Dynamics and composition of the chloroplast translation apparatus on thylakoid membranes in Chlamydomonas reinhardtii
Felix Willmund – Frederik Sommer/Michael Schroda – Timo Mühlhaus
Thylakoid membranes are highly organized and accommodate many essential multisubunit complexes involved in the light reactions of photosynthesis. All major complexes are of heterogenic origin, comprising subunits encoded in the nucleus and subunits originating from the plastidic genome itself. Targeting of nucleus-encoded proteins to the thylakoid membrane is posttranslational, and their translocation and integration into thylakoid membranes is well studied. Here, thylakoid import and membrane integration is mediated via the four machineries cpSec, cpTAT, spSRP and ALB31. In contrast, little is known about the analogous issues for plastid-encoded proteins which seems to occur cotranslational. For many years, chloroplast ribosomes were observed to associate with thylakoid membranes in a light-dependent manner. Recently it was shown, that translation initiates off the thylakoid membrane and that ribosomes synthesizing membrane proteins attach to the membrane once the first transmembrane segment in the nascent peptide emerges from the ribosome2. However, little is known about factors mediating this relocation. One obvious factor is cpSRP54, which was observed to bind nascent PsbA3 but the mild phenotype of cpSRP54 Arabidopsis mutants indicates that cpSRP54 is not the only essential factor for the targeting of plastid-encoded proteins4. Thus, many open questions remain: Which factors associate with translating chloroplast ribosomes to relocate them to thylakoid membranes? Which channels and receptor proteins are involved in cotranslational protein integration? How are the translational dynamics during thylakoid relocation?
1. Proteomics of thylakoid associated ribosomes: The goal is, to identify proteins assisting the cotranslation integration of chloroplast-encoded proteins on thylakoid membranes: We are currently in the progress to tag C. reinhardtii ribosomal proteins encoded within the chloroplast genome for specific affinity purification of active chloroplast ribosome-complexes and their subsequent proteomic analysis. With this tool we plan to identify (novel) factors that associate with translating ribosomes during protein synthesis of thylakoid membrane proteins. In addition, it is planned to crosslink cell-lysates and subsequently lyse thylakoid membranes with mild detergents, in order to identify potential dogging proteins and membrane pores that co-purify with ribosomes. Thus, possible channel and receptor proteins responsible for the insertion of newly-translated proteins into the membranes should co-purify. Here a close collaboration with theoretical groups within BioComp is important to dissect true ribosome associating factors from false positive proteins which might co-purify during the purification.
2. Ribosome-profiling in Chlamydomonas cells. We have already established ribosome profiling assays with tilling-microarrays which allows the detailed investigation of the chloroplast translation apparatus as the limited number of translated chloroplast transcripts can be easily spotted as short overlapping sequences on microarrays. The project aims to take the next step for the genome-wide study of plant acclimation by establishing deep-sequencing based ribosome profiling of whole-cellular Chlamydomonas ribosomes (Ribo-Seq). While many methodological steps are already established for the array approach, the bioinformatic analysis is by far more challenging. Here, millions of 28-30nt long ribosome-footprint reads need to be specifically mapped to the whole cellular transcriptome. For this evaluation, it is essential to closely collaborate with theoretical groups to elaborate this technique step by step. Initially the analysis of the dataset will be restricted to chloroplast translation with focus on translation of thylakoid membrane proteins. By this Ribo-Seq can be compared to the microarray data and add codon-resolution insight into the dynamics of chloroplast translation. For example, it will be extremely interesting to see if ribosomes pause during relocation, analog to the bacterial system. In subsequent steps, the evaluation will be stepwise extended to the analysis of the genome-wide dataset.
1. Celedon and Cline (2013) Biochim Biophys Acta 1833(2): 341–351.
2. Nilsson et al., (1999). EMBO J 18(3):733–742.
3. Zoschke and Barkan (2015) PNAS 112(13):E1678-87
4. Tzvetkova-Chevolleau et al. (2007) Plant Cell 19(5):1635–1648.