We used high-resolution two-dimensional polyacrylamide gel electrophoresis to evaluate the effect of aging on the heat shock response in Drosophila melanogaster. Although the aging process is not well understood at the molecular level, recent observations suggest that quantitative changes in gene expression occur as these fruit flies approach senescence. Such genetic alterations are in accord with our present data, which clearly show marked differences in the synthesis of heat shock proteins between young and old fruit flies. In 10-day-old flies, a heat shock of 20 min results in the expression of 14 new proteins as detectable by two-dimensional electrophoresis of [35Slmethionine-labeled polypeptides, whereas identical treatment of 45-day-old flies leads to the expression of at least 50 new or highly up-regulated proteins. In addition, there is also a concomitant increase in the rate of synthesis of a number of the normal proteins in the older animals. Microdensitometric determinations of the low molecular weight heat shock polypeptides on autoradiographs of five age groups revealed that their maximum expression occurs at 47 days for a population of flies with a mean life span of 33.7 days. Moreover, a heat shock effect similar to that observed in senescent flies occurs in young flies fed canavanine, an arginine analogue, before heat shock.Many of the modern theories of molecular aging imply, directly or indirectly, that age-dependent changes occur in the molecular configuration or expression ofproteins and that these changes contribute to the diminution in the viability of senescent cells (1). In essence, these theories can be divided into three broad categories: (i) those that predict posttranslational modifications such as protein crosslinkage (2) and deamidation or glycosylation (3); (ii) those that predict alterations in primary structure, such as the mutational theories (4-7) and the error-catastrophe hypothesis (8); and (iii) those that predict specific changes in the pattern or timing of protein expression, such as the genetic-program theories (9). Several investigations have demonstrated that posttranslational changes do indeed occur during senescence (10-12); however, unequivocal evidence showing an age-dependent effect on the fidelity of protein synthesis has not been reported (1, 13). Previous studies in this laboratory gave no evidence that aged Drosophila express altered polypeptides (1). Two-dimensional gel electrophoresis data indicated that the qualitative pattern of protein expression was identical in young and old flies, but there were significant quantitative changes in the expression of proteins with age in the unstressed imago of Drosophila melanogaster.The expression of "new" polypeptides was also observed in non-heat-shocked senescent flies after exposure to inhibitors of mitochondrial function such as nonactin (14). These results suggest that qualitative changes in gene expression can be induced in aged animals by a metabolic or chemical stress. Thus, general perturbations of hom...

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