Format

Send to

Choose Destination
Mol Cell Proteomics. 2017 Dec;16(12):2199-2218. doi: 10.1074/mcp.M116.066654. Epub 2017 Oct 5.

Capturing the Asc1p/Receptor for Activated C Kinase 1 (RACK1) Microenvironment at the Head Region of the 40S Ribosome with Quantitative BioID in Yeast.

Author information

1
From the ‡Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Göttingen Center for Molecular Biosciences (GZMB), Georg-August-University Göttingen, 37077 Göttingen, Germany.
2
§Department of Molecular Genetics, Institute of Microbiology and Genetics, GZMB, Georg-August-University Göttingen, 37077 Göttingen, Germany.
3
From the ‡Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Göttingen Center for Molecular Biosciences (GZMB), Georg-August-University Göttingen, 37077 Göttingen, Germany; ovaleri@gwdg.de.

Abstract

The Asc1 protein of Saccharomyces cerevisiae is a scaffold protein at the head region of ribosomal 40S that links mRNA translation to cellular signaling. In this study, proteins that colocalize with Asc1p were identified with proximity-dependent Biotin IDentification (BioID), an in vivo labeling technique described here for the first time for yeast. Biotinylated Asc1p-birA*-proximal proteins were identified and quantitatively verified against controls applying SILAC and mass spectrometry. The mRNA-binding proteins Sro9p and Gis2p appeared together with Scp160p, each providing ribosomes with nuclear transcripts. The cap-binding protein eIF4E (Cdc33p) and the eIF3/a-subunit (Rpg1p) were identified reflecting the encounter of proteins involved in the initiation of mRNA translation at the head region of ribosomal 40S. Unexpectedly, a protein involved in ribosome preservation (the clamping factor Stm1p), the deubiquitylation complex Ubp3p-Bre5p, the RNA polymerase II degradation factor 1 (Def1p), and transcription factors (Spt5p, Mbf1p) colocalize with Asc1p in exponentially growing cells. For Asc1R38D, K40Ep, a variant considered to be deficient in binding to ribosomes, BioID revealed its predominant ribosome localization. Glucose depletion replaced most of the Asc1p colocalizing proteins for additional ribosomal proteins, suggesting a ribosome aggregation process during early nutrient limitation, possibly concomitant with ribosomal subunit clamping. Overall, the characterization of the Asc1p microenvironment with BioID confirmed and substantiated our recent findings that the β-propeller broadly contributes to signal transduction influencing phosphorylation of colocalizing proteins (e.g. of Bre5p), and by that might affect nuclear gene transcription and the fate of ribosomes.

PMID:
28982715
PMCID:
PMC5724181
DOI:
10.1074/mcp.M116.066654
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center