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Protein N-terminal (Nt) acetylation is one of the most abundant modifications in eukaryotes, covering ∼50-80 % of the proteome, depending on species. Cells with defective Nt-acetylation display a wide array of phenotypes such as impaired growth, mating defects and increased stress sensitivity. However, the pleiotropic nature of these effects has hampered our understanding of the functional impact of protein Nt-acetylation. The main enzyme responsible for Nt-acetylation throughout the eukaryotic kingdom is the N-terminal acetyltransferase NatA. Here we employed a multi-dimensional proteomics approach to analyzeSaccharomyces cerevisiaelacking NatA activity, which caused global proteome remodeling. Pulsed-SILAC experiments revealed that NatA-deficient strains consistently increased degradation of ribosomal proteins compared to wild type. Explaining this phenomenon, thermal proteome profiling uncovered decreased thermostability of ribosomes in NatA-knockouts. Our data are in agreement with a role for Nt-acetylation in promoting stability for parts of the proteome by enhancing the avidity of protein-protein interactions and folding.TeaserA multidimensional proteomics approach reveals the effect of N-terminal acetylation onSaccharomyces cerevisiaecytosolic ribosomal proteins.

Dynamic change in subcellular localization of signaling proteins is a general concept that eukaryotic cells evolved for eliciting a coordinated response to stimuli. Mass spectrometry (MS)-based proteomics in combination with subcellular fractionation can provide comprehensive maps of spatio-temporal regulation of cells, but involves laborious workflows that does not cover the phospho-proteome level. Here we present a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions. We benchmarked the workflow by studying spatio-temporal EGFR phospho-signaling dynamics in-vitro in HeLa cells and in-vivo in mouse tissues. Finally, we investigated the spatio-temporal stress signaling, revealing cellular relocation of ribosomal proteins in response to hypertonicity and muscle contraction. Proteomics data generated in this study can be explored through https://SpatialProteoDynamics.github.io.

The 5′ cap structure of eukaryotic mRNA is critical for its processing, transport, translation, and stability. The many functions of the cap and the fact that most, if not all, mRNA carries the same type of cap makes it difficult to analyze cap function in vivo at individual steps of gene expression. We have used the lariat capping ribozyme (LCrz) from the myxomycete Didymium to replace the mRNA m7G cap of a single reporter mRNA species with a tiny lariat in which the first and the third nucleotide are joined by a 2′, 5′ phosphodiester bond. We show that the ribozyme functions in vivo in the budding yeast Saccharomyces cerevisiae presumably without cofactors and that lariat capping occurs cotranscriptionally. The lariat-capped reporter mRNA is efficiently exported to the cytoplasm where it is found to be oligoadenylated and evenly distributed. Both the oligoadenylated form and a lariat-capped mRNA with a templated poly(A) tail translates poorly, underlining the critical importance of the m7G cap in translation. Finally, the lariat-capped RNA exhibits a threefold longer half-life compared to its m7G-capped counterpart, consistent with a key role for the m7G cap in mRNA turnover. Our study emphasizes important activities of the m7G cap and suggests new utilities of lariat capping as a molecular tool in vivo.