Successive Amino‐Terminal Proteolysis of the Large Subunit of Ribulose 1,5‐bisphosphate Carboxylase/Oxygenase by Vacuolar Enzymes from French Bean Leaves

Abstract

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 % (masshol.) dimethyl sulfoxide, 0.05 % (masshol.) 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, 274-2791 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.

Senescence in wheat leaves: is a cysteine endopeptidase involved in the degradation of the large subunit of Rubisco?

“…A cysteine protease mediating the first step in the catabolism of LSU was described by Yoshida and Minamikawa (1996). We therefore tested the influence of the cysteine protease inhibitor E-64 in our system (Fig.…”

“…In segments incubated in darkness or in low light and in intact dark-incubated plants, a degradation product of about 50 kDa accumulated while LSU declined. Yoshida and Minamikawa (1996) described a similar fragment of 50 kDa in incubated French bean leaf extracts. In contrast to our results, they could not detect the fragment in incubated detached leaves.…”

“…Parrott et al (2005) also detected a band of this size in extracts of excised (but not of attched) barley leaves after intiating senescence. Yoshida and Minamikawa (1996) further reported that Rubisco LSU is degraded by at least two types of vacuolar proteases. A protease, which belongs to the same cysteine protease class as SH-EP, hydrolyzes LSU in the first step.…”

“…Consistent with this result, the cleavage product reported by Ishida et al (1997) also did not cross-react with anti-N-LSU. The cleavage site was further characterized and located at the 14th amino acid from the N-terminus (Yoshida and Minamikawa 1996;Navarre and Wolpert 1999). LSU seems to be especially vulnerable at this site.…”

“…It has been shown that Rubisco degrading proteases exist in the vacuole (Lin and Wittenbach 1981;Thayer and Huffaker 1984;Bhalla and Dalling 1986). Yoshida and Minamikawa (1996) suggested the involvement of at least two proteases in the degradation of purified Rubisco by vacuolar lysates. In the first step, a cysteine protease catalyzed the degradation at the N-terminus of LSU leading to a 48-kDa fragment through the 50 kDa intermediate, while in the second step, the 48-kDa polypeptide was converted by a serine protease through the 42-kDa intermediate to the 41-kDa product.…”