Conjugation of Ubiquitin to Proteins from Green Plant Tissues

Abstract

Conjugation of the polypeptide ubiquitin to endogenous proteins was studied in oat (Avena sativa L.) plants, and particularly in green tissues. Conjugating activity in leaf extracts was different from that in root extracts, and in both was less than in etiolated tissue. The conjugates were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and their formation was both time-and ATP-dependent and had a pH optimum of about 8.2. The assay had a high affinity for ATP with a probable Km of less than 50 micromolar. The ubiquitin conjugating system was also shown to be present in isolated chloroplasts, and ubiquitin could be conjugated to endogenous proteins of lyzed chloroplasts in which the ATP concentrations were reduced by preincubation or desalting. SDS-PAGE analysis led to the suggestion that the large and small subunits of ribulose-1,5-bisphosphate carboxylase (RuBPCase) may be able to be ubiquitinated, and we have shown that ubiquitin can stimulate the in vitro breakdown of 191-labeled RuBPCase. These results invite the speculation that ubiquitin may be involved in the regulation of protein turnover in green plants.

Isolation and characterization of tomato cDNA and genomic clones encoding the ubiquitin gene ubi3

“…If the proteins are ubiquitinated, it will be interesting to identify whether they are all nuclear-encoded. If chloroplast-encoded proteins are also ubiquitinated, it would be consistent with the recent report that lysed chloroplasts are capable of ubiquitinating chloroplast polypeptides [35]. The authors of this report speculated that a ubiquitin conjugating system may be involved in regulating protein turnover in chloroplasts.…”

Protein Synthesis and Breakdown during Heat Shock of Cultured Pear (Pyrus communis L.) Cells

“…The papers were then washed, twice with 10% cold TCA (10 min), twice with 5% cold TCA (5 min), and twice with cold ethanol(5 min), dried, and counted by liquid scintillation spectrophotometry as described above. The same TCA-precipitable cpm were loaded onto lanes of 13% polyacrylamide gels, and proteins were separated by SDS-PAGE, stained, and autoradiographed as described by Veierskov and Ferguson (1991).…”

The stroma of higher plant plastids contain ClpP and ClpC, functional homologs of Escherichia coli ClpP and ClpA: an archetypal two-component ATP-dependent protease.

“…The energy-dependem plastid protease responsible for degrading such newly synthesized but unassembled chloroplast polypeptides remains to be identified. One candidate was the ubiquitin system, because severa1 groups reported ubiquitin conjugation activity in chloroplast extracts (Wettern et al, 1990;Veierskov and Ferguson, 1991).…”