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Proteolysis is an essential process to maintain cellular homeostasis. One pathway that mediates selective protein degradation and which is in principle conserved throughout the kingdoms of life is the N-degron pathway, formerly called the ‘N-end rule’. In the cytosol of eukaryotes and prokaryotes, N-terminal residues can be major determinants of protein stability. While the eukaryotic N-degron pathway depends on the ubiquitin proteasome system, the prokaryotic counterpart is driven by the Clp protease system. Plant chloroplasts also contain such a protease network, which suggests that they might harbor an organelle specific N-degron pathway similar to the prokaryotic one. Recent discoveries indicate that the N-terminal region of proteins affects their stability in chloroplasts and provides support for a Clp-mediated entry point in an N-degron pathway in plastids. This review discusses structure, function and specificity of the chloroplast Clp system, outlines experimental approaches to test for an N-degron pathway in chloroplasts, relates these aspects into general plastid proteostasis and highlights the importance of an understanding of plastid protein turnover.

A new study shows that mitochondrial retrograde signaling relies on strongly compartmentalized individual pathways previously not taken into account. This involves a link between mitochondrial oxygen consumption and cytosolic oxygen sensing via the N-degron pathway.

A variety of methods for studying glandular leaf hairs (trichomes) as multicellular micro-organs are well established for synthetic biology platforms like tobacco or tomato but rather rare for nonglandular and usually single-celled trichomes of the model plant Arabidopsis thaliana. A thorough isolation of-ideally intact-trichomes is decisive for further biochemical and genomic analyses of primary and secondary metabolic compounds, enzymes, and especially transcripts to monitor initial success of an engineering approach. While isolation of tomato or tobacco trichomes is rather easy, by simply freezing whole plants in liquid nitrogen and brushing off trichomes, this approach does not work for Arabidopsis. This is mainly due to damage of trichome cells during the collection procedure and very low yield. Here, we provide a robust method for a virtually epithelial cell-free isolation of Arabidopsis trichomes. This method is then joined with an RNA isolation protocol to perform mRNA analysis on extracts of the isolated trichomes using a semi-quantitative RT-PCR setup.

Studying the stability of a protein dependent on its N-terminal residue requires a mechanism, which selectively exposes the amino acid at the N-terminus. Here, we describe the use of the tobacco etch virus (TEV) protease to generate a specific N-terminal amino acid in the stroma of the chloroplast. The established molecular reporter system further allows the quantification of the reporter protein half-life dependent on the identity of the N-terminal residue.

The N-degron pathway, formerly the N-end rule pathway, regulates functions of regulatory proteins. It impacts protein half-life and therefore directs the actual presence of target proteins in the cell. The current concept holds that the N-degron pathway depends on the identity of the amino (N)-terminal amino acid and many other factors, such as the follow-up sequence at the N terminus, conformation, flexibility, and protein localization. It is evolutionarily conserved throughout the kingdoms. One possible entry point for substrates of the N-degron pathway is oxidation of N-terminal Cys residues. Oxidation of N-terminal Cys is decisive for further enzymatic modification of various neo–N termini by arginylation that generates potentially neofunctionalized or instable proteoforms. Here, I focus on the posttranslational modifications that are encompassed by protein degradation via the Cys/Arg branch of the N-degron pathway—part of the PROTEOLYSIS 6 (PRT6)/N-degron pathway—as well as the underlying physiological principles of this branch and its biological significance in stress response.

Toxic proteins are prime targets for molecular farming (the generation of pharmacologically active or biotechnologically usable compounds in plants) and are also efficient tools for targeted cell ablation in genetics, developmental biology, and biotechnology. However, achieving conditional activity of cytotoxins and maintaining the toxin-expressing plants as stably transformed lines remain challenging. Here, we produce a switchable version of the highly cytotoxic bacterial RNase barnase by fusing the protein to a portable protein degradation cassette, the low-temperature degron cassette. This method allows conditional genetics based on conditional protein degradation via the N-end rule or N-degron pathway and has been used to vice versa accumulate and/or deplete a diverse variety of highly active, unstable or stable target proteins in different living multicellular organisms and cell systems. Moreover, we expressed the barnase fusion under control of the trichome-specific TRIPTYCHON promoter. This enabled efficient temperature-dependent control of protein accumulation in Arabidopsis (Arabidopsis thaliana) leaf hairs (trichomes). By tuning the levels of the protein, we were able to control the fate of trichomes in vivo. The on-demand formation of trichomes through manipulating the balance between stabilization and destabilization of barnase provides proof of concept for a robust and powerful tool for conditional switchable cell arrest. We present this tool as a potential strategy for the manufacture and accumulation of cytotoxic proteins and toxic high-value products in plants or for conditional genetic cell ablation.

Significance Cyclin-dependent kinases are the main drivers of the mitotic cell cycle. Here, we show that the activity of the main cell-cycle regulator in the model plant Arabidopsis, CDKA;1, also governs one of the most important processes in meiosis: the formation of meiotic cross-overs. We show that CDKA;1 activity especially affects the major class of meiotic cross-overs, known as class I cross-overs. We find that lowering kinase activity leads to a progressive loss of cross-overs, of which cross-overs near the chromosome ends are the last to disappear. Conversely, an increase of kinase activity increases the rate of cross-over formation., Little is known how patterns of cross-over (CO) numbers and distribution during meiosis are established. Here, we reveal that cyclin-dependent kinase A;1 (CDKA;1), the homolog of human Cdk1 and Cdk2, is a major regulator of meiotic recombination in Arabidopsis. Arabidopsis plants with reduced CDKA;1 activity experienced a decrease of class I COs, especially lowering recombination rates in centromere-proximal regions. Interestingly, this reduction of type I CO did not affect CO assurance, a mechanism by which each chromosome receives at least one CO, resulting in all chromosomes exhibiting similar genetic lengths in weak loss-of-function cdka;1 mutants. Conversely, an increase of CDKA;1 activity resulted in elevated recombination frequencies. Thus, modulation of CDKA;1 kinase activity affects the number and placement of COs along the chromosome axis in a dose-dependent manner.

Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilization of group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation. The PLANT CYSTEINE OXIDASEs (PCOs) have been identified as catalysts of this oxidation. ERF-VII stabilization in hypoxia presumably arises from reduced PCO activity. We directly demonstrate that PCO dioxygenase activity produces Cys-sulfinic acid at the N terminus of an ERF-VII peptide, which then undergoes efficient arginylation by an arginyl transferase (ATE1). This provides molecular evidence of N-terminal Cys-sulfinic acid formation and arginylation by N-end rule pathway components, and a substrate of ATE1 in plants. The PCOs and ATE1 may be viable intervention targets to stabilize N-end rule substrates, including ERF-VIIs, to enhance submergence tolerance in agriculture., The N-end rule pathway targets substrate proteins for proteasomal degradation. Here, White et al. show that Arabidopsis PLANT CYSTEINE OXIDASEs show dioxygenase activity producing Cys-sulfinic acid at the N-terminus of target proteins, which then act as direct substrates for arginyl transferase.

AtERF#111/ABR1 belongs to the group X of the ERF/AP2 transcription factor family (GXERFs) and is shoot specifically induced under submergence and hypoxia. It was described to be an ABA-response repressor, but our data reveal a completely different function. Surprisingly, AtERF#111 expression is strongly responsive to wounding stress. Expression profiling of ERF#111-overexpressing (OE) plants, which show morphological phenotypes like increased root hair length and number, strengthens the hypothesis of AtERF#111 being involved in the wounding response, thereby acting as a transcriptional activator of gene expression. Consistent with a potential function outside of oxygen signalling, we could not assign AtERF#111 as a target of the PRT6 N-degron pathway, even though it starts with a highly conserved N-terminal Met-Cys (MC) motif. However, the protein is unstable as it is degraded in an ubiquitin-dependent manner. Finally, direct target genes of AtERF#111 were identified by microarray analyses and subsequently confirmed by protoplast transactivation assays. The special roles of diverse members of the plant-specific GXERFs in coordinating stress signalling and wound repair mechanisms have been recently hypothesized, and our data suggest that AtERF#111 is indeed involved in these processes.

CDKA;1, the Arabidopsis orthologue of Cdk1 and Cdk2, controls microtubule organization in meiosis. Sofroni et al. find that reducing CDKA;1 activity converts simultaneous cytokinesis—the separation of all four meiotic products concomitantly—into two successive cytokineses after the first and second meiotic divisions, as found in many crop species., Precise control of cytoskeleton dynamics and its tight coordination with chromosomal events are key to cell division. This is exemplified by formation of the spindle and execution of cytokinesis after nuclear division. Here, we reveal that the central cell cycle regulator CYCLIN DEPENDENT KINASE A;1 (CDKA;1), the Arabidopsis homologue of Cdk1 and Cdk2, partially in conjunction with CYCLIN B3;1 (CYCB3;1), is a key regulator of the microtubule cytoskeleton in meiosis. For full CDKA;1 activity, the function of three redundantly acting CDK-activating kinases (CAKs), CDKD;1, CDKD;2, and CDKD;3, is necessary. Progressive loss of these genes in combination with a weak loss-of-function mutant in CDKA;1 allowed a fine-grained dissection of the requirement of cell-cycle kinase activity for meiosis. Notably, a moderate reduction of CDKA;1 activity converts the simultaneous cytokinesis in Arabidopsis, i.e., one cytokinesis separating all four meiotic products concurrently into two successive cytokineses with cell wall formation after the first and second meiotic division, as found in many monocotyledonous species.

The protein RPM1-INTERACTING PROTEIN4 (RIN4) is a central regulator of both layers of plant immunity systems, the so-called pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). RIN4 is targeted by several effectors, including thePseudomonas syringaeprotease effector AvrRpt2. Cleavage of RIN4 by AvrRpt2 generates unstable RIN4 fragments, whose degradation leads to the activation of the resistance protein RPS2 (RESISTANT TO P. SYRINGAE2). Hence, identifying the determinants of RIN4 degradation is key to understanding RPS2-mediated ETI, as well as virulence functions of AvrRpt2. In addition to RIN4, AvrRpt2 cleaves host proteins from the nitrate-induced (NOI) domain family. Although cleavage of NOI-domain proteins by AvrRpt2 may contribute to PTI regulation, the (in)stability of these proteolytic fragments and the determinants that regulate their stability have not been examined. Notably, a common feature of RIN4 and of many NOI-domain protein fragments generated by AvrRpt2 cleavage is the exposure of a new N-terminal residue that is destabilizing according to the N-end rule. Using antibodies raised against endogenous RIN4, we show that the destabilization of AvrRpt2-cleaved RIN4 fragments is independent of the N-end rule pathway (recently renamed N-degron pathway). By contrast, several NOI-domain protein fragments arebona fidesubstrates of the N-degron pathway. The discovery of this novel set of substrates considerably expands the number of proteins targeted for degradation by this ubiquitin-dependent pathway, for which very few physiological substrates are known in plants. Our results also open new avenues of research to understand the role of AvrRpt2 in promoting bacterial virulence.One sentence summaryAnalysis of RIN4/NOI fragments released after cleavage by the bacterial effector protease AvrRpt2 reveals a novel role of the N-end rule in the degradation of NOI-domain proteins, but not of RIN4.

© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust The N-end rule pathway is a highly conserved constituent of the ubiquitin proteasome system, yet little is known about its biological roles. Here we explored the role of the N-end rule pathway in the plant immune response. We investigated the genetic influences of components of the pathway and known protein substrates on physiological, biochemical and metabolic responses to pathogen infection. We show that the glutamine (Gln) deamidation and cysteine (Cys) oxidation branches are both components of the plant immune system, through the E3 ligase PROTEOLYSIS (PRT)6. In Arabidopsis thaliana Gln-specific amino-terminal (Nt)-amidase (NTAQ1) controls the expression of specific defence-response genes, activates the synthesis pathway for the phytoalexin camalexin and influences basal resistance to the hemibiotroph pathogen Pseudomonas syringae pv tomato (Pst). The Nt-Cys ETHYLENE RESPONSE FACTOR VII transcription factor substrates enhance pathogen-induced stomatal closure. Transgenic barley with reduced HvPRT6 expression showed enhanced resistance to Ps. japonica and Blumeria graminis f. sp. hordei, indicating a conserved role of the pathway. We propose that that separate branches of the N-end rule pathway act as distinct components of the plant immune response in flowering plants.

SUMMARYToxic proteins are prime targets for molecular farming and efficient tools for targeted cell ablation in genetics, developmental biology, and biotechnology. Achieving conditional activity of cytotoxins and their maintenance in form of stably transformed transgenes is challenging. We demonstrate here a switchable version of the highly cytotoxic bacterial ribonuclease barnase by using efficient temperature-dependent control of protein accumulation in living multicellular organisms. By tuning the levels of the protein, we were able to control the fate of a plant organin vivo. The on-demand-formation of specialized epidermal cells (trichomes) through manipulating stabilization versus destabilization of barnase is a proof-of-concept for a robust and powerful tool for conditional switchable cell arrest. We present this tool both as a potential novel strategy for the manufacture and accumulation of cytotoxic proteins and toxic high-value products in plants or for conditional genetic cell ablation.

International audience; Autophagy is essential for protein degradation, nutrient recycling, and nitrogen remobilization. Autophagy is induced during leaf ageing and in response to nitrogen starvation, and is known to play a fundamental role in nutrient recycling for remobilization and seed filling. Accordingly, ageing leaves of Arabidopsis autophagy mutants (atg) have been shown to over-accumulate proteins and peptides, possibly because of a reduced protein degradation capacity. Surprisingly, atg leaves also displayed higher protease activities. The work reported here aimed at identifying the nature of the proteases and protease activities that accumulated differentially (higher or lower) in the atg mutants. Protease identification was performed using shotgun LC-MS/MS proteome analyses and activity-based protein profiling (ABPP). The results showed that the chloroplast FTSH (FILAMENTATION TEMPERATURE SENSITIVE H) and DEG (DEGRADATION OF PERIPLASMIC PROTEINS) proteases and several extracellular serine proteases [subtilases (SBTs) and serine carboxypeptidase-like (SCPL) proteases] were less abundant in atg5 mutants. By contrast, proteasome-related proteins and cytosolic or vacuole cysteine proteases were more abundant in atg5 mutants. Rubisco degradation assays and ABPP showed that the activities of proteasome and papain-like cysteine protease were increased in atg5 mutants. Whether these proteases play a back-up role in nutrient recycling and remobilization in atg mutants or act to promote cell death is discussed in relation to their accumulation patterns in the atg5 mutant compared with the salicylic acid-depleted atg5/sid2 double-mutant, and in low nitrate compared with high nitrate conditions. Several of the proteins identified are indeed known as senescence- and stress-related proteases or as spontaneous cell-death triggering factors.

Despite a recent increase in interest towards phytoglobins and their importance in plants, much is still unknown regarding their biochemical/biophysical properties and physiological roles. The present study presents data on three recombinant Arabidopsis phytoglobins in terms of their UV-vis and Raman spectroscopic characteristics, redox state control, redox potentials and autoxidation rates. The latter are strongly influenced by pH for all three hemoglobins – (with a fundamental involvement of the distal histidine), as well as by added anion concentrations – suggesting either a process dominated by nucleophilic displacement of superoxide for AtHb2 or an inhibitory effect for AtHb1 and AtHb3. Reducing agents, such as ascorbate and glutathione, are found to either enhance– (presumably via direct electron transfer or via allosteric regulation) or prevent autoxidation. HbFe3+ reduction was possible in the presence of high (presumably not physiologically relevant) concentrations of NADH, glutathione and ascorbate, with differing behaviors for the three globins. The iron coordination sphere is found to affect the autoxidation, redox state interconversion and redox potentials in these three phytoglobins.

We applied an extended charge-based fractional diagonal chromatography (ChaFRADIC) workflow to analyze the N-terminal proteome of Arabidopsis thaliana seedlings. Using iTRAQ protein labeling and a multi-enzyme digestion approach including trypsin, GluC, and subtilisin, a total of 200 μg per enzyme, and measuring only one third of each ChaFRADIC-enriched fraction by LC-MS, we quantified a total of 2791 unique N-terminal peptides corresponding to 2249 different unique N-termini from 1270 Arabidopsis proteins. Our data indicate the power, reproducibility, and sensitivity of the applied strategy that might be applicable to quantify proteolytic events from as little as 20 μg of protein per condition across up to eight different samples. Furthermore, our data clearly reflect the methionine excision dogma as well as the N-end rule degradation pathway (NERP) discriminating into a stabilizing or destabilizing function of N-terminal amino acid residues. We found bona fide NERP destabilizing residues underrepresented, and the list of neo N-termini from wild type samples may represent a helpful resource during the evaluation of NERP substrate candidates. All MS data have been deposited in the ProteomeXchange with identifier PXD001855 (http://proteomecentral.proteomexchange.org/dataset/PXD001855).

Determination of the general capacity of proteolytic activity of a certain cell or tissue type can be crucial for an assessment of various features of an organism's growth and development and also for the optimization of biotechnological applications. Here, we describe the use of chimeric protein stability reporters that can be detected by standard laboratory techniques such as histological staining, selection using selective media or fluorescence microscopy. Dependent on the expression of the reporters due to the promoters applied, cell- and tissue-specific questions can be addressed. Here, we concentrate on methods which can be used for large-scale screening for protein stability changes rather than for detailed protein stability studies.

Conditional modulation of biological processes plays key roles in basic and applied research and in translation. It can be achieved on various levels via a multitude of approaches. One of the directions is manipulating target protein levels and activity by transcriptional, posttranscriptional, translational, and posttranslational control. Because in most of these techniques, the synthesis of the target proteins is adjusted to the needs, they all rely on the specific half-life of the target protein and its turn-over. Therefore, their time-of-action, in direct correlation to the desired reprogramming of molecular phenotypes caused by altering the target levels, is fixed and determined by the naturally inherent properties. We have introduced the low-temperature degron (lt-degron) to various intact multicellular organisms which allows to control target protein levels and therefore function and activity directly on the level of active protein. The lt-degron uses a combination of Ubiquitin-fusion technique linking target protein degradation to the N-end rule pathway of targeted proteolysis coupled with the use of cell- and tissue-specific promoters.

N-term 2017 was the first international meeting to bring together researchers from diverse disciplines with a shared interest in protein N-terminal modifications and the N-end rule pathway of ubiquitin-mediated proteolysis, providing a platform for interdisciplinary cross-kingdom discussions and collaborations, as well as strengthening the visibility of this growing scientific community.

ummary The N-end rule relates the stability of a protein to the identity of its N-terminal residue and some of its modifications. Since its discovery in the 1980s, the repertoire of N-terminal degradation signals has expanded, leading to a diversity of N-end rule pathways. Although some of these newly discovered N-end rule pathways remain largely unexplored in plants, recent discoveries have highlighted roles of N-end rule-mediated protein degradation in plant defense against pathogens and in cell proliferation during organ growth. Despite this progress, a bottleneck remains the proteome-wide identification of N-end rule substrates due to the prerequisite for endoproteolytic cleavage and technical limitations. Here, we discuss the recent diversification of N-end rule pathways and their newly discovered functions in plant defenses, stressing the role of proteases. We expect that novel proteomics techniques (N-terminomics) will be essential for substrate identification. We review these methods, their limitations and future developments.

Determination of the general capacity of proteolytic activity of a certain cell or tissue type can be crucial for an assessment of various features of an organism's growth and development and also for the optimization of biotechnological applications. Here, we describe the use of chimeric protein stability reporters that can be detected by standard laboratory techniques such as histological staining, selection using selective media or fluorescence microscopy. Dependent on the expression of the reporters due to the promoters applied, cell- and tissue-specific questions can be addressed. Here, we concentrate on methods which can be used for large-scale screening for protein stability changes rather than for detailed protein stability studies.

Conditional modulation of biological processes plays key roles in basic and applied research and in translation. It can be achieved on various levels via a multitude of approaches. One of the directions is manipulating target protein levels and activity by transcriptional, posttranscriptional, translational, and posttranslational control. Because in most of these techniques, the synthesis of the target proteins is adjusted to the needs, they all rely on the specific half-life of the target protein and its turn-over. Therefore, their time-of-action, in direct correlation to the desired reprogramming of molecular phenotypes caused by altering the target levels, is fixed and determined by the naturally inherent properties. We have introduced the low-temperature degron (lt-degron) to various intact multicellular organisms which allows to control target protein levels and therefore function and activity directly on the level of active protein. The lt-degron uses a combination of Ubiquitin-fusion technique linking target protein degradation to the N-end rule pathway of targeted proteolysis coupled with the use of cell- and tissue-specific promoters.

Targeted proteomics has become increasingly popular recently because of its ability to precisely quantify selected proteins in complex cellular backgrounds. Here, we demonstrated the utility of an LTQ-Orbitrap Velos Pro mass spectrometer in targeted parallel reaction monitoring (PRM) despite its unconventional dual ion trap configuration. We evaluated absolute specificity (>99%) and sensitivity (100 amol on column in 1 μg of total cellular extract) using full and mass range scans as survey scans together with data-dependent (DDA) and targeted MS/MS acquisition. The instrument duty cycle was a critical parameter limiting sensitivity, necessitating peptide retention time scheduling. We assessed synthetic peptide and recombinant peptide standards to predict or experimentally determine target peptide retention times. We applied optimized PRM to protein degradation in signaling regulation, an area that is receiving increased attention in plant physiology. We quantified relative abundance of selected proteins in plants that are mutant for enzymatic components of the N-end rule degradation (NERD) pathway such as the two tRNA-arginyl-transferases ATE1 and ATE2 and the two E3 ubiquitin ligases PROTEOLYSIS1 and 6. We found a number of upregulated proteins, which might represent degradation targets. We also targeted FLAGELLIN SENSITIVE2 (FLS2), a pattern recognition receptor responsible for pathogen sensing, in ubiquitin ligase mutants to assay the attenuation of plant immunity by degradation of the receptor.

Summary The N‐end rule pathway has emerged as a major system for regulating protein functions by controlling their turnover in medical, animal and plant sciences as well as agriculture. Although novel functions and enzymes of the pathway have been discovered, the ubiquitination mechanism and substrate specificity of N‐end rule pathway E3 ubiquitin ligases have remained elusive. Taking the first discovered bona fide plant N‐end rule E3 ligase PROTEOLYSIS1 (PRT1) as a model, we used a novel tool to molecularly characterize polyubiquitination live, in real time.We gained mechanistic insights into PRT1 substrate preference and activation by monitoring live ubiquitination using a fluorescent chemical probe coupled to artificial substrate reporters. Ubiquitination was measured by rapid in‐gel fluorescence scanning as well as in real time by fluorescence polarization.The enzymatic activity, substrate specificity, mechanisms and reaction optimization of PRT1‐mediated ubiquitination were investigated ad hoc instantaneously and with significantly reduced reagent consumption.We demonstrated that PRT1 is indeed an E3 ligase, which has been hypothesized for over two decades. These results demonstrate that PRT1 has the potential to be involved in polyubiquitination of various substrates and therefore pave the way to understanding recently discovered phenotypes of prt1 mutants.

The characteristic shapes and sizes of organs are established by cell proliferation patterns and final cell sizes, but the underlying molecular mechanisms coordinating these are poorly understood. Here we characterize a ubiquitin-activated peptidase called DA1 that limits the duration of cell proliferation during organ growth in Arabidopsis thaliana. The peptidase is activated by two RING E3 ligases, Big Brother (BB) and DA2, which are subsequently cleaved by the activated peptidase and destabilized. In the case of BB, cleavage leads to destabilization by the RING E3 ligase PROTEOLYSIS 1 (PRT1) of the N-end rule pathway. DA1 peptidase activity also cleaves the deubiquitylase UBP15, which promotes cell proliferation, and the transcription factors TEOSINTE BRANCED 1/CYCLOIDEA/PCF 15 (TCP15) and TCP22, which promote cell proliferation and repress endoreduplication. We propose that DA1 peptidase activity regulates the duration of cell proliferation and the transition to endoreduplication and differentiation during organ formation in plants by coordinating the destabilization of regulatory proteins.

SUMMARYThe N-end rule pathway has emerged as a major system for regulating protein functions by controlling their turn-over in medical, animal and plant sciences as well as agriculture. Although novel functions and enzymes of the pathway were discovered, ubiquitination mechanism and substrate specificity of N-end rule pathway E3 Ubiquitin ligases remained elusive. Taking the first discovered bona fide plant N-end rule E3 ligase PROTEOLYSIS1 (PRT1) as a model, we use a novel tool to molecularly characterize polyubiquitination live, in real-time.We gained mechanistic insights in PRT1 substrate preference and activation by monitoring live ubiquitination by using a fluorescent chemical probe coupled to artificial substrate reporters. Ubiquitination was measured by rapid in-gel fluorescence scanning as well as in real time by fluorescence polarization.Enzymatic activity, substrate specificity, mechanisms and reaction optimization of PRT1-mediated ubiquitination were investigated ad hoc in short time and with significantly reduced reagent consumption.We demonstrated for the first time that PRT1 is indeed an E3 ligase, which was hypothesized for over two decades. These results demonstrate that PRT1 has the potential to be involved in polyubiquitination of various substrates and therefore pave the way to understanding recently discovered phenotypes of prt1 mutants.

Western blot (WB) analysis is the most widely used method to monitor expression of proteins of interest in protein extracts of high complexity derived from diverse experimental setups. WB allows the rapid and specific detection of a target protein, such as non-tagged endogenous proteins as well as protein-epitope tag fusions depending on the availability of specific antibodies. To generate quantitative data from independent samples within one experiment and to allow accurate inter-experimental quantification, a reliable and reproducible method to standardize and normalize WB data is indispensable. To date, it is a standard procedure to normalize individual bands of immunodetected proteins of interest from a WB lane to other individual bands of so-called housekeeping proteins of the same sample lane. These are usually detected by an independent antibody or colorimetric detection and do not reflect the real total protein of a sample. Housekeeping proteins-assumed to be constitutively expressed mostly independent of developmental and environmental states-can greatly differ in their expression under these various conditions. Therefore, they actually do not represent a reliable reference to normalize the target protein's abundance to the total amount of protein contained in each lane of a blot.Here, we demonstrate the Smart Protein Layers (SPL) technology, a combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer via WB. SPL allows a rapid and highly sensitive protein visualization and quantification with a sensitivity comparable to conventional silver staining with a 1000-fold higher dynamic range. For normalization, standardization and quantification of protein gels and WBs, a sample-dependent bi-fluorescent standard reagent is applied and, for accurate quantification of data derived from different experiments, a second calibration standard is used. Together, the precise quantification of protein expression by lane-to-lane, gel-to-gel, and blot-to-blot comparisons is facilitated especially with respect to experiments in the area of proteostasis dealing with highly variable protein levels and involving protein degradation mutants and treatments modulating protein abundance.

In order to determine the stability of a protein or protein fragment dependent on its N-terminal amino acid, and therefore relate its half-life to the N-end rule pathway of targeted protein degradation (NERD), non-Methionine (Met) amino acids need to be exposed at their amino terminal in most cases. Per definition, at this position, destabilizing residues are generally unlikely to occur without further posttranslational modification of immature (pre-)proproteins. Moreover, almost exclusively, stabilizing, or not per se destabilizing residues are N-terminally exposed upon Met excision by Met aminopeptidases. To date, there exist two prominent protocols to study the impact of destabilizing residues at the N-terminal of a given protein by selectively exposing the amino acid residue to be tested. Such proteins can be used to study NERD substrate candidates and analyze NERD enzymatic components. Namely, the well-established ubiquitin fusion technique (UFT) is used in vivo or in cell-free transcription/translation systems in vitro to produce a desired N-terminal residue in a protein of interest, whereas the proteolytic cleavage of recombinant fusion proteins by tobacco etch virus (TEV) protease is used in vitro to purify proteins with distinct N-termini. Here, we discuss how to accomplish in vivo and in vitro expression and modification of NERD substrate proteins that may be used as stability tester or activity reporter proteins and to characterize potential NERD substrates.The methods to generate artificial substrates via UFT or TEV cleavage are described here and can be used either in vivo in the context of stably transformed plants and cell culture expressing chimeric constructs or in vitro in cell-free systems such as rabbit reticulocyte lysate as well as after expression and purification of recombinant proteins from various hosts.

The automated SPOT (synthetic peptide arrays on membrane support technique) synthesis technology has entrenched as a rapid and robust method to generate peptide libraries on cellulose membrane supports. The synthesis method is based on conventional Fmoc chemistry building up peptides with free N-terminal amino acids starting at their cellulose-coupled C-termini. Several hundreds of peptide sequences can be assembled with this technique on one membrane comprising a strong binding potential due to high local peptide concentrations. Peptide orientation on SPOT membranes qualifies this array type for assaying substrate specificities of N-recognins, the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Pioneer studies described binding capability of mammalian and yeast enzymes depending on a peptide's N-terminus. SPOT arrays have been successfully used to describe substrate specificity of N-recognins which are the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Here, we describe the implementation of SPOT binding assays with focus on the identification of N-recognin substrates, applicable also for plant NERD enzymes.

Western blot (WB) analysis is the most widely used method to monitor expression of proteins of interest in protein extracts of high complexity derived from diverse experimental setups. WB allows the rapid and specific detection of a target protein, such as non-tagged endogenous proteins as well as protein-epitope tag fusions depending on the availability of specific antibodies. To generate quantitative data from independent samples within one experiment and to allow accurate inter-experimental quantification, a reliable and reproducible method to standardize and normalize WB data is indispensable. To date, it is a standard procedure to normalize individual bands of immunodetected proteins of interest from a WB lane to other individual bands of so-called housekeeping proteins of the same sample lane. These are usually detected by an independent antibody or colorimetric detection and do not reflect the real total protein of a sample. Housekeeping proteins-assumed to be constitutively expressed mostly independent of developmental and environmental states-can greatly differ in their expression under these various conditions. Therefore, they actually do not represent a reliable reference to normalize the target protein's abundance to the total amount of protein contained in each lane of a blot.Here, we demonstrate the Smart Protein Layers (SPL) technology, a combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer via WB. SPL allows a rapid and highly sensitive protein visualization and quantification with a sensitivity comparable to conventional silver staining with a 1000-fold higher dynamic range. For normalization, standardization and quantification of protein gels and WBs, a sample-dependent bi-fluorescent standard reagent is applied and, for accurate quantification of data derived from different experiments, a second calibration standard is used. Together, the precise quantification of protein expression by lane-to-lane, gel-to-gel, and blot-to-blot comparisons is facilitated especially with respect to experiments in the area of proteostasis dealing with highly variable protein levels and involving protein degradation mutants and treatments modulating protein abundance.

In order to determine the stability of a protein or protein fragment dependent on its N-terminal amino acid, and therefore relate its half-life to the N-end rule pathway of targeted protein degradation (NERD), non-Methionine (Met) amino acids need to be exposed at their amino terminal in most cases. Per definition, at this position, destabilizing residues are generally unlikely to occur without further posttranslational modification of immature (pre-)proproteins. Moreover, almost exclusively, stabilizing, or not per se destabilizing residues are N-terminally exposed upon Met excision by Met aminopeptidases. To date, there exist two prominent protocols to study the impact of destabilizing residues at the N-terminal of a given protein by selectively exposing the amino acid residue to be tested. Such proteins can be used to study NERD substrate candidates and analyze NERD enzymatic components. Namely, the well-established ubiquitin fusion technique (UFT) is used in vivo or in cell-free transcription/translation systems in vitro to produce a desired N-terminal residue in a protein of interest, whereas the proteolytic cleavage of recombinant fusion proteins by tobacco etch virus (TEV) protease is used in vitro to purify proteins with distinct N-termini. Here, we discuss how to accomplish in vivo and in vitro expression and modification of NERD substrate proteins that may be used as stability tester or activity reporter proteins and to characterize potential NERD substrates.The methods to generate artificial substrates via UFT or TEV cleavage are described here and can be used either in vivo in the context of stably transformed plants and cell culture expressing chimeric constructs or in vitro in cell-free systems such as rabbit reticulocyte lysate as well as after expression and purification of recombinant proteins from various hosts.

SummaryCyclin-dependent kinases (CDKs) are at the heart of eukaryotic cell-cycle control. The yeast Cdc2/CDC28 PSTAIRE kinase and its orthologs such as the mammalian Cdk1 have been found to be indispensable for cell-cycle progression in all eukaryotes investigated so far. CDKA;1 is the only PSTAIRE kinase in the flowering plant Arabidopsis and can rescue Cdc2/CDC28 mutants. Here, we show that cdka;1 null mutants are viable but display specific cell-cycle and developmental defects, e.g., in S phase entry and stem cell maintenance. We unravel that the crucial function of CDKA;1 is the control of the plant Retinoblastoma homolog RBR1 and that codepletion of RBR1 and CDKA;1 rescued most defects of cdka;1 mutants. Our work further revealed a basic cell-cycle control system relying on two plant-specific B1-type CDKs, and the triple cdk mutants displayed an early germline arrest. Taken together, our data indicate divergent functional differentiation of Cdc2-type kinases during eukaryote evolution.

Recent studies have shown that cyclin-dependent kinase (CDK) inhibitors can have a tremendous impact on cell cycle progression in plants. In animals, CDK inhibitors are tightly regulated, especially by posttranslational mechanisms of which control of nuclear access and regulation of protein turnover are particularly important. Here we address the posttranslational regulation of INHIBITOR/INTERACTOR OF CDK 1 (ICK1)/KIP RELATED PROTEIN 1 (KRP1), an Arabidopsis (Arabidopsis thaliana) CDK inhibitor. We show that ICK1/KRP1 exerts its function in the nucleus and its presence in the nucleus is controlled by multiple nuclear localization signals as well as by nuclear export. In addition, we show that ICK1/KRP1 localizes to different subnuclear domains, i.e. in the nucleoplasm and to the chromocenters, hinting at specific actions within the nuclear compartment. Localization to the chromocenters is mediated by an N-terminal domain, in addition we find that this domain may be involved in cyclin binding. Further we demonstrate that ICK1/KRP1 is an unstable protein and degraded by the 26S proteasome in the nucleus. This degradation is mediated by at least two domains indicating the presence of at least two different pathways impinging on ICK1/KRP1 protein stability.

Conditional gene expression and modulating protein stability under physiological conditions are important tools in biomedical research. They led to a thorough understanding of the roles of many proteins in living organisms. Current protocols allow for manipulating levels of DNA, mRNA, and of functional proteins. Modulating concentrations of proteins of interest, their post-translational processing, and their targeted depletion or accumulation are based on a variety of underlying molecular modes of action. Several available tools allow a direct as well as rapid and reversible variation right on the spot, i.e., on the level of the active form of a gene product. The methods and protocols discussed here include inducible and tissue-specific promoter systems as well as portable degrons derived from instable donor sequences. These are either constitutively active or dormant so that they can be triggered by exogenous or developmental cues. Many of the described techniques here directly influencing the protein stability are established in yeast, cell culture and in vitro systems only, whereas the indirectly working promoter-based tools are also commonly used in higher eukaryotes. Our major goal is to link current concepts of conditionally modulating a protein of interest’s activity and/or abundance and approaches for generating cell and tissue types on demand in living, multicellular organisms with special emphasis on plants.

Almost two billion years of evolution have generated a vast and amazing variety of eukaryotic life with approximately 8.7 million extant species. Growth and reproduction of all of these organisms depend on faithful duplication and distribution of their chromosomes to the newly forming daughter cells in a process called the cell cycle. However, most of what is known today about cell cycle control comes from a few model species that belong to the unikonts; that is, to only one of five 'supergroups' that comprise the eukaryotic kingdom. Recently, analyzing species from distantly related clades is providing insights into general principles of cell cycle regulation and shedding light on its evolution. Here, referring to animal and fungal as opposed to non-unikont systems, especially flowering plants from the archaeplastid supergroup, we compare the conservation of central cell cycle regulator functions, the structure of network topologies, and the evolutionary dynamics of substrates of core cell cycle kinases.

The decision to replicate its DNA is of crucial importance for every cell and, in many organisms, is decisive for the progression through the entire cell cycle. A comparison of animals versus yeast has shown that, although most of the involved cell-cycle regulators are divergent in both clades, they fulfill a similar role and the overall network topology of G1/S regulation is highly conserved. Using germline development as a model system, we identified a regulatory cascade controlling entry into S phase in the flowering plant Arabidopsis thaliana, which, as a member of the Plantae supergroup, is phylogenetically only distantly related to Opisthokonts such as yeast and animals. This module comprises the Arabidopsis homologs of the animal transcription factor E2F, the plant homolog of the animal transcriptional repressor Retinoblastoma (Rb)-related 1 (RBR1), the plant-specific F-box protein F-BOX-LIKE 17 (FBL17), the plant specific cyclin-dependent kinase (CDK) inhibitors KRPs, as well as CDKA;1, the plant homolog of the yeast and animal Cdc2+/Cdk1 kinases. Our data show that the principle of a double negative wiring of Rb proteins is highly conserved, likely representing a universal mechanism in eukaryotic cell-cycle control. However, this negative feedback of Rb proteins is differently implemented in plants as it is brought about through a quadruple negative regulation centered around the F-box protein FBL17 that mediates the degradation of CDK inhibitors but is itself directly repressed by Rb. Biomathematical simulations and subsequent experimental confirmation of computational predictions revealed that this regulatory circuit can give rise to hysteresis highlighting the here identified dosage sensitivity of CDK inhibitors in this network., Author Summary In order to grow, multicellular organisms need to multiply their cells. Cell proliferation is achieved through a complex order of events called the cell cycle, during which the nuclear DNA is duplicated and subsequently distributed to the newly forming daughter cells. The decision to replicate the nuclear DNA is in many organisms crucial to progress through the entire cell cycle. Alterations of the cell cycle, especially at the entry point, can cause severe developmental defects and are often causal for maladies, such as cancer. Substantial work in yeast and animals has revealed the regulatory steps controlling S-phase entry. In contrast, relatively little is known about the plant cell cycle despite plants being one of the largest classes of living organisms and despite the importance of plants for human life, for instance as the basis of human nutrition. Our work presents a molecular framework of core cell-cycle regulation for entry into the DNA replication phase in the model plant Arabidopsis. We report here the identification of a regulatory cascade that likely functions in many plant cells and organisms. With this, we also provide an important basis for comparative analyses of cell-cycle control between different eukaryotes, such as yeast and mammals.

Major progress has been made in unravelling of regulatory mechanisms in eukaryotic cells. Modification of target protein properties by reversible phosphorylation events has been found to be one of the most prominent cellular control processes in all organisms. The phospho-status of a protein is dynamically controlled by protein kinases and counteracting phosphatases. Therefore, monitoring of kinase and phosphatase activities, identification of specific phosphorylation sites, and assessment of their functional significance are of crucial importance to understand development and homeostasis. Recent advances in the area of molecular biology and biochemistry, for instance, mass spectrometry-based phosphoproteomics or fluorescence spectroscopical methods, open new possibilities to reach an unprecidented depth and a proteome-wide understanding of phosphorylation processes in plants and other species. In addition, the growing number of model species allows now deepening evolutionary insights into signal transduction cascades and the use of kinase/phosphatase systems. Thus, this is the age where we move from an understanding of the structure and function of individual protein modules to insights how these proteins are organized into pathways and networks. In this introductory chapter, we briefly review general definitions, methodology, and current concepts of the molecular mechanisms of protein kinase function as a foundation for this methods book. We briefly review biochemistry and structural biology of kinases and provide selected examples for the role of kinases in biological systems.

Enzyme-substrate interactions are weak and occur only transiently and thus, a faithful analysis of these interactions typically requires elaborated biochemical methodology. The bimolecular-fluorescence complementation (BiFC) assay, also referred to as split YFP assay, is a powerful and straightforward tool to test protein-protein interactions. This system is commonly used due to many advantages and especially due to its simple ease of use. BIFC relies on the reconstitution of an N-terminal and C-terminal half of YFP into a functional, i.e., fluorescent protein. Noteworthy, the dissociation constant of the two YFP halves is much lower than the association constant leading to a stabilization of the protein-protein interaction to be monitored. Whereas this property is sometimes critical, it also increases the sensitivity of the detection system by stabilizing transient interactions. Here, we exploit this property to detect and monitor interaction between a kinase and its substrate. In particular, we characterize with the BiFC system kinase-variants that show an altered substrate binding.

Biological information is often transmitted by phosphorylation cascades. However, the biological relevance of specific phosphorylation events is often difficult to determine. An invaluable tool to study the effect of kinases and/or phosphatases is the use of phospho- and dephospho-mimetic substitutions in the respective target proteins. Here, we present a generally applicable procedure of how to design, set-up, and carry out phosphorylation modulation experiments and subsequent monitoring of protein activities, taking -cyclin-dependent kinases (CDKs) as a case study. CDKs are key regulators of cell cycle progression in all eukaryotic cells. Consequently, CDKs are controlled at many levels and phosphorylation of CDKs -themselves is used to regulate their kinase activity. We describe in detail complementation experiments of a mutant in CDKA;1, the major cell cycle kinase in Arabidopsis, with phosphorylation-site variants of CDKA;1. CDKA;1 versions were generated either by mimicking a phosphorylated amino acid by replacing the respective residue with a negatively charged amino acid, e.g., aspartate or glutamate, or by mutating it to a non-phoshorylatable amino acid, such as alanine, valine, or phenylalanine. The genetic complementation studies were accompanied by the isolation of these kinase variants from plant extract and subsequent kinase assays to determine changes in their activity levels. This work allowed us to judge the importance of -posttranslational regulation of CDKA;1 in plants and has shown that the molecular mechanistics of CDK function are apparently conserved across the kingdoms. However, the regulatory wiring of CDKs is -strikingly different between plants, animals, and yeast.

Biological information is often transmitted by phosphorylation cascades. However, the biological relevance of specific phosphorylation events is often difficult to determine. An invaluable tool to study the effect of kinases and/or phosphatases is the use of phospho- and dephospho-mimetic substitutions in the respective target proteins. Here, we present a generally applicable procedure of how to design, set-up, and carry out phosphorylation modulation experiments and subsequent monitoring of protein activities, taking -cyclin-dependent kinases (CDKs) as a case study. CDKs are key regulators of cell cycle progression in all eukaryotic cells. Consequently, CDKs are controlled at many levels and phosphorylation of CDKs -themselves is used to regulate their kinase activity. We describe in detail complementation experiments of a mutant in CDKA;1, the major cell cycle kinase in Arabidopsis, with phosphorylation-site variants of CDKA;1. CDKA;1 versions were generated either by mimicking a phosphorylated amino acid by replacing the respective residue with a negatively charged amino acid, e.g., aspartate or glutamate, or by mutating it to a non-phoshorylatable amino acid, such as alanine, valine, or phenylalanine. The genetic complementation studies were accompanied by the isolation of these kinase variants from plant extract and subsequent kinase assays to determine changes in their activity levels. This work allowed us to judge the importance of -posttranslational regulation of CDKA;1 in plants and has shown that the molecular mechanistics of CDK function are apparently conserved across the kingdoms. However, the regulatory wiring of CDKs is -strikingly different between plants, animals, and yeast.

Major progress has been made in unravelling of regulatory mechanisms in eukaryotic cells. Modification of target protein properties by reversible phosphorylation events has been found to be one of the most prominent cellular control processes in all organisms. The phospho-status of a protein is dynamically controlled by protein kinases and counteracting phosphatases. Therefore, monitoring of kinase and phosphatase activities, identification of specific phosphorylation sites, and assessment of their functional significance are of crucial importance to understand development and homeostasis. Recent advances in the area of molecular biology and biochemistry, for instance, mass spectrometry-based phosphoproteomics or fluorescence spectroscopical methods, open new possibilities to reach an unprecidented depth and a proteome-wide understanding of phosphorylation processes in plants and other species. In addition, the growing number of model species allows now deepening evolutionary insights into signal transduction cascades and the use of kinase/phosphatase systems. Thus, this is the age where we move from an understanding of the structure and function of individual protein modules to insights how these proteins are organized into pathways and networks. In this introductory chapter, we briefly review general definitions, methodology, and current concepts of the molecular mechanisms of protein kinase function as a foundation for this methods book. We briefly review biochemistry and structural biology of kinases and provide selected examples for the role of kinases in biological systems.