Fig. 5.

AtRAF1 stimulated assembly of Rubisco. ³⁵S-Met “pulse” unlabeled-Met “chase” analysis of hybrid L₈^AS₈^t Rubisco synthesis and turnover relative to tobacco L₈S₈ Rubisco performed on young attached leaves under constant illumination (∼500 µmol quanta m²/s) (Fig. S5). (A) Autoradiography signals of ndPAGE separated soluble protein from 6 mm² of leaf taken 15, 30, and 45 min after infiltration with [³⁵S]methionine showing increasing ³⁵S incorporation into L₈S₈ Rubisco. Plotted are the average densitometry signals for L₈S₈ Rubisco at each time point (n = 3 ± SD) relative to the average of the 45-min wild-type sample signals. Rates of L₈S₈ synthesis extrapolated from linear fits to the normalized data were 27 × 10⁻⁴ (r² = 0.999), 78 × 10⁻⁴ (r² = 0.997), and 229 × 10⁻⁴ (r² = 1.000) for the tob^AtL (●), tob^AtL-R1 (○), and wild-type (□) leaves, respectively. (B) ndPAGE analyses made on soluble protein from the same leaves taken 2, 4, and 7 h after a “chase” infiltration with 10-mM unlabeled-methionine. No discernible changes in the densitometry of either hybrid L₈^AS₈^t or wild-type L₈S₈ Rubisco autoradiography signals were detected indicative of little or no Rubisco turnover during this period.