MODULATION OF THE HEAT SHOCK UBIQUITIN POOL IN SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1

Abstract

ABSTRACTImmunoblotting experiments performed with an anti‐ubiquitin antibody revealed that Skeletonema costatum (Grev.) Cleve cells contained free ubiquitin as well as ubiquitin conjugated to various endogenous proteins. A temperature shift from 18° to 30°C greatly increased the total amount of ubiquitin and particularly the ubiquitin fraction in high molecular mass conjugates. A solid‐phase immunoassay indicated values of 0.031 ± 0.004 pmol·10−6 cells for free ubiquitin and 0.046 ± 0.004 pmol·10−6 cells for conjugated ubiquitin for cells grown at 18°C, and 0.056 ± 0.008pmol·10−6cells and 0.21 ± 0.03 pmol·10−6cells, respectively, after a temperature increase from 18° to 30°C. Cell‐free extracts of S. costatum were equally able to form thiol ester linkages with 125I‐ubiquitin in an adenosine triphosphate–dependent manner at 18° C and at 30°C. Cell‐free extracts were also able to conjugate 125I‐ubiquitin to endogenous proteins, but the ubiquitin conjugation rate at 30°C was lower than at 18°C. Incubation of S. costatum for 3 h at 30°C and then for 3 h at 18°C resulted in the formation of high amounts of ubiquitin conjugates, suggesting that partially inactive or denaturated proteins accumulate during heat stress. These denaturated proteins are then conjugated to ubiquitin very efficiently when the physiological temperature is restored. Thus, S. costatum cells contain ubiquitin and an active ubiquitin conjugation system responding to stress conditions (temperature stress). The intracellular concentration of ubiquitin conjugates is most likely limited by the availability of protein substrates to be conjugated rather than by ubiquitin‐conjugating activity.