Spatial distribution of protein translation and of protein translocation in the yeast Saccharomyces cerevisiae

• Applicant: Professor Dr. Nils Johnsson
• Subject Area: Cell Biology
• Term: from 2017 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 343927390

Spatial distribution of protein translation and of protein translocation in the yeast Saccharomyces cerevisiaeThe concept that proteins are synthesized and possibly translocated at the places where they later act is still quite new but applies to a growing list of proteins. The best known and best understood pathways of local translation are those that recognize short motifs in mRNAs to deliver them by active transport to the sites of protein synthesis. However, there might exist other mechanisms of localized translation that do not depend on the positional information in mRNAs. The proof of the existence of these pathways is difficult as the newly made proteins will immediately mix with the already existing pool of old molecules. In this proposal we plan to develop three Split-Ubiquitin (Ub)-based strategies to measure translation and translocation in single cells with high spatial and temporal resolution. In one approach protein synthesis will be measured by tagging the machinery of translation or translocation with the C-terminal half of Ub (Cub) and the nascent polypeptide chain with the N-terminal half of Ub (Nub). Cub will be sandwiched by the autofluorescent mCherry and GFP (CCG) thus prompting the CCG fusion to emit yellow light under the fluorescence microscope. Once the Cub-labelled machine is engaged in the synthesis or translocation of its Nub-labelled substrate the generated proximity between the two Ub-peptides will force them to fold into the native like Ub. The GFP will be instantaneously cleaved off from the Cub and degraded leaving behind translation or translocation proteins that switch their colour from yellow to red. A distinct cellular distribution of red- and yellow-labelled proteins will be specific for the investigated protein and reflect hotspots of its synthesis and/or translocation.In an alternative approach we will measure the proximity of the nascent chain to its own mRNA. The CCG module will be fused to a phage coat protein (MS2CCG) that binds with high affinity to RNA loop sequences that will be integrated into the 3UTR of a mRNA encoding a Nub-fusion protein. Once the Nub is translated it will induce the cleavage of the GFP from the MS2CCG that is bound to its own ribosome-engaged mRNA. Again a local increase in the ratio of mCherry/GFP indicates the site of the synthesis of the Nub fusion protein. A slight reconfiguration of the assay will also enable us to measure the interactions between mRNA and mRNA-binding proteins thus opening a complementary perspective on the targeting and translation of mRNA. By including the results of all three assays we aim to establish a map of translation and translocation that highlights gene-specific hotspots of protein biogenesis.We will establish the method in yeast where the ease of deleting genes will greatly facilitate the identification the determinants involved in local synthesis. However, the technique should be readily applicable to cells of higher eukaryotes.

• DFG Programme: Research Grants