Proteolysis is crucial for the maturation, regulation and recycling of the chloroplast proteome. Although several dozen chloroplast proteases are known, information concerning their substrates and functions is limited. In particular, little is known about the structural features of substrates that trigger their proteolysis. Most chloroplast proteins are nuclear encoded and are targeted through an N-terminal chloroplast transit peptide (cTP) that is removed by stromal processing peptidase (SPP).

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... To better understand proteolytic maturation, the soluble N-terminal proteome of the Arabidopsis thaliana chloroplast was characterized. A cTP cleavage motif was observed that suggests other peptidases, in addition to SPP, are involved in chloroplast protein maturation. There was a clear preference for small uncharged amino acids at the processed protein N-terminus suggesting the existence of a chloroplast specific 'N-end rule'. The soluble chloroplast peptidases PREP and OOP have been shown to degrade small polypeptides in vitro and are thought to be responsible for removal of cTP fragments and other degradation products. The CLP protease system can degrade intact protein substrates with the aid of ATP dependent (AAA+) CLPC chaperones that unfold and feed substrates into the CLP proteolytic core. An array of proteomic tools were used to compare Arabidopsis mutants deficient in the above peptidases with wild type. ii Degradation products, including cTPs, were found to accumulate in peptidase mutants indicative, of rate-limited or blocked degradation pathways. Incomplete or altered Nterminal maturation for chloroplast proteins was dependent on the type and severity of the peptidase deficiency. These results provide molecular details to help explain dwarf, chlorotic mutant phenotypes and demonstrate the interplay between protein import, proteolytic processing and the downstream degradation of damaged or unwanted proteins in the chloroplast. Substrate and sequence cleavage specificity was determined for soluble chloroplast glutamyl-endopeptidase (CGEP) and the plastoglobule localized metallopeptidase PGM48. Structural models were used to predict peptidase substrate binding mechanisms. iii BIOGRAPHICAL SKETCH