Most chloroplast and mitochondrial proteins are synthesized in the cytosol of the plant cell and have to be imported into the organelles post-translationally. Molecular chaperones play an important role in preventing ...Most chloroplast and mitochondrial proteins are synthesized in the cytosol of the plant cell and have to be imported into the organelles post-translationally. Molecular chaperones play an important role in preventing protein aggregation of freshly translated preproteins and assist in maintaining the preproteins in an import competent state. Pre- proteins can associate with HSP70, HSP90, and 14-3-3 proteins in the cytosol. In this study, we analyzed a large set of wheat germ-translated chloroplast preproteins with respect to their chaperone binding. Our results demonstrate that the formation of distinct 14-3-3 or HSP90 containing preprotein complexes is a common feature in post-translational protein transport in addition to preproteins that seem to interact solely with HSP70. We were able to identify a diverse and extensive class of preproteins as HSP90 substrates, thus providing a tool for the investigation of HSP90 client protein association. The analyses of chimeric HSP90 and 14-3-3 binding preproteins with exchanged transit peptides indicate an involvement of both the transit peptide and the mature part of the proteins, in HSP90 binding. We identified two partner components of the HSP90 cycle, which were present in the preprotein containing high-molecular-weight complexes, the HSP70/HSP90 organizing protein HOP, as well as the immunophilin FKBP73. The results establish chloroplast preproteins as a general class of HSP90 client proteins in plants using HOP and FKBP as novel cochaperones.展开更多
A new method for screening gene mutations by using a Coupled In Vitro Transcription and Translation System is reported in this paper. It includes the following steps: (1) The target DNA fragments with T7T1 sequence a...A new method for screening gene mutations by using a Coupled In Vitro Transcription and Translation System is reported in this paper. It includes the following steps: (1) The target DNA fragments with T7T1 sequence at its 5 prime end are amplified (T7T1: GGATCCTAATACGACTCTATAGGGAG ACCACCATG); (2) The RNA and peptide are synthesized and labeled from the PCR product in the coupled In Vitro Transcription-Translation System; (3) The produced peptides are analyzed by using SDS-PAGE and pH Gradient gel focusing electrophoresis. Four peptide products from 4 HB patients with nonsense mutation in Exon H of F IX gene show truncated protein bands with speeded migration in the autoradiography of the SDS-PAGE. Ten out of 11 HB patients with different missense mutations show abnormal patterns in the autoradiography of a pH 4-7 gradient gel focusing electrophoresis. Conclusion: The PCR and the Coupled In Vitro Transcription-Translation System/SDS-PAGE is a good method for proteins truncation test. The PCR and the Transcription-Translation System combined with pH gradient gel focusing electrophoresis is an efficient method for screening the abnormal protein products from DNA fragments with missense mutations.展开更多
文摘Most chloroplast and mitochondrial proteins are synthesized in the cytosol of the plant cell and have to be imported into the organelles post-translationally. Molecular chaperones play an important role in preventing protein aggregation of freshly translated preproteins and assist in maintaining the preproteins in an import competent state. Pre- proteins can associate with HSP70, HSP90, and 14-3-3 proteins in the cytosol. In this study, we analyzed a large set of wheat germ-translated chloroplast preproteins with respect to their chaperone binding. Our results demonstrate that the formation of distinct 14-3-3 or HSP90 containing preprotein complexes is a common feature in post-translational protein transport in addition to preproteins that seem to interact solely with HSP70. We were able to identify a diverse and extensive class of preproteins as HSP90 substrates, thus providing a tool for the investigation of HSP90 client protein association. The analyses of chimeric HSP90 and 14-3-3 binding preproteins with exchanged transit peptides indicate an involvement of both the transit peptide and the mature part of the proteins, in HSP90 binding. We identified two partner components of the HSP90 cycle, which were present in the preprotein containing high-molecular-weight complexes, the HSP70/HSP90 organizing protein HOP, as well as the immunophilin FKBP73. The results establish chloroplast preproteins as a general class of HSP90 client proteins in plants using HOP and FKBP as novel cochaperones.
文摘A new method for screening gene mutations by using a Coupled In Vitro Transcription and Translation System is reported in this paper. It includes the following steps: (1) The target DNA fragments with T7T1 sequence at its 5 prime end are amplified (T7T1: GGATCCTAATACGACTCTATAGGGAG ACCACCATG); (2) The RNA and peptide are synthesized and labeled from the PCR product in the coupled In Vitro Transcription-Translation System; (3) The produced peptides are analyzed by using SDS-PAGE and pH Gradient gel focusing electrophoresis. Four peptide products from 4 HB patients with nonsense mutation in Exon H of F IX gene show truncated protein bands with speeded migration in the autoradiography of the SDS-PAGE. Ten out of 11 HB patients with different missense mutations show abnormal patterns in the autoradiography of a pH 4-7 gradient gel focusing electrophoresis. Conclusion: The PCR and the Coupled In Vitro Transcription-Translation System/SDS-PAGE is a good method for proteins truncation test. The PCR and the Transcription-Translation System combined with pH gradient gel focusing electrophoresis is an efficient method for screening the abnormal protein products from DNA fragments with missense mutations.