Ribosomal Asc1p/RACK1 in the phosphorylation signaling network of Saccharomyces cerevisiae
von Kerstin Schmitt
Datum der mündl. Prüfung:2016-02-17
Betreuer:Dr. Oliver Valerius
Gutachter:Dr. Oliver Valerius
Gutachter:Prof. Dr. Heike Krebber
EnglischThe WD40 scaffold protein Asc1 of Saccharomyces cerevisiae solely consists of a seven-bladed β propeller. The protein is highly conserved among eukaryotes and known as RACK1 in metazoans and plants. Asc1p/RACK1 localizes to the head of the 40S ribosomal subunit next to the mRNA exit channel and associates with proteins of signal transduction pathways, thus providing a link between cellular signaling and mRNA translation. In this work, phosphorylation of Asc1p was analyzed by mass spectrometry resulting in the identification of at least three previously unknown phosphorylation sites. To analyze the impact of phosphorylation on the functionality of the protein, Asc1p phospho-site mutant strains were generated for these residues as well as for five additional phospho-sites known from high-throughput studies. Phenotypic characterization showed slightly increased sensitivity to translation inhibitors caused by dephospho-mimics at T143 and Y250, whereas all other phospho-site mutations caused no obvious effect. However, phosphorylation of T12, T96, T99, and especially T143 is required to maintain the abundance of the ribosome binding-compromised Asc1DEp variant. According to the reduced level of the dephospho-mimicking DE-variants, the respective mutant strains show asc1- phenotypes. Mutation of Y250 did not change Asc1DEp abundance, but also resulted in asc1- phenotypes with respect to cycloheximide sensitivity. Asc1p has a strong impact on the cellular phospho-proteome. A SILAC-based LC MS approach identified almost 300 Asc1p-sensitive phosphorylation sites in more than 200 proteins that are mainly involved in translation and signal transduction but also other processes. Identification of Asc1p-associated proteins, including, for instance, mRNA-binding proteins, further corroborate Asc1p’s involvement in some of these processes, such as mRNA translation. With RNASeq analysis of polysome-derived mRNAs the translatomes of wild-type ASC1 and asc1- strains were compared. Among 73 transcripts of differentially translated mRNAs, the YHR177W mRNA is very remarkable as the total level of the transcript significantly increases, whereas its polysome association decreases. Yhr177wp belongs to the fungal-specific family of WOPR transcription factors that regulate morphology and development. Altogether, this work verifies the conserved β-propeller Asc1p as an important rack-wheel within cellular adaptation, signaling, and process coordination.
Keywords: Asc1; RACK1; Saccharomyces cerevisiae; protein phosphorylation; quantitative proteomics; ribosome; signal transduction network