AbstractWe examined the effect of aging on the expression of ubiquitin RNA and the binding of the ubiquitin polypeptide to proteins following heat shock inDrosophila melanogaster.Heat‐shocked adult flies transcribe two major RNA species‐one of 4.4 kb and one of about 6 kb that hybridize to the polyubiquitin‐encoding probe. Several less abundant RNAs were also observed but the 4.4‐kb band was present as the major RNA species in both stressed and nonstressed flies of both ages. The 6‐kb fragment was more abundant in heat shocked aged flies than in younger flies. The quantitative expression of the polyubiquitin gene increased in proportion to the duration of the heat stress. Moreover, the induction of the polyubiquitin RNA was markedly elevated during aging following heat shock. Hybridization of Northern blots with the monoubiquitin gene probe revealed a band of 0.9 kb that was not significantly affected by heat stress.We also investigated the relationship between the changes in polyubiquitin gene expression and the formation of ubiquitin‐protein complexes in aging heat‐shocked flies. Heat shock to old flies results in a significant increase in the level of proteins immunoprecipitated by anti‐ubiquitin antibodies. In the case of proteins synthesized 2 h before heat shock, most of the ubiquitinated proteins were of high molecular weight. For those proteins synthesized during a 30‐min heat shock and the 2 h following heat shock, two major immunoprecipitated bands were observed: an 80‐kD and a 70‐kD polypeptide. The ubiquitination of a 60 kD protein was also observed in nonstressed flies, but its for mation was drastically reduced following heat shock. For proteins synthesized during and after heat shock from both age groups, the major ubiquitinated polypeptide is 70 kD. In all age groups, more ubiquitin complexes were formed with proteins synthesized before heat shock, than with proteins synthesized either during or after heat shock. This suggests that cellular proteins synthesized at physiological temperatures are more sensitive to heat induced damage than those synthesized during stress. These data support the hypothesis that in aging flies, heat shock induces an unusually high concentration of abnormal proteins which are targeted for degradation by the ubiquitin‐dependent proteolytic system