Naslov
Protease networks and proteostasis in plant chloroplasts and mitochondria: an Omics approach to organellar protein maturation, stability and turnover

Autori
Van Wijk, Klaas ; Rowland, Elden ; Majsec, Kristina ; Bhuiyan, Nazmul H. ; Kim, Jitae ; Kumar, Vivek ; Kumari, Sunita ; Waare, Doreen ; Sun, Qi

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, ostalo, znanstveni

Skup
65th ASMS Conference

Mjesto i datum
Indianapolis (IN), Sjedinjene Američke Države, 04.07.2017. - 08.07.2017

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
TAILS ; in vitro cleavage essays ; co-expression networks ; Chloroplasts and mitochondrial proteostasis networks

Sažetak
Introduction Plastids and mitochondria are key cell organelles with dynamic proteomes in photosynthetic eukaryotes. To maintain organelle protein homeostasis (proteostasis), there exist proteostasis networks containing protein chaperones, peptidases and their substrate recognition factors, but many peptidases and their functional connections and substrates are poorly characterized. Multi-disciplinary ‘Omics’ approaches are needed to address the complexity of these proteostasis networks and identify underlying general principles for protein maturation, stability and degradation. Mass-spectrometry is ideally suited for characterization of maturation events, identification of N-terminal stability rules and degradation products and for tracking the dynamic responses of the proteostasis networks at the proteome level. Methods Cleavage specificity was determined by mass spectrometry in vitro using recombinant chloroplast peptidases. In vivo N-termini were determined using a TAILS approach and Nano LC-MS/MS. Quantitative proteomics was supported by spectral counting and spectral intensity measurement. Co-expression networks were constructed from normalized and quality-controlled public microarray datasets. Annotations for protein locations and functions were based on extensive manual curation and the Plant Proteome Database housed by the van Wijk lab at http://ppdb.tc.cornell.edu. Loss-of function mutants and over-expresser lines were generated in Arabidopsis thaliana. Preliminary Data This study provides systematic insight in organellar proteostasis by combining large scale mRNA-based co-expression analysis of some 100 organellar proteolytic components, molecular genetics, quantitative comparative proteomics, TAIL-based N-terminomics and using different protease loss-of-function mutants in Arabidopsis. The distribution of experimentally confirmed plastid and mitochondrial peptidases across MEROPS clans and families was compared to the total peptidase complement and showed striking biases, such as the (near) absence of cysteine and aspartic peptidases, and peptidase inhibitors. In contrast, other protease families were exclusively organellar. Based on mRNA-based co-expression analysis and statistical thresholds for co-localization, we inferred additional plastid peptidases and associate mitochondrial and plastid peptidases with specific processes and developmental states. Protein maturation and protein degradation products were identified by TAILS and MS/MS and provided general rules for N-terminal stability and cleavage site patterns. Single and higher order plastid protease mutants were generated and used to probe the proteostasis network. Novel genetic interactions suggest hierarchical peptidase activities across multiple chloroplast protease systems.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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