# Editorial: Plant protein termini: Their generation, modification and function

Patrick Willems, Pitter F. Huesgen, Iris Finkemeier, Emmanuelle Graciet, Thierry Meinnel, Frank van Breusegem

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Patrick Willems, Pitter F. Huesgen, Iris Finkemeier, Emmanuelle Graciet, Thierry Meinnel, et al. Editorial: Plant protein termini: Their generation, modification and function. Frontiers in Plant Science, 2022, 13, pp.1040392. 10.3389/fpls.2022.1040392. hal-03797034

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OPEN ACCESS

EDITED AND REVIEWED BY
Ning Li,
Hong Kong University of Science and
Technology, Hong Kong, SAR China

*CORRESPONDENCE
Patrick Willems
patwille.willems@ugent.be

SPECIALTY SECTION
This article was submitted to Plant Proteomics and Protein Structural Biology, a section of the journal Frontiers in Plant Science

RECEIVED 09 September 2022
ACCEPTED 15 September 2022
PUBLISHED 30 September 2022

CITATION
Willems P, Huesgen PF, Finkemeier I, Graciet E, Meinnel T and Van Breusegem F (2022) Editorial: Plant protein termini: Their generation, modification and function.
*Front. Plant Sci.* 13:1040392.
doi: 10.3389/fpls.2022.1040392

COPYRIGHT
© 2022 Willems, Huesgen, Finkemeier, Graciet, Meinnel and Van Breusegem. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Editorial: Plant protein termini: Their generation, modification and function

Patrick Willems<sup>1,2,3,4*</sup>, Pitter F. Huesgen<sup>5,6,7</sup>, Iris Finkemeier<sup>8</sup>, Emmanuelle Graciet<sup>9,10</sup>, Thierry Meinnel<sup>11</sup> and Frank Van Breusegem<sup>1,2</sup>

<sup>1</sup>Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium, <sup>2</sup>Vlaams Instituut voor Biotechnologie (VIB) Center for Plant Systems Biology, Vlaams Instituut voor Biotechnologie (VIB), Ghent, Belgium, <sup>3</sup>Department of Biomolecular Medicine, Ghent University, Ghent, Belgium, <sup>4</sup>Vlaams Instituut voor Biotechnologie (VIB) Center for Medical Biotechnology, Vlaams Instituut voor Biotechnologie (VIB), Ghent, Belgium, <sup>5</sup>Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Jülich, Germany, <sup>6</sup>Cologne Excellence Cluster on Stress Responses in Ageing-Associated Diseases, CECAD, Medical Faculty and University Hospital, University of Cologne, Cologne, Germany, <sup>7</sup>Institute of Biochemistry, Department for Chemistry, University of Cologne, Cologne, Germany, <sup>8</sup>Plant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Münster, Germany, <sup>9</sup>Department of Biology, Maynooth University, Maynooth, Ireland, <sup>10</sup>Kathleen Lonsdale Institute for Human Health Research, Maynooth University, Maynooth, Ireland, <sup>11</sup>Université Paris-Saclay, Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre National de la Recherche Scientifique (CNRS), Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France

KEYWORDS

protein N-termini, N-terminal modifications, N-degron, Arabidopsis thaliana, alternative splicing, alternative translation initiation

## Editorial on the Research Topic

### Plant protein termini: Their generation, modification and function

A single gene can give rise to multiple protein species, or proteoforms, by taking advantage of biological processes occurring before, during or after protein synthesis (Figure 1). These processes often yield altered protein starts that can influence protein function. For instance, alternative splicing or translation initiation can lead to distinct protein N-termini, while nascent polypeptide chains can be trimmed and N-terminally acetylated during translation. Also, after protein synthesis, protein termini can be recognized by modifying enzymes or by E3 ubiquitin ligases, the latter marking proteins for ubiquitin-dependent degradation by the N-degron pathways. Alternatively, protease processing can generate novel protein fragments (with new N- and C-termini) that can acquire specialized functions. A central method within protein N-terminal biology is positional proteomics, where peptides matching protein N-termini can be enriched and identified, thereby informing on the identity, modification status and intensity of protein N-termini within cells. N-terminal proteomics can be used to delineate alternative proteoforms (e.g. shaped by alternative splicing or translation
<figure><img src="image_3.png" /><figcaption>FIGURE 1</figcaption></figure>

Biological processes shaping distinct N-terminal proteoforms. Expression of alternative transcripts with differing exon-intron structures can encode novel protein N-termini. Alternative translation initiation sites can be selected, thereby shaping truncated and extended N-terminal proteoforms. During translation, initiator Met (green) can be cleaved by Met aminopeptidases, exposing the second residue (here Ser, orange). In addition, protein N-termini can be N-terminally modified, e.g. such as co-translational N-terminal acetylation catalyzed by N-terminal acetyltransferases. Lastly, post-translational cleavage of proteins by proteases yields proteolytic fragments that can acquire novel functions or be further degraded, e.g. *via* the ubiquitin proteasome pathway. Ac-CoA, acetyl coenzyme A; Ub, ubiquitin.

initiation) or identify substrates of N-terminal modifying enzymes, proteases or N-degron pathways.

In plants, our understanding of the extent and functional implications of protein termini and their corresponding proteoforms is still in its infancy. This Research Topic focuses on the closely intertwined research disciplines studying the generation, modification and function of protein N-termini in plants.

## Alternative splicing and translation initiation

While alternative splicing is a ubiquitous process in plants, their true contribution to proteome complexity remains a debatable topic (Yu et al., 2016). In this Research Topic, Linnenbrügger et al. report on alternatively splicing of glucose-6-phosphate dehydrogenase (G6PDH), which produces N-terminally extended proteoforms, G6PDF5.4 and G6PDF5.5, that localize at the cytosolic side of the endoplasmic reticulum (ER). G6PDH catalyzes the first step of the oxidative pentose-phosphate pathway (OPPP), and OPPP activity at ER subdomains was further supported by the co-localization and

co-purification of G6PDF5.4 with downstream OPPP enzymes 6-phosphogluconolactonase PGL2 and 6-phosphogluconate dehydrogenase PGD2. While PGD2 was identified as S-palmitoylated (Kumar et al., 2022), ER targeting for PGL2 was shown to be dependent on a C-terminal CaaX prenylation motif. Taken together, these enzymatic OPPP reactions could provide NAPDH for biosynthetic reactions at the cytosolic side of the ER. Evidence for the existence of multiple gene proteoforms can be systematically gained by using OMICS technologies such as N-terminal proteomics and ribosome footprinting (ribo-seq). Here, Willems et al. provide complementary evidence for ~90 proteoform pairs stemming from alternative translation initiation sites (aTIS) in Arabidopsis by combining N-terminal proteomics and translation initiation ribosome footprinting. Next to aTIS mutagenesis validation experiments, N-termini from truncated proteins initiated from aTIS downstream of the canonical AUG start codon resulted were compliant with co-translational modification rules. Genes giving rise to full-length and truncated N-terminal proteoforms often result in alternative targeting of the proteins. For example, the full-length proteins are often targeted to mitochondria and/or chloroplasts, while downstream aTIS sites seem to act as a
mechanism to bypass N-terminal targeting peptides and
provide cytosolic protein copies.

N-terminal acetylation and N-
degron pathways

Yeast serves as a key model species to study eukaryotic
biological systems. In this Research Topic, a novel assay in yeast
is described to study the modification and recognition of plant
protein N-termini. Meeting the need for a straightforward and
more sensitive assay to test and identify putative plant N-degron
substrates, Kozlic et al. introduced the Arabidopsis PRT6/N-
degron pathway within a *Saccharomyces cerevisiae* strain
deficient in its endogenous E3 ubiquitin ligase UBR1, which is
also termed N-recognin because of its ability to bind so-called
destabilizing N-terminal residues. Co-expression of the
Arabidopsis N-recognin PRT6 and ubiquitin conjugating
enzyme AtUBC2 successfully reconstituted an active N-degron
pathway in yeast. To facilitate high-throughput assays, a GFP
fluorescence-based read-out was developed. This confirmed
PRT6/N-degron dependent turnover of known substrates in
plants, such as HRE2 and ZPR2 (Gibbs et al., 2011; Weits
et al., 2019), as well as other tested proteins such as BBX31.
Results in yeast differed from *in planta* assays for others, such as
the basal immunity regulator RIN4 (Goslin et al., 2019). Also in
yeast, the E3 ligase Doa10 was shown to recognize acetylated
protein N-termini as N-degron (Ac/N-degrons) and target for
ubiquitin-dependent degradation (Hwang et al., 2010). Such Ac/
N-degrons can be conditionally exposed in misfolded proteins
and functions within ER-associated protein degradation.
Another link to ER stress was studied in this Research Topic
by Huber et al. in Arabidopsis plants deficient in the NatB
complex or in Doa10, which is an ER-membrane resident E3
ubiquitin ligase. The NatB mutants are hypersensitive to salt and
osmotic stress (Huber et al., 2020), which both trigger protein
misfolding and ER stress. To induce protein misfolding,
dithiothreitol (DTT) and tunamycin (TM) were administered
to impair structural disulfides and glycoprotein folding,
respectively. *NatB* mutants were solely hypersensitive to DTT
treatment, excluding a major function for in ER stress. Instead,
impaired redox balance likely underlies the observed phenotype,
as lower levels of reactive oxygen species were observed and
DTT-responsive transcripts were induced in *NatB* mutants. The
role of DOA10 in Arabidopsis remains ambiguous, as *doa10*
mutants were insensitive to both DTT and TM treatment, ruling
out DOA10 as sole N-recognin of Ac/N-degron in plants. To
improve the enzymatic characterization of acetyltransferases,
Asensio et al. developed a continuous, non-radioactive assay
using the *Schizosaccharomyces pombe* SpNatA N-terminal
acetyltransferase. Here, the release of coenzyme A (CoA) by

NAT activity was coupled to the reaction of pyruvate
dehydrogenase that re-generates acetyl-CoA and releases
NADH that can be monitored spectrometrically. The assay
was first validated using SpNatA, which uncovered a hitherto
unknown role for remote basic residues that could interact with
a negatively charged region at the extremity of the SpNatA
peptide binding site. Showcasing its capacity to characterize new
GNAT enzymes, the rice plastidial OsGNAT2 was shown to
have a similar activity and substrate specificity as its Arabidopsis
orthologue AtGNAT2 (Bienvenut et al., 2020). The assay shows
great promise for high-throughput measurements of GNATs,
and the identification of their substrates and inhibitors.

Limited proteolysis

In contrast to protein degradation, protein processing or
limited proteolysis can yield protein fragments with acquired
functions. In this Research Topic, Heidorn-Czarna et al. review
protein processing events within mitochondria in plants, yeast
and mammals. In addition to the primary processing of N-
terminal presequences, several secondary protein processing
events take place in mitochondria. While the action of some
proteases is evolutionary conserved, limited proteolysis
orchestrated by other mitochondrial proteases such as
rhomboid proteases or the ATP23 metalloprotease seem to
have evolutionarily diverged. The study of mitochondrial
processing events in plants is scarce, necessitating additional
studies using N-terminal proteomics to expand the substrate
repertoire and study the function of mitochondrial proteases in
plants. Staying on the topic of limited proteolysis, De Backer
et al. review membrane-bound transcription factors (MB-TFs)
that can be released by proteolytic processing or via alternative
splicing that eliminates the transmembrane domain (TMDs).
While a total of 64 high-confidence MB-TFs are present in
Arabidopsis (Yao et al., 2017), activation mechanisms are only
elucidated for three bZIP TFs. In silico analyses pointed out 18
MB-TFs with alternative transcripts lacking TMDs and 11 MB-
TFs helix-breaking motifs inside the TMD, which are a typical
structural feature facilitating regulated intramembrane
proteolysis. For identification of responsible proteases,
multiplexed gene editing, N-terminal proteomics, chemical
biology, and proximity-based labeling form promising avenues
to explore in the future.

Author contributions

All authors jointly defined the content of the Research Topic
and participated in the editing process. All authors listed have
made a substantial, direct, and intellectual contribution to the work and approved it for publication.

## Funding

This work was supported by the Research Foundation-Flanders (Junior Postdoctoral fellowship grant no. 12T1722N to PW).

## Acknowledgments

We genuinely acknowledge the contribution of every author, reviewer, and editor that made this Research Topic possible.

## References

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Gibbs, D. J., Lee, S. C., Isa, N. M., Gramuglia, S., Fukao, T., Bassel, G. W., et al. (2011). Homeostatic response to hypoxia is regulated by the n-end rule pathway in plants. *Nature* 479 (7373), 415–418. doi: 10.1038/nature10534

Goslin, K., Eschen-Lippold, L., Naumann, C., Linster, E., Sorel, M., Klecker, M., et al. (2019). Differential n-end rule degradation of RIN4/NOI fragments generated by the AvrRpt2 effector protease. *Plant Physiol.* 180 (4), 2272–2289. doi: 10.1104/ pp.19.00251

Huber, M., Bienvenut, W. V., Linster, E., Stephan, I., Armbruster, L., Sticht, C., et al. (2020). NatB-mediated n-terminal acetylation affects growth and biotic stress responses. *Plant Physiol.* 182 (2), 792–806. doi: 10.1104/ pp.19.00792

## Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

## Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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