Does the Ubiquitination Degradation Pathway Really Reach inside of the Chloroplast? A Re-Evaluation of Mass Spectrometry-Based Assignments of Ubiquitination

Abstract

A recent paper in Science Advances by Sun et al. claims that intrachloroplast proteins in the model plant Arabidopsis can be polyubiquitinated and then extracted into the cytosol for subsequent degradation by the proteasome. Most of this conclusion hinges on several sets of mass spectrometry (MS) data. If the proposed results and conclusion are true, this would be a major change in the proteolysis/proteostasis field, breaking the long-standing dogma that there are no polyubiquitination mechanisms within chloroplast organelles (nor in mitochondria). Given its importance, we reanalyzed their raw MS data using both open and closed sequence database searches and encountered many issues not only with the results but also discrepancies between stated methods (e.g., use of alkylating agent iodoacetamide (IAA)) and observed mass modifications. Although there is likely enrichment of ubiquitination signatures in a subset of the data (probably from ubiquitination in the cytosol), we show that runaway alkylation with IAA caused extensive artifactual modifications of N termini and lysines to the point that a large fraction of the desired ubiquitination signatures is indistinguishable from artifactual acetamide signatures, and thus, no intra-chloroplast polyubiquitination conclusions can be drawn from these data. We provide recommendations on how to avoid such perils in future work.

PDX proteins from Arabidopsis thaliana as novel substrates of cathepsin B: implications for vitamin B6 biosynthesis regulation

“…With the current findings, it remains unclear whether PDX proteins are actually ubiquitinated in planta since our data did not support the UPP as primary reason for PDX instabilities. Recently, van Wijk et al [77] showed that alkylating agents, such as iodoacetamide, can result in artifactual acetamide signatures that are not distinguishable from ubiquitination signatures in mass spectrometry-based analysis. Since both Manzano et al [23] and Saracco et al [24] used iodoacetamide in their works, it opens up the possibility that at least in part some of the proteins identified are false positives and artifacts of the experimental approach.…”