There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. Sequence analysis...There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. Sequence analysis of 43 dual targeted proteins in comparison with 385 mitochondrial proteins and 567 chloroplast proteins ofArabidopsis thaliana revealed an overall significant increase in phenylalanines, leucines, and serines and a decrease in acidic amino acids and glycine in dual targeting peptides （dTPs）. The N-terminal portion of dTPs has significantly more serines than mTPs. The number of arginines is similar to those in mTPs, but almost twice as high as those in cTPs. We have investigated targeting determinants of the dual targeting peptide of Thr-tRNA synthetase （ThrRS-dTP） studying organellar import of N- and C-terminal deletion constructs of ThrRS-dTP coupled to GFR These results show that the 23 amino acid long N-terminal portion of ThrRS-dTP is crucial but not sufficient for the organellar import. The C-terminal deletions revealed that the shortest peptide that was capable of conferring dual targeting was 60 amino acids long. We have purified the ThrRS- dTP（2-60） to homogeneity after its expression as a fusion construct with GST followed by CNBr cleavage and ion exchange chromatography. The purified ThrRS-dTP（2-60） inhibited import of pF1β into mitochondria and of pSSU into chloroplasts at μM concentrations showing that dual and organelle-specific proteins use the same organellar import pathways. Furthermore, the CD spectra of ThrRS-dTP（2-60） indicated that the peptide has the propensity for forming α-helical structure in membrane mimetic environments; however, the membrane charge was not important for the amount of induced helical structure. This is the first study in which a dual targeting peptide has been purified and investigated by biochemical and biophysical means.展开更多
Transfer RNA plays a fundamental role in the protein biosynthesis as an adaptor molecule by functioning as a biological link between the genetic nucleotide sequence in the mRNA and the amino acid sequence in the prote...Transfer RNA plays a fundamental role in the protein biosynthesis as an adaptor molecule by functioning as a biological link between the genetic nucleotide sequence in the mRNA and the amino acid sequence in the protein.To perform its role in protein biosynthesis,it has to be accurately recognized by aminoacyl-tRNA synthetases(aaRSs)to generate aminoacyl-tRNAs(aa-tRNAs).The correct pairing between an amino acid with its cognate tRNA is crucial for translational quality control.Production and utilization of mis-charged tRNAs are usually detrimental for all the species,resulting in cellular dysfunctions.Correct aa-tRNAs formation is collectively controlled by aaRSs with distinct mechanisms and/or other trans-factors.However,in very limited instances,mis-charged tRNAs are intermediate for specific pathways or essential components for the translational machinery.Here,from the point of accuracy in tRNA charging,we review our understanding about the mechanism ensuring correct aa-tRNA generation.In addition,some unique mis-charged tRNA species necessary for the organism are also briefly described.展开更多
Aminoacyl-tRNA synthetases(aaRSs)are ubiquitously expressed,essential enzymes,synthesizing aminoacyl-tRNAs for protein synthesis.Functional defects of aaRSs frequently cause various human disorders.Human KARS encodes ...Aminoacyl-tRNA synthetases(aaRSs)are ubiquitously expressed,essential enzymes,synthesizing aminoacyl-tRNAs for protein synthesis.Functional defects of aaRSs frequently cause various human disorders.Human KARS encodes both cytosolic and mitochondrial lysyl-tRNA synthetases(LysRSs).Previously,two mutations(c.1129 G>A and c.517 T>C)were identified that led to hearing impairment;however,the underlying biochemical mechanism is unclear.In the present study,we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol,but affect the cytosolic LysRS level,its tertiary structure,and cytosolic tRNA aminoacylation in vitro.As for mitochondrial translation,the two mutations have little effect on the steady-state level,mitochondrial targeting,and tRNA binding affinity of mitochondrial LysRS.However,they exhibit striking differences in charging mitochondrial tRNALys,with the c.517T>C mutant being completely deficient in vitro and in vivo.We constructed two yeast genetic models,which are powerful tools to test the in vivo aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels.Overall,our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129G>A and c.517T>C mutations and provided yeast genetic bases to investigate other KARS mutations in the future.展开更多
文摘There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. Sequence analysis of 43 dual targeted proteins in comparison with 385 mitochondrial proteins and 567 chloroplast proteins ofArabidopsis thaliana revealed an overall significant increase in phenylalanines, leucines, and serines and a decrease in acidic amino acids and glycine in dual targeting peptides （dTPs）. The N-terminal portion of dTPs has significantly more serines than mTPs. The number of arginines is similar to those in mTPs, but almost twice as high as those in cTPs. We have investigated targeting determinants of the dual targeting peptide of Thr-tRNA synthetase （ThrRS-dTP） studying organellar import of N- and C-terminal deletion constructs of ThrRS-dTP coupled to GFR These results show that the 23 amino acid long N-terminal portion of ThrRS-dTP is crucial but not sufficient for the organellar import. The C-terminal deletions revealed that the shortest peptide that was capable of conferring dual targeting was 60 amino acids long. We have purified the ThrRS- dTP（2-60） to homogeneity after its expression as a fusion construct with GST followed by CNBr cleavage and ion exchange chromatography. The purified ThrRS-dTP（2-60） inhibited import of pF1β into mitochondria and of pSSU into chloroplasts at μM concentrations showing that dual and organelle-specific proteins use the same organellar import pathways. Furthermore, the CD spectra of ThrRS-dTP（2-60） indicated that the peptide has the propensity for forming α-helical structure in membrane mimetic environments; however, the membrane charge was not important for the amount of induced helical structure. This is the first study in which a dual targeting peptide has been purified and investigated by biochemical and biophysical means.
基金supported by the National Natural Science Foundation of China(31270852,31000355)the National Key Basic Research Program of China(2012CB911000)
文摘Transfer RNA plays a fundamental role in the protein biosynthesis as an adaptor molecule by functioning as a biological link between the genetic nucleotide sequence in the mRNA and the amino acid sequence in the protein.To perform its role in protein biosynthesis,it has to be accurately recognized by aminoacyl-tRNA synthetases(aaRSs)to generate aminoacyl-tRNAs(aa-tRNAs).The correct pairing between an amino acid with its cognate tRNA is crucial for translational quality control.Production and utilization of mis-charged tRNAs are usually detrimental for all the species,resulting in cellular dysfunctions.Correct aa-tRNAs formation is collectively controlled by aaRSs with distinct mechanisms and/or other trans-factors.However,in very limited instances,mis-charged tRNAs are intermediate for specific pathways or essential components for the translational machinery.Here,from the point of accuracy in tRNA charging,we review our understanding about the mechanism ensuring correct aa-tRNA generation.In addition,some unique mis-charged tRNA species necessary for the organism are also briefly described.
基金supported by the National Key Research and Development Program of China(2017YFA0504000)the National Natural Science Foundation of China(91940302,31500644,31570792,31822015,81870896,31670801)+1 种基金the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB19010203)Shanghai Key Laboratory of Embryo Original Diseases(Shelab201904)。
文摘Aminoacyl-tRNA synthetases(aaRSs)are ubiquitously expressed,essential enzymes,synthesizing aminoacyl-tRNAs for protein synthesis.Functional defects of aaRSs frequently cause various human disorders.Human KARS encodes both cytosolic and mitochondrial lysyl-tRNA synthetases(LysRSs).Previously,two mutations(c.1129 G>A and c.517 T>C)were identified that led to hearing impairment;however,the underlying biochemical mechanism is unclear.In the present study,we found that the two mutations have no impact on the incorporation of LysRS into the multiple-synthetase complex in the cytosol,but affect the cytosolic LysRS level,its tertiary structure,and cytosolic tRNA aminoacylation in vitro.As for mitochondrial translation,the two mutations have little effect on the steady-state level,mitochondrial targeting,and tRNA binding affinity of mitochondrial LysRS.However,they exhibit striking differences in charging mitochondrial tRNALys,with the c.517T>C mutant being completely deficient in vitro and in vivo.We constructed two yeast genetic models,which are powerful tools to test the in vivo aminoacylation activity of KARS mutations at both the cytosolic and mitochondrial levels.Overall,our data provided biochemical insights into the potentially molecular pathological mechanism of KARS c.1129G>A and c.517T>C mutations and provided yeast genetic bases to investigate other KARS mutations in the future.
