Degradation of PsbO by Synechocystis Deg proteases

IRMA N. ROBERTS

Umea

Workshop; Umeå Renewable Energy Workshop; 2010

KBC, Universidad de Umea

Degradation of PsbO by Synechocystis Deg proteases The unicellular cyanobacterium Synechocystis sp. PCC6803 contains three Deg proteases (HhoA, HhoB and HtrA) with high sequence homology to the luminal located Deg proteases of Arabidopsis (Kieselbach & Funk 2003, Huesgen et al. 2005). As yet, their physiological role in oxygenic photosynthetic organisms is unclear, although it has been widely speculated that they participate in the PSII repair cycle (Kapri-Pardes et al. 2007). Accumulating evidence suggests that the reduction/oxidation (redox) regulation by the ferredoxin/thioredoxin system plays an important role in the light-mediated regulation of the activity of chloroplasts enzymes. This redox regulation operates through the modification of the redox state of thiol groups of cysteine residues in target proteins. Both PsbO1 and PsbO2 from Arabidopsis have been demonstrated to be protein targets for thioredoxin in vitro (Marchand et al. 2006). Here, in order to investigate the ability of the Synechocystis Deg proteases to degrade PsbO according to its redox state we performed an in vitro degradation assay in which a sample of purified PsbO or PSII complex was incubated together with one of the three Synechocystis Deg proteases in reducing conditions. The reducing media was conferred by the E. coli thioredoxin/thioredoxin reductase system. The results obtained showed that HhoA is able to hydrolyze PsbO under reducing conditions while HhoB and HtrA are not. Four major degradation fragments were detected by SDS-PAGE and western blot analysis already after 2 h of incubation when PsbO isolated from spinach leaves was used as a substrate. No degradation fragments were detected when the substrate and the enzymes were incubated in the absence of the thioredoxin/thioredoxin reductase system or in the presence of an incomplete version of it. HhoA was active against purified PsbO (from spinach or Synechocystis) as well as PsbO attached to the PSII complex. These results confirm the character of PsbO as a protein target of thioredoxin in the thylakoid lumen of Synechocystis and show that its conversion to a reduced form is a prerequisite for it to be recognized and degraded by the HhoA.