AtULP1 family members show differing peptidase activity for AtSUMO substrates. [³⁵S]AtSUMO-1–HA, AtSUMO-2–HA, AtSUMO-3–HA, AtSUMO-5–HA, AtSUMO-3-Gly-Gly-X₁₈ and AtSUMO-5-Gly-Gly-X₅ were in vitro translated in an RRL and then incubated with a buffer (50 mM Tris, pH 8.0, 150 mM NaCl and 0.1% 2-mercaptoethanol) or 0.5 mg/ml of FL AtULP1A, AtULP1C, AtULP1D and AtESD4, and the catalytic core (Δ) of AtULP1A, AtULP1C, AtULP1D and AtESD4, or yeast Ulp1 for 1 h at 30 °C. The samples were then resolved on SDS/17% polyacrylamide gels and visualized by autoradiography. Roman numerals indicate each peptidase assay number as referred to in the text. The ‘+’ symbol indicates total cleavage of the SUMO–HA, the ‘+/−’ symbol indicates partial cleavage of SUMO–HA and the ‘−’ symbol indicates no detectable cleavage of SUMO–HA by each ULP1. The asterisk (*) indicates that when AtSUMO-2–HA is in vitro translated in RRL, background peptidase activity is detected (lane 1). This peptidase activity is probably due to a rabbit peptidase present in the RRL.

Figure 4. Specificity of plant, animal and yeast ULP1 family members for their SUMO substrates

AtULP1 family members show differing peptidase activity for M-SUMO, yeast SUMO and T-SUMO substrates. [³⁵S]M-SUMO-1–HA, M-SUMO-2–HA, M-SUMO-4–HA, yeast Smt3–HA and T-SUMO–HA were in vitro translated in an RRL and then incubated with a buffer (50 mM Tris, pH 8.0, 150 mM NaCl and 0.1% 2-mercaptoethanol) or 0.5 mg/ml of FL AtULP1A, AtULP1C, AtULP1D and AtESD4, and the catalytic core (Δ) of AtULP1A, AtULP1C, AtULP1D and AtESD4, or yeast Ulp1 for 1 h at 30 °C. The samples were then resolved on SDS/17% polyacrylamide gels and visualized by autoradiography. The ‘+’ symbol indicates total cleavage of the SUMO–HA, the ‘+/−’ symbol indicates partial cleavage of SUMO–HA and the ‘−’ symbol indicates no detectable cleavage of SUMO–HA by each ULP1.

Figure 5. AtULP1 family members exhibit isopeptidase activity in vitro

(A) Schematic diagram of in vitro SUMO isopeptidase assay. (B) The ULP1 family of proteases shows specificity for the SUMO moiety of SUMOylated substrates. [³⁵S]Mammalian RanGAP was in vitro translated in an RRL. In the RRL, some of the RanGAP is SUMOylated by endogenous SUMOylation machinery. The in vitro translated product was then used in an in vitro SUMOylation assay using purified recombinant GST–M-SUMO-1, GST–T-SUMO, GST–AtSUMO-1, GST–AtSUMO-2, GST–AtSUMO-3 and GST–AtSUMO-5 to produce GST–SUMO-modified RanGAP. The SUMO-modified RanGAP reaction was then incubated with a buffer (50 mM Tris, pH 8.0, 150 mM NaCl and 0.1% 2-mercaptoethanol) or 0.5 mg/ml of FL AtULP1A, AtULP1C, AtULP1D and AtESD4, and the catalytic core (Δ) of AtULP1A, AtULP1C, AtULP1D, AtESD4, or yeast Ulp1 for 1 h at 30 °C. The samples were then resolved on SDS/8% polyacrylamide gels and visualized by autoradiography. Roman numerals indicate each isopeptidase assay number as referred to in the text. Asterisks (*) indicate partial cleavage of GST–T-SUMO-RanGAP by FL AtULP1A and GST–M-SUMO-1-RanGAP by FL AtULP1D. (C) The GST moiety attached to AtSUMO-3 and AtSUMO-5 is not responsible for the lack of ULP1 activity towards these two SUMO substrates. As in (B), [³⁵S]mammalian RanGAP was in vitro translated in an RRL where some of the RanGAP is SUMOylated by endogenous SUMOylation machinery. The in vitro translated product was then used in an in vitro SUMOylation assay using purified recombinant AtSUMO-3 and AtSUMO-5 (the GST tag was removed from these proteins using TEV protease) to produce AtSUMO-modified RanGAP. This AtSUMO-modified RanGAP migrates just below the RRL-SUMO-modified RanGAP. The SUMO-modified RanGAP reaction was then incubated with a buffer (50 mM Tris, pH 8.0, 150 mM NaCl and 0.1% 2-mercaptoethanol) or 0.5 mg/ml of FL AtULP1A, AtULP1C, AtULP1D and AtESD4, and the catalytic core (Δ) of AtULP1A, AtULP1C, AtULP1D and AtESD4 for 1 h at 30 °C. The samples were then resolved on SDS/8% polyacrylamide gels and visualized by autoradiography.

Figure 6. Classification of SUMOs and ULP1 family members based on activity

(A) Summary of results from peptidase and isopeptidase assays. The peptidase and isopeptidase activities of each ULP1 protein for each specific SUMO substrate are shown by a ‘+’ symbol, indicating total cleavage of the SUMO–HA, a ‘+/−’ symbol, indicating partial cleavage of SUMO–HA, and a ‘−’ symbol indicating no detectable cleavage of SUMO–HA by each ULP1. ^aFL AtULP1D shows isopeptidase activity for GST–M-SUMO-1 conjugated to RanGAP. (B) SUMOs are grouped based on the ULP1 family members that cleave them as described in the present paper. The classification is based on FL and/or catalytic core ability to demonstrate peptidase and/or isopeptidase activity for SUMO substrates. ^aAtULP1D shows peptidase activity for AtSUMO-1, AtSUMO-2, T-SUMO, and Smt3, yet it only shows isopeptidase activity for GST–M-SUMO-1 conjugated to RanGAP. ^bAtULP1C shows peptidase activity for AtSUMO-1, AtSUMO-2, T-SUMO, Smt3, M-SUMO-1 and M-SUMO-2, yet it only shows isopeptidase activity for RRL-SUMO conjugated to RanGAP.