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Molecular & Cellular Proteomics 8:1789-1810, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Large Scale Comparative Proteomics of a Chloroplast Clp Protease Mutant Reveals Folding Stress, Altered Protein Homeostasis, and Feedback Regulation of Metabolism^*,

Boris Zybailov, Giulia Friso, Jitae Kim, Andrea Rudella^,, Verenice Ramírez Rodríguez^,¶, Yukari Asakura, Qi Sun^|| and Klaas J. van Wijk^,**

From the Department of Plant Biology and
||Computation Biology Service Unit, Cornell University, Ithaca, New York 14853

The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex. To understand the role of Clp proteases in plastid biogenesis and homeostasis, leaf proteomes of young seedlings of clpr2-1 and wild type were compared using large scale mass spectrometry-based quantification using an LTQ-Orbitrap and spectral counting with significance determined by G-tests. Virtually only chloroplast-localized proteins were significantly affected, indicating that the molecular phenotype was confined to the chloroplast. A comparative chloroplast stromal proteome analysis of fully developed plants was used to complement the data set. Chloroplast unfoldase ClpB3 was strongly up-regulated in both young and mature leaves, suggesting widespread and persistent protein folding stress. The importance of ClpB3 in the clp2-1 mutant was demonstrated by the observation that a CLPR2 and CLPB3 double mutant was seedling-lethal. The observed up-regulation of chloroplast chaperones and protein sorting components further illustrated destabilization of protein homeostasis. Delayed rRNA processing and up-regulation of a chloroplast DEAD box RNA helicase and polynucleotide phosphorylase, but no significant change in accumulation of ribosomal subunits, suggested a bottleneck in ribosome assembly or RNA metabolism. Strong up-regulation of a chloroplast translational regulator TypA/BipA GTPase suggested a specific response in plastid gene expression to the distorted homeostasis. The stromal proteases PreP1,2 were up-regulated, likely constituting compensation for reduced Clp protease activity and possibly shared substrates between the ClpP and PreP protease systems. The thylakoid photosynthetic apparatus was decreased in the seedlings, whereas several structural thylakoid-associated plastoglobular proteins were strongly up-regulated. Two thylakoid-associated reductases involved in isoprenoid and chlorophyll synthesis were up-regulated reflecting feedback from rate-limiting photosynthetic electron transport. We discuss the quantitative proteomics data and the role of Clp proteolysis using a "systems view" of chloroplast homeostasis and metabolism and provide testable hypotheses and putative substrates to further determine the significance of Clp-driven proteolysis.

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