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EIF2 interactions with initiator tRNA and eIF2B are regulated by post-translational modifications and conformational dynamics.

ABSTRACT: Translation of messenger RNA (mRNA) into proteins is key to eukaryotic gene expression and begins when initiation factor-2 (eIF2) delivers methionyl initiator tRNA (Met-tRNAi (Met)) to ribosomes. This first step is controlled by eIF2B mediating guanine nucleotide exchange on eIF2. We isolated eIF2 from yeast and used mass spectrometry to study the intact complex, and found that eIF2? is the most labile of the three subunits (eIF2?/?/?). We then compared conformational dynamics of the ternary complex eIF2:GTP:Met-tRNAi (Met) with apo eIF2 using comparative chemical cross-linking. Results revealed high conformational dynamics for eIF2? in apo eIF2 while in the ternary complex all three subunits are constrained. Novel post-translational modifications identified here in both eIF2 and eIF2B were combined with established sites, and located within protein sequences and homology models. We found clustering at subunit interfaces and highly phosphorylated unstructured regions, at the N-terminus of eIF2?, and also between the eIF2B? core and catalytic domains. We propose that modifications of these unstructured regions have a key role in regulating interactions between eIF2 and eIF2B, as well as other eIFs.

SUBMITTER: Beilsten-Edmands V

PROVIDER: S-EPMC4860841 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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eIF2 interactions with initiator tRNA and eIF2B are regulated by post-translational modifications and conformational dynamics.

Beilsten-Edmands Victoria V Gordiyenko Yuliya Y Kung Jocky Ck JC Mohammed Shabaz S Schmidt Carla C Robinson Carol V CV

Cell discovery 20150811

Translation of messenger RNA (mRNA) into proteins is key to eukaryotic gene expression and begins when initiation factor-2 (eIF2) delivers methionyl initiator tRNA (Met-tRNAi (Met)) to ribosomes. This first step is controlled by eIF2B mediating guanine nucleotide exchange on eIF2. We isolated eIF2 from yeast and used mass spectrometry to study the intact complex, and found that eIF2β is the most labile of the three subunits (eIF2α/β/γ). We then compared conformational dynamics of the ternary com ...[more]

PMID: 27462419

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Project description:The ubiquitin-proteasome system (UPS) is one of the major protein degradation pathways in eukaryotic cells. Abnormal functioning of this system has been observed in cancer and neurological diseases. The 20S proteasomes, essential components of the UPS, are present not only within the cells but also in the extracellular space, and their concentration in blood plasma has been found to be elevated and dependent upon the disease state, being of prognostic significance in patients suffering from cancer, liver diseases, and autoimmune diseases. However, functions of extracellular proteasomes and mechanisms of their release by cells remain largely unknown. The main mechanism of proteasome activity regulation is provided by modulation of their composition and post-translational modifications (PTMs). Moreover, diverse PTMs of proteins are known to participate in the loading of specific elements into extracellular vesicles. Since previous studies have revealed that the transport of extracellular proteasomes may occur via extracellular vesicles, we have set out to explore the PTMs of extracellular proteasomes in comparison to cellular counterparts. In this work, cellular and extracellular proteasomes were affinity purified and separated by SDS-PAGE for subsequent trypsinization and matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) analysis. In total, we could identify 64 and 55 PTM sites in extracellular and cellular proteasomes, respectively, including phosphorylation, ubiquitination, acetylation, and succinylation. We observed novel sites of acetylation at K238 and K192 of the proteasome subunits β2 and β3, respectively, that are specific for extracellular proteasomes. Moreover, cellular proteasomes show specific acetylation at K227 of α2 and ubiquitination at K201 of β3. Interestingly, succinylation of β6 at the residue K228 seems not to be present exclusively in extracellular proteasomes, whereas both extracellular and cellular proteasomes may also be acetylated at this site. The same situation takes place at K201 of the β3 subunit where ubiquitination is seemingly specific for cellular proteasomes. Moreover, crosstalk between acetylation, ubiquitination, and succinylation has been observed in the subunit α3 of both proteasome populations. These data will serve as a basis for further studies, aimed at dissection of the roles of extracellular proteasome-specific PTMs in terms of the function of these proteasomes and mechanism of their transport into extracellular space.

Dataset Information

EIF2 interactions with initiator tRNA and eIF2B are regulated by post-translational modifications and conformational dynamics.

Publications

eIF2 interactions with initiator tRNA and eIF2B are regulated by post-translational modifications and conformational dynamics.

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