Reactivation of ubiquitination in Artemia franciscana embryos during recovery from anoxia-induced quiescence

Encysted gastrulae of Artemia franciscana are known to enter a reversible state of quiescence promoted by anoxia, during which the half-life of cytochrome oxidase is prolonged up to 77-fold. This observation suggests that proteolytic pathways within mitochondria are inhibited, and indeed the suppression of the initial step in ubiquitin-mediated proteolysis under anoxia has been reported. Given that active embryos require efficient degradation of macromolecules, we investigated the reactivation of ubiquitination during recovery from anoxia and aerobic acidosis (elevated CO2 levels under aerobic conditions). During 6 h of recovery from anoxia, the levels of ubiquitin-conjugated proteins rose 6.5-fold, reaching 78 % of the pre-anoxia (control) values. Concomitant with the elevation in ubiquitin conjugates was a sharp decline in AMP level, a rise in ATP level and an alkalization of intracellular pH. Our results suggest that the reinitiation of ubiquitin conjugation is partially dependent on decreasing AMP and/or increasing ATP levels. However, when anoxic embryos were transferred to aerobic acidosis, which promotes a return to control (aerobic) levels of adenylates yet maintains the acidic intracellular pH, 71 % of the total suppression of ubiquitination still remained. This observation reveals a predominant role of intracellular alkalization in the reactivation of ubiquitination during recovery. We suggest that the rapid reversibility of the factors regulating ubiquitin conjugation allows Artemia embryos readily to reinitiate the degradation of proteins via the ubiquitin-mediated pathway during recovery.