Successive amino-terminal proteolysis of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase by vacuolar enzymes from French bean leaves

Mainly using the protein immunoblot technique, we observed the decrease in amounts of the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in detached primary leaves of French bean plants during senescence under the light or in darkness, but detected no significant degradation products of these subunits. Treatment of the detached leaves with 0.6% (mass/vol.) dimethyl sulfoxide, 0.05% (mass/vol.) Tween 80 considerably promoted the senescence, as estimated by the reduction in content of the soluble protein, and also in the amounts of LSU and SSU, but no degradation product of either subunit was found. When extracts prepared from the primary leaves were incubated at pH 5.4 or pH 7.4, the amount of LSU of 53 kDa decreased and concurrently 50-kDa and 42-kDa polypeptides were formed. Since the results suggested that Rubisco may be degraded by vacuolar enzymes, we incubated Rubisco with vacuolar lysates prepared from the senescing primary leaves and found that the LSU, but not SSU, was degraded to a 41-kDa polypeptide through three intermediates of 50 kDa, 48 kDa and 42 kDa. Determination of amino-terminal amino acid sequences of these fragments indicated that each of the proteolysis steps occurred by removal of a small amino-terminal peptide. Experiments with various inhibitors of proteases as well as with a purified Vigna mungo vacuolar protease, termed SH-EP [Mitsuhashi, W. & Minamikawa, T. (1989) Plant Physiol. 89, 274-279] suggested the involvement of two types of proteases in these steps: a cysteine protease that is the same type of enzyme as SH-EP catalyzes the steps from the LSU to the 48-kDa polypeptide through the 50-kDa polypeptide, and a serine protease catalyzes the steps from the 48-kDa polypeptide to the 41-kDa polypeptide through the 42-kDa polypeptide.