Changes in the Expression and the Enzymic Properties of the 20S Proteasome in Sugar-Starved Maize Roots. Evidence for an in Vivo Oxidation of the Proteasome

Abstract

The 20S proteasome (multicatalytic proteinase) was purified from maize (Zea mays L. cv DEA 1992) roots through a five-step procedure. After biochemical characterization, it was shown to be similar to most eukaryotic proteasomes. We investigated the involvement of the 20S proteasome in the response to carbon starvation in excised maize root tips. Using polyclonal antibodies, we showed that the amount of proteasome increased in 24-h-carbon-starved root tips compared with freshly excised tips, whereas the mRNA levels of ␣3 and ␤6 subunits of 20S proteasome decreased. Moreover, in carbon-starved tissues, chymotrypsin-like and caseinolytic activities of the 20S proteasome were found to increase, whereas trypsin-like activities decreased. The measurement of specific activities and kinetic parameters of 20S proteasome purified from 24-h-starved root tips suggested that it was subjected to posttranslational modifications. Using dinitrophenylhydrazine, a carbonyl-specific reagent, we observed an increase in carbonyl residues in 20S proteasome purified from starved root tips. This means that 20S proteasome was oxidized during starvation treatment. Moreover, an in vitro mild oxidative treatment of 20S proteasome from non-starved material resulted in the activation of chymotrypsin-like, peptidyl-glutamyl-peptide hydrolase and caseinolytic-specific activities and in the inhibition of trypsin-like specific activities, similar to that observed for proteasome from starved root tips. Our results provide the first evidence, to our knowledge, for an in vivo carbonylation of the 20S proteasome. They suggest that sugar deprivation induces an oxidative stress, and that oxidized 20S proteasome could be associated to the degradation of oxidatively damaged proteins in carbon starvation situations.Living organisms are subjected to numerous biotic or abiotic stresses. Daily, at a cellular level, changing environmental growth conditions trigger the synthesis of new sets of proteins necessary for the acclimation response, and the degradation of regulatory proteins, damaged proteins, and proteins that have become useless. Thus, in plant cells subjected to carbon starvation, the activity of enzymes involved in sugar metabolism and respiration (Journet et al., 1986;Brouquisse et al., 1991;Irving and Hurst, 1993), nitrogen reduction and assimilation Peeters and Van Laere, 1992), regulation of cell division and growth (Chevalier et al., 1996), or protein synthesis (Webster and Henry, 1987;Tassi et al., 1992) decreases and, in most cases, the corresponding proteins are likely subjected to proteolysis. In contrast, the activity of enzymes related to the catabolism of proteins (Tassi et al., 1992;James et al., 1993James et al., , 1996Chevalier et al., 1995;Moriyasu and Ohsumi, 1996), amino acids , or lipids (Dieuaide et al., 1992;Ismail et al., 1997) increases. Genes encoding enzymes involved in protein and lipid catabolism have been shown to be induced by sugar depletion (Koch, 1996), and it is clear that the selective synthesis and degradation of...

Arabidopsis thaliana26S Proteasome Subunits RPT2a and RPT5a Are Crucial for Zinc Deficiency-Tolerance

“…Oxidized proteins are known to be a substrate for Ub-independent 20SP proteolysis. 1,11,16) In our study, 20SP activity was elevated under the Zn-deficient condition both in the wild-type and in the rpt mutants, and the increase in activity was more evident in the rpt mutant (Fig. 5A).…”

“…1) Oxidation of 20SP elevates the proteolytic activity of 20SP. 11) It is also known that under carbon starvation, the activity of 20SP is elevated together with an accumulation of oxidatively damaged proteins. 11) Zinc (Zn) deficiency induces oxidative stress [12][13][14][15] as in other forms of nutrient starvations, and under these conditions, it is likely that protein oxidation is accelerated.…”

Evidence for the Existence in Arabidopsis thaliana of the Proteasome Proteolytic Pathway

“…Both the 20 S and 26 S proteasomes were characterized at molecular (21,22) and biochemical levels (23) in Arabidopsis thaliana. As observed in animal and yeast cells, plant proteasome has been found to be up-regulated, at transcriptional or translational levels, in oxidative conditions such as carbon starvation (24) or cadmium stress (25)(26)(27)(28), and its involvement in the response to oxidative stress has been postulated. In plants, the preferential involvement of the 20 S proteasome in the degradation of oxidized proteins was recently supported by in vivo experiments using A. thaliana mutants (29).…”

“…Immunoprecipitation Experiments-Polyclonal antibodies raised against the maize 20 S proteasome (24) and tomato LAP (35) were used for immunoprecipitation experiments. Serum IgG fractions were purified using 1 ml of HiTrap protein A column (Amersham Biosciences) as recommended by the manufacturer.…”

“…Immunoprecipitation protocol was adapted from Ref. 24. Briefly, 100 l of either clarified extract or partially purified fractions were incubated 1 h at 4°C with increasing volumes of purified immune or preimmune serum.…”