Endogenous substrates of the Euglena chloroplast chaperonin 60 at permissive and bleaching temperatures

Cultures of Euglena gracilis grown at the moderately elevated temperature o f 33 degrees C experience an irreversible loss of chlorophyll and chloropla st function known as heat-bleaching. Since molecular chaperones play an imp ortant role in protein folding, we considered whether or not chloroplast ch aperonin GO (Cpn60) binds to nascent polypeptides and newly imported polype ptides, its likely targets for folding. In this report, we address the effe ct of growth temperature on the ability of chloroplast Cpn60 to bind endoge nous substrate in vivo using pulse-labeling and co-immunoprecipitation with antibody against Cpn60. Four major polypeptides and about a half-dozen min or polypeptides appear to be the targets of the chaperonin complex at the p ermissive temperature of 23 degrees C. Among the principal targets are the large subunit of Rubisco (RbcL) and elongation factor-Tu (EF-Tu). By contra st, only RbcL co-immunoprecipitates with Cpn60 in stromal extracts from par tially bleached Euglena but at a much reduced level. Associations between t he chaperonin and its target polypeptides resume in a partially bleached cu lture when returned to the permissive temperature. These observations sugge st that chloroplast chaperonin targets only a small, select group of protei ns for folding. In addition, the activity of the chaperonin complex in Eugl ena is sensitive to even a modest increase in growth temperature.