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FUNCTIONAL CHARACTERIZATION OF THE CLP PROTEASE SYSTEM IN ARABIDOPSIS CHLOROPLASTS THROUGH REVERSE GENETICS AND PROTEOMICS
Proteases play an important role in regulating protein maturation, activity and life-time. The Clp protease system in Arabidopsis thaliana plastids accumulates at relatively high levels and consists of a proteolytic core and associated chaperones. The core is an assembly of five different catalytic ClpP subunits, four non-catalytic ClpR subunits, and two ClpS proteins with unknown function. ClpR,S are unique to photosynthetic organisms. Three ATP-dependent chaperones, ClpC1,C2,D, are expected to deliver substrates to the ClpPRS core. Control of Clp activity is not understood and Clp substrates are unknown. Arabidopsis T-DNA insertion Clp mutants were isolated and genotyped. Null mutants for ClpP4,P5 are embryo-lethal under both auto- and heterotrophic conditions. Mutants of ClpP3,R4 did not form seedlings under autotrophic conditions but developed albino seedlings under heterotrophic conditions, displaying limited greening under low light. Null mutants for the chaperones ClpC1 and ClpD have pale-green and wild-type phenotypes, respectively. ClpP,R core subunits are likely essential, while there are redundancies in the ClpC,D subfamily. Two mutants with partial loss of gene expression for ClpR1 and ClpR2 (clpr2-1) exhibited pale-green phenotypes, with clpr2-1 having a stronger phenotype. ClpR2 protein accumulation in clpr2-1 chloroplasts was 5-fold reduced, while the ClpPRS core was 3-fold downregulated, suggesting an induction of core composition heterogeneity. Stromal chaperones were upregulated several fold and ClpC was recruited to the thylakoid membrane. Thylakoid protein homeostasis was unbalanced as deduced from increased accumulation of thylakoid proteases, plastoglobules, protein precursors and degradation products. Clpr2-1 chloroplasts were smaller, with 30% less thylakoids than wild-type. Clearly, ClpR2 is not a redundant member of the Clp family and reduced CLPR2 gene expression has adverse effects on plastid and plant development. A comparative proteome analysis using differential stable isotope labeling of clpr2-1 and wild-type stroma identified 298 proteins, and 113 were quantified. The Calvin cycle was down-regulated, explaining the slower development of clpr2-1. The most striking response was the high accumulation of the chloroplast protein translation machinery and chaperones. This suggests that the ClpPRS core complex may be involved in regulation of plastid gene expression, providing a first understanding of the functional role of the Clp family in plastids.
Clp protease; Arabidopsis thaliana; comparative proteomics; reverse genetics
dissertation or thesis