Exceptional sensitivity of rubisco activase to thermal denaturation in vitro and in vivo

Heat stress inhibits photosynthesis by reducing the activation of Rubisco b y Rubisco activase. To determine if loss of activase function is caused by protein denaturation, the thermal stability of activase was examined in vit ro and in vivo and compared with the stabilities of two other soluble chlor oplast proteins. Isolated activase exhibited a temperature optimum for ATP hydrolysis of 44 degreesC compared with greater than or equal to 60 degrees C for carboxylation by Rubisco. Light scattering showed that unfolding/aggr egation occurred at 45 degreesC and 37 degreesC for activase in the presenc e and absence of ATP gammaS, respectively, and at 65 degreesC for Rubisco. Addition of chemically denatured rhodanese to heat-treated activase trapped . partially folded activase in an insoluble complex at treatment temperatur es that were similar to those that caused increased light scattering and lo ss of activity. To examine thermal stability in vivo, heat-treated tobacco (Nicotiana rustica cv Pulmila) protoplasts and chloroplasts were lysed with detergent in the presence of rhodanese and the amount of target protein th at aggregated was determined by immunoblotting. The results of these experi ments showed that thermal denaturation of activase in vivo occurred at temp eratures similar to those that denatured isolated activase and far below th ose required to denature Rubisco or phosphoribulokinase. Edman degradation analysis of aggregated proteins from tobacco and pea (Pisum sativum cv "Lit tle Marvel") chloroplasts showed that activase was the major protein that d enatured in response to heat stress. Thus, loss of activase activity during heat stress is caused by an exceptional sensitivity of the protein to ther mal denaturation and is responsible, in part, for deactivation of Rubisco.