To elucidate SUMO-1 functions in vivo, we targeted by homologous recombination the last three exons of the murine Sumo-1 gene. Sumo-1 mRNA abundance was reduced to one-half in heterozygotes and was undetectable in Sumo-1 ؊/؊ mice, and SUMO-1-conjugated RanGAP1 was detectable in wild-type mouse embryo fibroblasts (MEFs) but not in Sumo-1 ؊/؊ MEFs, indicating that gene targeting yielded Sumo-1-null mice. Sumo-1 mRNA is expressed in all tissues of wild-type mice, and its abundance is highest in the testis, brain, lungs, and spleen. Sumo-2 and Sumo-3 mRNAs are also expressed in all tissues, but their abundance was not upregulated in Sumo-1-null mice. The development and function of testis are normal in the absence of Sumo-1, and Sumo-1 Small ubiquitin-related modifier 1 (SUMO-1) is a member of the ubiquitin-related protein family (15,16,19,23,24). SUMO proteins are ubiquitously expressed throughout the eukaryotic kingdom. The yeast Caenorhabditis elegans and Drosophila melanogaster have a single SUMO gene, but other organisms, such as vertebrates and plants, have several SUMO genes. Four SUMO family members, SUMO-1 to SUMO-4, have been found in mammals. SUMO-4 precursor cannot be processed for substrate conjugation in vivo, and it may function exclusively through noncovalent interactions (27). SUMO-2 to SUMO-4 share greater amino acid sequence identity with each other than with SUMO-1. SUMO-1 to SUMO-3 have a wide tissue distribution, whereas SUMO-4 expression is limited to the kidneys, lymph nodes, and spleen (10, 21). SUMO-1, a 97-amino-acid-residue mature polypeptide, shares 18% sequence identity with ubiquitin, and the two proteins have similar three-dimensional structures. Like ubiquitin, SUMO is covalently conjugated to substrates by an isopeptide bond through the carboxyl terminus.A consensus SUMO acceptor site, comprising the sequence ⌿KXE (where "⌿" is a large hydrophobic amino acid and K is the SUMO attachment residue) has been identified, although nonconsensus acceptor sites are also used for SUMO conjugation (15, 16). In addition, biological functions have been assigned to SUMO paralogues that are not covalently attached to substrate proteins (13). Many nuclear receptors, including androgen, progesterone, and glucocorticoid receptors, contain SUMO acceptor sites, suggesting a role for sumoylation in their signaling (18). Sumoylation is an evolutionarily conserved pathway from yeast to humans. SUMO is first activated by an E1 activating enzyme, the Aos1/Uba2 heterodimer, subsequently transferred to the unique E2 conjugating enzyme Ubc9, and conjugated to substrates in a reaction facilitated by different E3 ligases, including members of the PIAS family, RanBP2 nucleoporin, and polycomb protein Pc2. SUMO modification is a dynamic, reversible process, and distinct enzymes are responsible for SUMO conjugation and deconjugation. Several SUMO-specific proteases, including SENP1 (SuPr-2), SENP2 (SuPr-1), SENP3 (SMT3IP1), SENP5, and SENP6 (SUSP1), have been identified and localized (16,17).Sumoylation is an impor...