Exceptional Sensitivity of Rubisco Activase to Thermal Denaturation in vitro and In vivo

Heat stress inhibits photosynthesis by reducing the activation of Rubisco by Rubisco activase. To determine if loss of activase function is caused by protein denaturation, the thermal stability of activase was examined in vitro and in vivo and compared with the stabilities of two other soluble chlor...

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Bibliographic Details
Published in:	Plant physiology (Bethesda) 2001-11, Vol.127 (3), p.1053-1064
Main Authors:	Salvucci, Michael E., Osteryoung, Katherine W., Crafts-Brandner, Steven J., Vierling, Elizabeth
Format:	Article
Language:	eng
Subjects:	Adenosine Triphosphate - metabolism Aggregation Agronomy. Soil science and plant productions Analysis Bioenergetics and Photosynthesis Biological and medical sciences Carboxylation Chloroplasts Chloroplasts - metabolism Degradation Economic plant physiology Enzyme Activation Enzyme inhibition Fundamental and applied biological sciences. Psychology Heat stress disorders High temperature Hot Temperature Hydrolysis Immunoblotting Light Light scattering Metabolism Net assimilation, photosynthesis, carbon metabolism. Photorespiration, respiration, fermentation (anoxia, hypoxia) Nicotiana - metabolism Nutrition. Photosynthesis. Respiration. Metabolism Peas Phosphotransferases (Alcohol Group Acceptor) - metabolism Photosynthesis Photosynthesis - physiology Photosynthesis, respiration. Anabolism, catabolism Phytochemicals Phytochemistry Pisum sativum - metabolism Plant embryology Plant physiology and development Plant Proteins - chemistry Plant Proteins - metabolism Plants Protein Denaturation Proteins Protoplasts Ribulose-Bisphosphate Carboxylase - chemistry Ribulose-Bisphosphate Carboxylase - metabolism Thermal stability Thermodynamic stability Thiosulfate Sulfurtransferase - pharmacology
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Summary:	Heat stress inhibits photosynthesis by reducing the activation of Rubisco by Rubisco activase. To determine if loss of activase function is caused by protein denaturation, the thermal stability of activase was examined in vitro and in vivo and compared with the stabilities of two other soluble chloroplast proteins. Isolated activase exhibited a temperature optimum for ATP hydrolysis of 44°C compared with ≥60°C for carboxylation by Rubisco. Light scattering showed that unfolding/aggregation occurred at 45°C and 37°C for activase in the presence and absence of ATPγS, respectively, and at 65°C for Rubisco. Addition of chemically denatured rhodanese to heat-treated activase trapped partially folded activase in an insoluble complex at treatment temperatures that were similar to those that caused increased light scattering and loss 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 that aggregated was determined by immunoblotting. The results of these experiments showed that thermal denaturation of activase in vivo occurred at temperatures similar to those that denatured isolated activase and far below those required to denature Rubisco or phosphoribulokinase. Edman degradation analysis of aggregated proteins from tobacco and pea (Pisum sativum cv "Little Marvel") chloroplasts showed that activase was the major protein that denatured in response to heat stress. Thus, loss of activase activity during heat stress is caused by an exceptional sensitivity of the protein to thermal denaturation and is responsible, in part, for deactivation of Rubisco.
ISSN:	0032-0889 1532-2548