Study On The Degradation Pathway Of Ribosomal Protein L3

Objective:The ribosome is an organelle that is used to synthesize proteins.It is a macromolecular complex composed of ribosomal proteins and ribosomal RNAs.In the fast-growing yeast cells.5500 nt RNA and 79 ribosomal proteins undergo complex maturation processes and then result in approximately 2000 ribosomes per minute.There exist approximately 200,000 ribosomes each cell in budding yeast,whose proteins accounts for about 50% of the total proteins.Such a large amount of ribosomes and ribosomal proteins are produced and exist in cells,to maintain their homeostasis,there must be a mechanism to degrade excess ribosomes.Autophagy and the ubiquitinproteasome system are the major pathways for protein degradation in eukaryotic cells.The ubiquitin-proteasome system,the proteasome degrades proteins by recognizing the ubiquitin tag.Autophagy pathway,many organelles,macromolecular complexes and proteins are degraded by the autophagy pathway.Originally,the ribosome is considered to be randomly wrapped into the autophagosome,and then fused with the lysosome to form autolysosome and undergo further degradation.Claudine Kraft et al found that there exists selective autophagy of ribosomes in budding yeast,called Ribophagy.In related research,there are three major factors involved in the pathway,ubiquitination modification and deubiquitination,induction of specific reagent and autophagy receptors.Rpl3 is a ribosomal large subunit protein,which can promote translational elongation fidelity and is a not only the key but conserved ribosomal protein.In this experiment,we will study the degradation pathway of the ribosomal protein L3(Rpl3)in fission yeast.Methods:The ribosomal protein L3(Rpl3)was tagged with GFP(green fluorescent protein)with homologous recombination method.Spot assay was used to determine the assembly situation of the labeled proteins.Proteasome inhibitor,MG132,and protein synthesis inhibitor,Cycloheximide(CHX),were used to determine the regulation of the ubiquitinproteasome system.Autophagy was induced by starvation and detected by Western blot or fluorescence microscopy.The tetrad dissection experiment was used to gain strains which combined the knockout of autophagy core genes(atg5?,atg1?)and rrp4? with Rpl3-GFP.A molecular cloning method was used to construct a plasmid,and the plasmid with site-directed mutation was gained by PCR,E.coli transformation and DNA sequencing.A linearized plasmid treated with NotI was used to transfer the gene sequence of interest into the genome of yeast with lithium acetate transformation method.Pil1 co-tethering assay,co-immunoprecipitation and yeast two-hybrid assay were used to identify protein interaction.Mass spectrometry,Pil1 co-tethering assay,and iLIR v1.0 WWW Server were used to seek autophagy receptors of ribosomes.Results:The result of Spot assay showed that Rpl3-GFP can correctly assemble into ribosomes.Yeast cells were treated with MG132 and Cycloheximide(CHX),the result showed that the ubiquitin-proteasome system does not participate in the regulation of Rpl3.Autophagy induced by starvation could degrade Rpl3.While the core gene,atg5,was knocked out,starvation induction did not cause Rpl3 to be degraded,indicating that autophagy is involved in the regulation of Rpl3,The comparison and observation of Rpl3 with site mutant on ubiquitination and wild type Rpl3 showed that the ubiquitination modification does not affect the autophagy degradation of Rpl3.DTT induction and the absence of amino acids or nucleotides can induce autophagy in fission yeast,leading to the degradation of Rpl3.Knockout of rrp14 affects protein content,but does not affect the autophagy regulation of Rpl3.Rsa1,homolog protein of NUFIP1 in fission yeast,and Rpf2 and other proteins are not ribosomal receptors.The ribosomal protein L3 is degraded in a ubiquitin-independent autophagy pathway.Conclusion:1.In fission yeast,Rpl3 is not degraded by ubiquitin-proteasome system,but regulated by autophagy system.2.Ubiquitination modification does not affect the autophagic degradation of Rpl3.DTT and lack of amino acids or nucleotides can cause the autophagic degradation of Rpl3.3.Pol5 and Rsa1 are not ribosome receptors,and the ribosome receptor could be found by comprehensive analysis and experiments.