Ubiquitination and ATP levels in garden pea seeds

Abstract

Developing and germinating pea seeds contain high levels of ubiquitin conjugated to proteins as detected on western blots. In contrast, the level of dry seed protein‐ubiquitin conjugates in vivo appears low, with mainly free ubiquitin present. The ubiquitination of endogenous dry pea seed proteins is observed in vitro, relying only on already present endogenous ubiquitin, suggesting the enzymatic machinery for ubiquitination is present in the dry seed. Energy source in the form of ATP increased the formation of large molecular mass conjugates, although some conjugation took place without added ATP. The usefulness of dry seeds, having low levels of ATP which can then be manipulated in the in vitro reaction is discussed. ATP and ubiquitin degrading activities are detected in the crude in vitro system, pointing to the need to purify the individual components, or to seek specific inhibitors of the undesirable secondary reactions.