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.
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 alpha 3 and beta 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.
Basset
Gilles
G
Unité de Physiologie Végétale, Institut National de la Recherche Agronomique, Centre de Recherche de Bordeaux, Boîte Postale 81, 33883 Villenave d'Ornon cedex, France.
Raymond
Philippe
P
Malek
Lada
L
Brouquisse
Renaud
R
D002244:Carbon / Q000172:deficiency* / Q000494:pharmacology; D002918:Chymotrypsin / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D006834:Hydrazines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D018517:Plant Roots / Q000201:enzymology* / Q000235:genetics; D046988:Proteasome Endopeptidase Complex; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014357:Trypsin / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
D006834:Hydrazines; D009097:Multienzyme Complexes; D012333:RNA, Messenger; C446799:dinitrophenylhydrazine; D002244:Carbon; D002918:Chymotrypsin; D014357:Trypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex