| dc.description.abstracteng | The conserved ribosome-associated Asc1 protein of Saccharomyces cerevisiae forms a seven bladed β-propeller and binds to the head region of the 40S ribosomal subunit in close proximity to the mRNA exit tunnel. Asc1p is considered a scaffold protein organizing the local microenvironment near the area of translation initiation at the ribosome to link mRNA translation with cellular signaling. In this study, proteins in the direct proximity of Asc1p were discovered with the in vivo protein labeling technique proximity-dependent Biotin IDentification (BioID) in combination with high-resolution mass spectrometry. The RNA polymerase II degradation factor Def1p and the ribosomal clamping factor Stm1p as well as mRNA-binding proteins and translational/transcriptional regulators appeared as Asc1p-proximal proteins during exponential cell growth. In addition to mRNA-binding proteins close to Asc1p at exponential growth such as Scp160p, Sro9p and Gis2p, mild heat stress increasingly attracts further mRNA-binding proteins, namely Hek2p, New1p, and Psp2p into the Asc1p proximity. Phenotypes caused by the synthetic deletion of the SCP160 or STM1 gene in asc1- cells also revealed genetic interactions. Additionally, proteins required for mRNP granule formation and activity reflect Asc1p’s impact on P-body homeostasis. Starvation for glucose caused severe changes in the Asc1p-neighborhood: Ribosomal proteins of the small and the large ribosomal subunit, and the ubiquinone biosynthetic enzyme Coq5p accumulated in the Asc1p proximity and replaced most of the proteins present during exponential growth. These changes might reflect a general aggregation of ribosomes at glucose starvation, and might indicate a channeling of translation capacities towards mitochondria. The Asc1R38D K40E protein variant was described to be ribosome-binding compromised, however, was revealed here as capable to bind the ribosome in vivo. Additional proximal proteins suggest either a subpopulation of Asc1R38D K40Ep apart from the ribosome or spatial flexibility of the protein at the ribosome. To study physical Asc1p-protein interactions in further detail, ASC1 alleles were constructed for the site-specific incorporation of the photo-reactive amino acid Bpa as cross-linker into the Asc1 protein at different sites. Future in vitro cross-linking experiments of these Asc1Bpap variants with some of the identified proximal proteins will lead to detailed knowledge about the nature of the interactions. Overall, the Asc1p microenvironment suggests that the
β-propeller not only coordinates protein biosynthesis with cellular signaling, but beyond that synchronizes these processes with nuclear mRNA synthesis. | de |