On the Intracellular Trafficking of Mouse S5 Ribosomal Protein from Cytoplasm to Nucleoli
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| Title: | On the Intracellular Trafficking of Mouse S5 Ribosomal Protein from Cytoplasm to Nucleoli |
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| Authors: | H. Papachristou, Theodora Choli-Papadopoulou, Ioannis S. Vizirianakis, Georgios Papadopoulos, T. Papamarcaki, Ch. Matragkou, Z. Karetsou, Asterios S. Tsiftsoglou |
| Source: | Journal of Molecular Biology. 392:1192-1204 |
| Publisher Information: | Elsevier BV, 2009. |
| Publication Year: | 2009 |
| Subject Terms: | Ribosomal Proteins, 0301 basic medicine, Cytoplasm, Cell Nucleus/*chemistry, Polymerase Chain Reaction/methods, Recombinant Fusion Proteins, Blotting, Western, Green Fluorescent Proteins, Polymerase Chain Reaction, Mice, 03 medical and health sciences, Genes, Reporter, Animals, Humans, Phosphorylation, Cytoplasm/*chemistry, Green Fluorescent Proteins/analysis/genetics, Cell Nucleus, 0303 health sciences, Microscopy, Confocal, Ribosomal Proteins/*analysis/*genetics, Recombinant Fusion Proteins/analysis/genetics, Protein Transport, Mutagenesis, Cell Nucleolus/*chemistry, Cell Nucleolus, HeLa Cells |
| Description: | The non-ribosomal functions of mammalian ribosomal proteins have recently attracted worldwide attention. The mouse ribosomal protein S5 (rpS5) derived from ribosomal material is an assembled non-phosphorylated protein. The free form of rpS5 protein, however, undergoes phosphorylation. In this study, we have (a) investigated the potential role of phosphorylation in rpS5 protein transport into the nucleus and then into nucleoli and (b) determined which of the domains of rpS5 are involved in this intracellular trafficking. In vitro PCR mutagenesis of mouse rpS5 cDNA, complemented by subsequent cloning and expression of rpS5 truncated recombinant forms, produced in fusion with green fluorescent protein, permitted the investigation of rpS5 intracellular trafficking in HeLa cells using confocal microscopy complemented by Western blot analysis. Our results indicate the following: (a) rpS5 protein enters the nucleus via the region 38-50 aa that forms a random coil as revealed by molecular dynamic simulation. (b) Immunoprecipitation of rpS5 with casein kinase II and immobilized metal affinity chromatography analysis complemented by in vitro kinase assay revealed that phosphorylation of rpS5 seems to be indispensable for its transport from nucleus to nucleoli; upon entering the nucleus, Thr-133 phosphorylation triggers Ser-24 phosphorylation by casein kinase II, thus promoting entrance of rpS5 into the nucleoli. Another important role of rpS5 N-terminal region is proposed to be the regulation of protein's cellular level. The repetitively co-appearance of a satellite C-terminal band below the entire rpS5 at the late stationary phase, and not at the early logarithmic phase, of cell growth suggests a specific degradation balancing probably the unassembled ribosomal protein molecules with those that are efficiently assembled to ribosomal subunits. Overall, these data provide new insights on the structural and functional domains within the rpS5 molecule that contribute to its cellular functions. |
| Document Type: | Article |
| Language: | English |
| ISSN: | 0022-2836 |
| DOI: | 10.1016/j.jmb.2009.07.049 |
| Access URL: | https://pubmed.ncbi.nlm.nih.gov/19631221 https://www.ncbi.nlm.nih.gov/pubmed/19631221 https://www.sciencedirect.com/science/article/pii/S0022283609009024 https://europepmc.org/abstract/MED/19631221 https://core.ac.uk/display/132813328 https://pubmed.ncbi.nlm.nih.gov/19631221/ http://olympias.lib.uoi.gr/jspui/handle/123456789/19638 |
| Rights: | Elsevier TDM |
| Accession Number: | edsair.doi.dedup.....6b7bdba1865a42c8c800df5a21ecf8fb |
| Database: | OpenAIRE |
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Nájsť tento článok vo Web of Science | Abstract: | The non-ribosomal functions of mammalian ribosomal proteins have recently attracted worldwide attention. The mouse ribosomal protein S5 (rpS5) derived from ribosomal material is an assembled non-phosphorylated protein. The free form of rpS5 protein, however, undergoes phosphorylation. In this study, we have (a) investigated the potential role of phosphorylation in rpS5 protein transport into the nucleus and then into nucleoli and (b) determined which of the domains of rpS5 are involved in this intracellular trafficking. In vitro PCR mutagenesis of mouse rpS5 cDNA, complemented by subsequent cloning and expression of rpS5 truncated recombinant forms, produced in fusion with green fluorescent protein, permitted the investigation of rpS5 intracellular trafficking in HeLa cells using confocal microscopy complemented by Western blot analysis. Our results indicate the following: (a) rpS5 protein enters the nucleus via the region 38-50 aa that forms a random coil as revealed by molecular dynamic simulation. (b) Immunoprecipitation of rpS5 with casein kinase II and immobilized metal affinity chromatography analysis complemented by in vitro kinase assay revealed that phosphorylation of rpS5 seems to be indispensable for its transport from nucleus to nucleoli; upon entering the nucleus, Thr-133 phosphorylation triggers Ser-24 phosphorylation by casein kinase II, thus promoting entrance of rpS5 into the nucleoli. Another important role of rpS5 N-terminal region is proposed to be the regulation of protein's cellular level. The repetitively co-appearance of a satellite C-terminal band below the entire rpS5 at the late stationary phase, and not at the early logarithmic phase, of cell growth suggests a specific degradation balancing probably the unassembled ribosomal protein molecules with those that are efficiently assembled to ribosomal subunits. Overall, these data provide new insights on the structural and functional domains within the rpS5 molecule that contribute to its cellular functions. |
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| ISSN: | 00222836 |
| DOI: | 10.1016/j.jmb.2009.07.049 |