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

A recent
paper in Science Advances by Sun et al. claims that intra-chloroplast
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.