Modification of the Proteolytic Fragmentation Pattern upon Oxidation of Cysteines from Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

Abstract

The proteolytic susceptibility of the native CO(2)-fixing photosynthetic enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco) has been shown to increase in vitro after oxidative treatments that affect cysteine thiols. A limited incubation of oxidized (pretreated with the disulfide cystamine) Rubisco from Chlamydomonas reinhardtii with subtilisin or proteinase K generated fragments of molecular mass about 53 kDa (band I in SDS-PAGE) and 47 kDa (band II) derived from the large subunit (55 kDa) of the enzyme. In contrast, proteolysis of the reduced Rubisco (pretreated with the free thiol cysteamine) produced only the 53 kDa band. The same fragmentation pattern was reproduced with Rubiscos from other algae and higher plants, as well as with other chemical modifications of protein cysteines. N-terminal sequencing of the fragments showed that band I arised from clipping the unstructured N-terminal stretch of the large subunit up to Lys18. Band II was generated by a cleavage close to Val69. The increased susceptibility of the oxidized form resulted from proteases gaining access to a loop (from Ser61 to Thr68) located between stretches of secondary structure that form the N-terminal domain. Native electrophoresis and kinetic analysis of fragment accumulation during subtilisin digestion demonstrated that subunit dissociation was induced by the proteolytic processing at the Ser61-Thr68 loop, which is characteristic of the oxidized Rubisco. Holoenzyme dissasembly was readily followed by the full degradation of the released subunits. In contrast, the limited processing to band I observed with the reduced enzyme did not compromise the quaternary structure of the Rubisco hexadecamer, thus preventing further proteolysis.

Degradation of Rubisco SSU during oxidative stress triggers aggregation of Rubisco particles in Chlamydomonas reinhardtii

“…2 bottom panel, Rubisco-fractions 27-11, ribosome-fractions 21-13), suggesting its possible aggregation. We also observed that under oxidizing conditions, the LSU of Rubisco breaks down to smaller fragments, as previously noted (Penarrubia and Moreno 1990;Ishida et al 1997;Marin-Navarro and Moreno 2003). In addition, when the band intensities of LSU and SSU (Fig.…”

“…The fragments of the LSU were shown to co-exist as parts of the holoenzyme (Ishida et al 1999;Marin-Navarro and Moreno 2003). Indeed, the fragments of the LSU comigrate with the holoenzyme in a sucrose gradient, and are not released following proteolysis.…”

Comparative proteomics illustrates the complexity of drought resistance mechanisms in two wheat (Triticum aestivum L.) cultivars under dehydration and rehydration

“…2 and the rapidly down-regulated OEE 2 (spot 4113) in Longchun 23 under drought stress suggested that drought-tolerant cultivar have more stability of oxygen-evolving activity of PS II. Rubisco is a key rate-limiting enzyme responsible for photosynthetic carbon assimilation [83, 84]. There were several down-regulated Rubisco proteins found in two wheat cultivars, including Ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBisCO) large subunit (spot 6713), Ribulose1,5-bisphosphate carboxylase activase isoform 1 (spots 3402 and 2504), RuBisCO large subunit-binding protein subunit alpha (spot 1604) and subunit beta (spot 2908) in Xihan No.…”

Rubisco large-subunit translation is autoregulated in response to its assembly state in tobacco chloroplasts

“…The regulatory mechanisms involved, however, may differ. In addition, in vivo data from Chlamydomonas (35) and cucumber (36), and in vitro data from wheat and barley (37,38) suggest that LS undergoes fragmentation upon light and oxidative stress, followed by proteolysis. One may speculate that CES is involved, and might be enhanced if these cleavages expose the LS repressor motif.…”