Cytosolic HSP90 Cochaperones HOP and FKBP Interact with Freshly Synthesized Chloroplast Preproteins of Arabidopsis

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.

Yeast knockout library allows for efficient testing of genomic mutations for cell-free protein synthesis

Cell-free protein synthesis(CFPS)systems from crude lysates have benefitted from modifications to their enzyme composition.For example,functionally deleting enzymes in the source strain that are deleterious to CFPS can improve protein synthesis yields.However,making such modifications can take substantial time.As a proof-of-concept to accelerate prototyping capabilities,we assessed the feasibility of using the yeast knockout collection to identify negative effectors in a Saccharomyces cerevisiae CFPS platform.We analyzed extracts made from six deletion strains that targeted the single deletion of potentially negative effectors(e.g.,nucleases).We found a statistically significant increase in luciferase yields upon loss of function of GCN3,PEP4,PPT1,NGL3,and XRN1 with a maximum increase of over 6-fold as compared to the wild type.Our work has implications for yeast CFPS and for rapidly prototyping strains to enable cell-free synthetic biology applications.

Cell-free synthesis of stable isotope-labeled internal standards for targeted quantitative proteomics

High-sensitivity mass spectrometry approaches using selected reaction monitoring(SRM)or multiple reaction monitoring(MRM)methods are powerful tools for targeted quantitative proteomics-based investigation of dynamics in specific biological systems.Both high-sensitivity detection of lowabundance proteins and their quantification using this technique employ stable isotope-labeled peptide internal standards.Currently,there are various ways for preparing standards,including chemical peptide synthesis,cellular protein expression,and cell-free protein or peptide synthesis.Cell-free protein synthesis(CFPS)or in vitro translation(IVT)systems in particular provide high-throughput and low-cost preparation methods,and various cell types and reconstituted forms are now commercially available.Herein,we review the use of such systems for precise and reliable protein quantification.