Characterization Of Acid Ribosomal Protein P1 From Euplotes Octocarinatus

In eukaryotes,acidic ribosomal proteins are called P proteins,which include PO,P1,and P2.The acidic ribosomal proteins and the conserved domain of 26S/28S rRNA constitute a major part of the GTPase-associated center in eukaryotic ribosomes.They form a lateral protuberance on the 60S ribosomal subunit(the so-called ribosomal stalk).The ribosomal stalk is involved in protein synthesis regulation through interaction with soluble translation factors and in GTP hydrolysis control during elongation steps.The isoelectric point of the acidic ribosomal protein is located in the acidic range(3-5),which can be phosphorylated by several kinases as CK2 and PK60S when associated to the ribosome.It is considered that the primary function of the stalk is recruitment of translational GTPases and stimulation of factor-dependent GTP hydrolysis.The ciliated protozoan Euplotes octocarinatus is a classical single-celled eukaryote organism,which has two types of nuclei:micronucleus and macronucleus.Besides the dimorphic nucleus,another striking characteristic on gene expression and regulation in E.octocarinatus is its deviation from the universal genetic code:the codon UAA and UAG are functioning as termination signals,while the codon UGA is translated into cysteine or selenocysteine.To understand the assembling and function of ribosomal stalk in lower eukaryotic organisms and the involved mechanisms,E.octocarinatus was used in this study.Two PI genes were identified from the transcriptome and the genome of E.octocarinatus.Two recombinant plasmids(pET28a-P1A and pGEX-6P-1-P1B)were constructed and used to transform E.coli BL21 for expression.Recombinant proteins of EoP1A and EoP1B were purified by affinity chromatography.The interaction between EoP1A and EoP1B was tested by pull-down assay.Also,the phosphorylation of EoP1A was studied using NMR.Below is a summary of key results.1.Cloning of PI genes from Euplotes octocarinatus.The gene EoP1A and EoP1B were obtained by PCR.The full length is 803 bp for EoP1A and 641 bp for EoP1B.Bioinformation analysis and PCR sequencing showed that both of them have no intron and contain an ORF of 357 bp encoding a protein of 118 amino acids.The identity of the encoding region sequences between two genes is 76%,and the amino acid sequence similarity is 81%.The multiple sequence alignment showed the presence of the conserved amino acids as other species at N domain which can form a-helix.There also have additional 8 amino acids insertion at N-terminal domain.The sequence data also clearly showed that the C-terminal domain of P1 proteins from E.octocarinatus are very different from those in other eukaryotes which contains a highly conserved 11 amino acids sequence.The C-terminal domain of EoPl are 5-7aa longer than other eucaryotics and the last three amino acids are DEY.2.Expression and purification of fusion proteins His-EoP1A and GST-EoP1B.The recombinant plasmids pET28a-EoP1A and pGEX-6P-1-EoP1B were constructed and expressed in E.coli BL21.Recombinant proteins of EoP1A and EoP1B were purified by Ni-NTA or GST affinity chromatography.Both the purified proteins were detected by SDS-PAGE and confirmed by Western blotting.3.Assay of interaction between EoP1A and EoP1B.The interaction between EoP1A and EoP1B were studied by pull-down assay.The result showed two P1 proteins can interact with each other in vitro.4.Phosphorylation detection of EoP1A.The potential phosphorylation sites of EoP1A were predicted using software NetPhos 2.0.According to the prediction result,the mutatants EoP1A-S8 and EoP1A-?Y were constructed by site-directed mutagenesis method.Each of these protein mutants was expressed and purified.Also,the protein kinase CK2 includes CK2a and CK2?were expressed and purified.NMR results showed that EoP1A-WT,EoP1A-AY were phosphorylated in vitro by CK2 except EoP1A-S8,suggesting that EoP1A was phosphorylated in vitro at Ser8.5.Assay of interaction among P proteins from Euplotes octocarinatus.The interaction between these P proteins were studied by pull-down assay.The results showed that both EoP1A and EoP1B could not interact with EoP2 in vitro.To conclude,two PI genes were identified from the transcriptome and the genome of E.octocarinatus.Sequence analysis showed that additional 8 amino acids inserted at N-terminal domain and the C-terminal regions of P1 proteins from E.octocarinatus are very different from those in other eukaryotes.Interaction between EoP1A and EoP1B was detected by pull-down assay.The analysis of phosphorylation site of EoP1A showed that position Ser8 was phosphorylated in vitro.However,interaction between both EoP1A and EoP1B with EoP2 was not detected by pull-down assay,suggesting that the interaction between P proteins in E.octocarinatus is more complex.The current study on P1 proteins from E.octocarinatus lays the foundation for further understand the assembling and function of ribosomal stalk in lower eukaryotic organisms.