Capturing the Asc1p/Receptor for Activated C Kinase 1 (RACK1) Microenvironment at the Head Region of the 40S Ribosome with Quantitative BioID in Yeast
Capturing the Asc1p/Receptor for Activated C Kinase 1 (RACK1) Microenvironment at the Head Region of the 40S Ribosome with Quantitative BioID in Yeast
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DOI：
10.1074/mcp.m116.066654
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发表时间：
2017-12-01
影响因子：
7
通讯作者：
Valerius, Oliver
中科院分区：
文献类型：
--
作者：
Opitz, Nadine;Schmitt, Kerstin;Valerius, Oliver
酿酒酵母的Asc1蛋白是位于核糖体40S头部的一种骨架蛋白，它将mRNA翻译与细胞信号联系起来。在这项研究中，与Asc1p共定位的蛋白质用邻近依赖的生物素鉴定(BioID)进行了鉴定，这是一种首次用于酵母的体内标记技术。生物素化Asc1p-BIRA(STAR)-近端蛋白被鉴定，并与对照应用SILAC和质谱学进行定量验证。MRNA结合蛋白Sro9p和Gis2p与Scp160p一起出现，每一个都为核糖体提供核转录。鉴定了帽结合蛋白eIF4E(Cdc33p)和eIF3/a亚基(Rpg1p)，反映了参与核糖体40S头部mRNA翻译的蛋白质的相遇。出乎意料的是，参与核糖体保存的蛋白质(钳制因子Stm1p)、去泛素化复合体Ubp3p-Bre5p、RNA聚合酶II降解因子1(Def1p)和转录因子(Spt5p、Mbf1p)在指数级生长的细胞中与Asc1p共存。对于Asc1(R38D，K40E)p，一个被认为缺乏与核糖体结合的变体，BioID揭示了其主要的核糖体定位。葡萄糖耗竭取代了大多数Asc1p共定位蛋白而产生了额外的核糖体蛋白，这表明在营养限制的早期，核糖体的聚集过程可能伴随着核糖体亚基的钳制。总体而言，用BioID对Asc1p微环境的表征证实并证实了我们最近的发现，即β-推进器广泛地参与了影响共定位蛋白(例如Bre5p)磷酸化的信号转导，并由此可能影响核基因转录和核糖体的命运。
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(star)-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 Asc1(R38D,K40E) p, a variant considered to be deficient in bindingto 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 beta-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.