The study compares the functional differences between SUMO-1 and SUMO-2/3 in vertebrate cells. SUMO-1 is a small ubiquitin-like modifier that is covalently conjugated to various cellular proteins, playing a role in processes such as nuclear transport, cell cycle control, oncogenesis, inflammation, and viral response. SUMO-2 and -3, which are closely related to SUMO-1, form a distinct subfamily and are also involved in protein modification. The study found that SUMO-2/3 constitutes a larger percentage of total cellular protein modification than SUMO-1. A significant pool of free, non-conjugated SUMO-2/3 was detected, and its conjugation to high molecular mass proteins was induced by protein-damaging stimuli such as acute temperature fluctuations. In contrast, SUMO-2/3 conjugated poorly to a major SUMO-1 substrate, RanGAP1, suggesting distinct regulatory mechanisms between the two SUMO pathways. These findings support the idea that SUMO-2/3 may play a role in cellular responses to environmental stress. The study also developed a specific antibody to detect SUMO-2/3 and demonstrated that SUMO-2/3 conjugation is up-regulated by various protein-damaging stimuli. The results suggest that SUMO-2/3 modification is distinct from SUMO-1 modification and may be involved in the cellular response to stress. The study highlights the functional heterogeneity between SUMO-1 and SUMO-2/3, indicating that they may have different roles in cellular processes.The study compares the functional differences between SUMO-1 and SUMO-2/3 in vertebrate cells. SUMO-1 is a small ubiquitin-like modifier that is covalently conjugated to various cellular proteins, playing a role in processes such as nuclear transport, cell cycle control, oncogenesis, inflammation, and viral response. SUMO-2 and -3, which are closely related to SUMO-1, form a distinct subfamily and are also involved in protein modification. The study found that SUMO-2/3 constitutes a larger percentage of total cellular protein modification than SUMO-1. A significant pool of free, non-conjugated SUMO-2/3 was detected, and its conjugation to high molecular mass proteins was induced by protein-damaging stimuli such as acute temperature fluctuations. In contrast, SUMO-2/3 conjugated poorly to a major SUMO-1 substrate, RanGAP1, suggesting distinct regulatory mechanisms between the two SUMO pathways. These findings support the idea that SUMO-2/3 may play a role in cellular responses to environmental stress. The study also developed a specific antibody to detect SUMO-2/3 and demonstrated that SUMO-2/3 conjugation is up-regulated by various protein-damaging stimuli. The results suggest that SUMO-2/3 modification is distinct from SUMO-1 modification and may be involved in the cellular response to stress. The study highlights the functional heterogeneity between SUMO-1 and SUMO-2/3, indicating that they may have different roles in cellular processes.