Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.

Abstract

Two adjacent N-terminal tryptic peptides of the large subunit of ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from spinach, wheat, tobacco, and muskmelon were removed by limited tryptic proteolysis. Characterization by peptide sequencing, amino acid composition, and tandem mass spectrometry revealed that the N-terminal residue from the large subunit of the enzyme from each plant species was acetylated proline. The sequence of the penultimate N-terminal tryptic peptide from the large subunit of the spinach and wheat enzyme was consistent with previous primary structure determinations. However, the penultimate N-terminal peptide from the large subunit of both the tobacco and muskmelon enzymes, while identical, differed from the corresponding peptide from spinach and wheat by containing a trimethyllysyl residue at position 14. Thus, tryptic proteolysis occurred at lysine-18 rather than lysine-14 as with the spinach and wheat enzymes. A comparison of the DNA sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase indicates that the N terminus has been post-translationally processed by removal of methionine-1 and serine-2 followed by acetylation of proline-3. In addition, for the enzyme from tobacco and muskmelon a third post-translational modification occurs at lysine-14 in the form of NE-trimethylation.Ribulose bisphosphate carboxylase/oxygenase [RbuP2 carboxylase, 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] is a hexadecameric plant protein with eight large subunits (LSs) and eight small subunits (SSs) (for a recent review, see ref.

Protein Methylation in Pea Chloroplasts

“…Hydrolysis of aspartate and glutamate methylesters produces methanol. Base-stable methyl-accepting sites are arginine (30), histidine (18), and lysine (12,19). We found both base-labile and base-stable linkages; under our labeling conditions 20% or less of the incorporated methyl groups are base labile.…”

“…Black et al (5) reported that 60 to 70% of the label incorporated into spinach chloroplast proteins was base labile. This discrepancy in the amounts of base-labile and base-stable linkages in spinach and pea chloroplast proteins may result from the lack of methylated lysine residues in the N-terminal region of spinach RuBPCase (12), from the different methods used to measure the amount of base-labile and base-stable linkages, or from the different labeling and washing methods used.…”

“…Houtz et al. (12) reported the methylation of specific lysine residues in the N-terminal region of the large subunit in several other species. The stromal fraction also contains a polypeptide ofmolecular mass about 24 kD that is methylated more in the light than in the dark (Fig.…”

“…At low concentrations of AdoMet, however, they concluded that the methyl-transfer reactions appear to be more specific with respect to their substrates. The only identified substrates for posttranslational methylation in chloroplasts are the large and small subunits of RuBPCase as described by Black et al (5) and Houtz et al (12). The role of specific protein methylation in plants is not known.…”

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

“…LSU seems to be especially vulnerable at this site. Other investigators described that during in vitro treatment of Rubisco with trypsin and the endoproteinase Lys C, proteolysis occurred at Lys 14 (Gutteridge et al 1986;Mulligan et al 1988;Houtz et al 1989). This proteolytic event caused a loss of carboxylase and oxygenase activities without destroying the quaternary structure and without disrupting the substrate binding (Gutteridge et al 1986;Mulligan et al 1988).…”