SUMO proteases of the SENP/Ulp family are master regulators of both sumoylation and desumoylation and regulate SUMO homeostasis in eukaryotic cells. SUMO conjugates rapidly increase in response to cellular stress, including nutrient starvation, hypoxia, osmotic stress, DNA damage, heat shock, and other proteotoxic stressors. Nevertheless, little is known about the regulation and targeting of SUMO proteases during stress. To this end we have undertaken a detailed comparison of the SUMO-binding activity of the budding yeast protein Ulp1 (ScUlp1) and its ortholog in the thermotolerant yeast Kluyveromyces marxianus, KmUlp1. We find that the catalytic UD domains of both ScUlp1 and KmUlp1 show a high degree of sequence conservation, complement a ulp1Δ mutant in vivo, and process a SUMO precursor in vitro. Next, to compare the SUMO-trapping features of both SUMO proteases we produced catalytically inactive recombinant fragments of the UD domains of ScUlp1 and KmUlp1, termed ScUTAG and KmUTAG respectively. Both ScUTAG and KmUTAG were able to efficiently bind a variety of purified SUMO isoforms and bound immobilized SUMO1 with nanomolar affinity. However, KmUTAG showed a greatly enhanced ability to bind SUMO and SUMO-modified proteins in the presence of oxidative, temperature and other stressors that induce protein misfolding. We also investigated whether a SUMO-interacting motif (SIM) in the UD domain of KmULP1 that is not conserved in ScUlp1 may contribute to the SUMO-binding properties of KmUTAG. In summary, our data reveal important details about how SUMO proteases target and bind their sumoylated substrates, especially under stress conditions. We also show that the robust pan-SUMO binding features of KmUTAG can be exploited to detect and study SUMO-modified proteins in cell culture systems.

Purple coneflower [Echinacea purpurea (L.) Moench] is an increasingly popular crop because of its value in the U.S. botanical industry. At present, there is no rapid method of analyzing it for chicoric acid, the predominant phenolic acid associated with immunostimulation in humans. The objective of this study was to quantify chicoric acid in purple coneflower roots by near infrared (NIR) reflectance spectroscopy. One hundred sixty‐nine plants were harvested and their root tissues analyzed for chicoric acid by high performance liquid chromatography (HPLC). Root samples were scanned between 1100 and 2498 nm and reflectance data recorded. The HPLC data were regressed against infrared spectra to develop empirical prediction equations. The optimum prediction equation produced coefficients of determination for calibration and cross validation of 0.90 and 0.86, respectively. The mean chicoric acid concentration was 8.29 g kg DM−1, and the standard errors of calibration and cross validation were 0.89 and 1.06 g kg DM−1, respectively. We conclude that NIR reflectance spectroscopy can accurately quantitate chicoric acid in purple coneflower.