a-new-approach-for-comparative-analysis-multiprotein-complexes-based

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on <sup>15</sup>N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

University of Münster

Carnegie Institution for Science

The versatile metabolism of the green alga Chlamydomonas reinhardtii is reflected in its complex response to anaerobic conditions. The anaerobic response is also remarkable in the context of renewable energy because C. reinhardtii is able to produce hydrogen under anaerobic conditions. To identify proteins involved during anaerobic acclimation as well as to localize proteins and pathways to the powerhouses of the cell, chloroplasts and mitochondria from C. reinhardtii in aerobic and anaerobic (induced by 8 h of argon bubbling) conditions were isolated and analyzed using comparative proteomics. A total of 2315 proteins were identified. Further analysis based on spectral counting clearly localized 606 of these proteins to the chloroplast, including many proteins of the fermentative metabolism. Comparative quantitative analyses were performed with the chloroplast-localized proteins using stable isotopic labeling of amino acids ([(13)C(6)]arginine/[(12)C(6)]arginine in an arginine auxotrophic strain). The quantitative data confirmed proteins previously characterized as induced at the transcript level as well as identified several new proteins of unknown function induced under anaerobic conditions. These proteins of unknown function provide new candidates for further investigation, which could bring insights for the engineering of hydrogen-producing alga strains.

Characterizing the anaerobic response of Chlamydomonas reinhardtii by quantitative proteomics.

The use and development of post-genomic tools naturally depends on large-scale genome sequencing projects. The usefulness of post-genomic applications is dependent on the accuracy of genome annotations, for which the correct identification of intron-exon borders in complex genomes of eukaryotic organisms is often an error-prone task. Although automated algorithms for predicting intron-exon structures are available, supporting exon evidence is necessary to achieve comprehensive genome annotation. Besides cDNA and EST support, peptides identified via MS/MS can be used as extrinsic evidence in a proteogenomic approach. We describe an improved version of the Genomic Peptide Finder (GPF), which aligns de novo predicted amino acid sequences to the genomic DNA sequence of an organism while correcting for peptide sequencing errors and accounting for the possibility of splicing. We have coupled GPF and the gene finding program AUGUSTUS in a way that provides automatic structural annotations of the Chlamydomonas reinhardtii genome, using highly unbiased GPF evidence. A comparison of the AUGUSTUS gene set incorporating GPF evidence to the standard JGI FM4 (Filtered Models 4) gene set reveals 932 GPF peptides that are not contained in the Filtered Models 4 gene set. Furthermore, the GPF evidence improved the AUGUSTUS gene models by altering 65 gene models and adding three previously unidentified genes.

Concerted action of the new Genomic Peptide Finder and AUGUSTUS allows for automated proteogenomic annotation of the Chlamydomonas reinhardtii genome.

The unicellular green alga Chlamydomonas reinhardtii has emerged to be an important model organism for the study of oxygenic eukaryotic photosynthesis as well as other processes occurring in the chloroplast. However, the chloroplast proteome in C. reinhardtii has only recently been comprehensively characterized, made possible by proteomics emerging as an accessible and powerful tool over the last decade. In this review, we introduce a compiled list of 996 experimentally chloroplast-localized proteins for C. reinhardtii, stemming largely from our previous proteomic dataset comparing chloroplasts and mitochondria samples to localize proteins. In order to get a taste of some cellular functions taking place in the C. reinhardtii chloroplast, we will focus this review particularly on metabolic differences between chloroplasts of C. reinhardtii and higher plants. Areas that will be covered are photosynthesis, chlorophyll biosynthesis, carbon metabolism, fermentative metabolism, ferredoxins and ferredoxin-interacting proteins.

The chloroplast proteome: a survey from the Chlamydomonas reinhardtii perspective with a focus on distinctive features.

Cyclic photosynthetic electron flow (CEF) is crucial to photosynthesis because it participates in the control of chloroplast energy and redox metabolism, and it is particularly induced under adverse environmental conditions. Here we report that down-regulation of the chloroplast localized Ca(2+) sensor (CAS) protein by an RNAi approach in Chlamydomonas reinhardtii results in strong inhibition of CEF under anoxia. Importantly, this inhibition is rescued by an increase in the extracellular Ca(2+) concentration, inferring that CEF is Ca(2+)-dependent. Furthermore, we identified a protein, anaerobic response 1 (ANR1), that is also required for effective acclimation to anaerobiosis. Depletion of ANR1 by artificial microRNA expression mimics the CAS-depletion phenotype, and under anaerobic conditions the two proteins coexist within a large active photosystem I-cytochrome b(6)/f complex. Moreover, we provide evidence that CAS and ANR1 interact with each other as well as with PGR5-Like 1 (PGRL1) in vivo. Overall our data establish a Ca(2+)-dependent regulation of CEF via the combined function of ANR1, CAS, and PGRL1, associated with each other in a multiprotein complex.

Mia Terashima

Institute of Plant Biology and Biotechnology

University of Münster

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on ¹⁵N Metabolic Labeling and Quantitative Mass Spectrometry

Mia Terashima

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Plant Biology and Biotechnology

University of Münster

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

Institute of Plant Biology and Biotechnology

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on ¹⁵N Metabolic Labeling and Quantitative Mass Spectrometry