In vivo homopropargylglycine incorporation enables sampling, isolation and characterization of nascent proteins from Arabidopsis thaliana

Abstract

Summary Determining which proteins are actively synthesized at a given point in time and extracting a representative sample for analysis is important to understand plant responses. Here we show that the methionine (Met) analogue homopropargylglycine (HPG) enables Bio‐Orthogonal Non‐Canonical Amino acid Tagging (BONCAT) of a small sample of the proteins being synthesized in Arabidopsis plants or cell cultures, facilitating their click‐chemistry enrichment for analysis. The sites of HPG incorporation could be confirmed by peptide mass spectrometry at Met sites throughout protein amino acid sequences and correlation with independent studies of protein labelling with 15N verified the data. We provide evidence that HPG‐based BONCAT tags a better sample of nascent plant proteins than azidohomoalanine (AHA)‐based BONCAT in Arabidopsis and show that the AHA induction of Met metabolism and greater inhibition of cell growth rate than HPG probably limits AHA incorporation at Met sites in Arabidopsis. We show HPG‐based BONCAT provides a verifiable method for sampling, which plant proteins are being synthesized at a given time point and enriches a small portion of new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis. Enriched nascent polypeptides samples were found to contain significantly fewer common post‐translationally modified residues than the same proteins from whole plant extracts, providing evidence for age‐related accumulation of post‐translational modifications in plants.

Bioorthogonal Chemistry and Its Applications

“…Tivendale et al compared the effects of azidohomoalanine (AHA) incorporation and homopropargylglycine (HPG) incorporation in Arabidopsis thaliana tissues or cell cultures to better understand how AHA affects the cells and to understand more fully the utility of BONCAT for sampling nascent proteins in plant science . Seedlings or cell cultures were pulse labeled with AHA or HPG.…”

“…90 Tivendale et al compared the effects of azidohomoalanine (AHA) incorporation and homopropargylglycine (HPG) incorporation in Arabidopsis thaliana tissues or cell cultures to better understand how AHA affects the cells and to understand more fully the utility of BONCAT for sampling nascent proteins in plant science. 91 Seedlings or cell cultures were pulse labeled with AHA or HPG. After a suitable incubation time, labeled proteins were extracted and were captured via CuAAC reaction using a suitable reaction partner bound to a chromatographic resin obtained from a commercially available kit (Click-iT Cell buffer kit, Thermo-Fisher Scientific).…”

How is auxin linked with cellular energy pathways to promote growth?

“…Links between proteases of mitochondrial machinery and auxin might be explained by these phenomena (Zhang et al ., 2014; Liu et al ., 2019). Analysis of changes in translation rates in response to auxin, through riboseq (Aspden et al ., 2014; Erhard et al ., 2018) and/or direct measures of protein synthesis (Tivendale et al ., 2021b), would enable this hypothesis to be tested.…”

Enzymes degraded under high light maintain proteostasis by transcriptional regulation inArabidopsis

“…The increased turnover of redox shuttling systems, namely glutathione- and thioredoxin-linked systems and the MDH enzymes involved in malate shuttles throughout the cell ( Fig. 4 ), may be due to increased flux through these pathways and thus a consequence of an increased rate of wear-out damage of these enzymes ( 60 , 64 ).…”