Protein degrons and degradation: Exploring substrate recognition and pathway selection in plants.

Proteome composition is dynamic and influenced by many internal and external cues, including developmental signals, light availability, or environmental stresses. Protein degradation, in synergy with protein biosynthesis, allows cells to respond to various stimuli and adapt by reshaping the proteome. Protein degradation mediates the final and irreversible disassembly of proteins, which is important for protein quality control and to eliminate misfolded or damaged proteins, as well as entire organelles. Consequently, it contributes to cell resilience by buffering against protein or organellar damage caused by stresses. Moreover, protein degradation plays important roles in cell signaling, as well as transcriptional and translational events. The intricate task of recognizing specific proteins for degradation is achieved by specialized systems that are tailored to the substrate's physicochemical properties and subcellular localization. These systems recognize diverse substrate cues collectively referred to as "degrons", which can assume a range of structural configurations. They are molecular surfaces recognized by E3 ligases of the ubiquitin-proteasome system, but can also be considered as general features recognized by other degradation systems, including autophagy or even organellar proteases. Here we provide an overview of the newest developments in the field, delving into the intricate processes of protein recognition and elucidating the pathways through which they are recruited for degradation.

Erasing marks: Functions of plant deubiquitylating enzymes in modulating the ubiquitin code.

Plant cells need to respond to environmental stimuli and developmental signals accurately and promptly. Ubiquitylation is a reversible posttranslational modification that enables the adaptation of cellular proteostasis to internal or external factors. The different topologies of ubiquitin linkages serve as the structural basis for the ubiquitin code, which can be interpreted by ubiquitin-binding proteins or readers in specific processes. The ubiquitylation status of target proteins is regulated by ubiquitylating enzymes or writers, and deubiquitylating enzymes (DUBs) or erasers. DUBs can remove ubiquitin molecules from target proteins. Arabidopsis (A. thaliana) DUBs belong to seven protein families and exhibit a wide range of functions and play an important role in regulating selective protein degradation processes, including proteasomal-, endocytic-, and autophagic protein degradation. DUBs also shape the epigenetic landscape and modulate DNA damage repair processes. In this review, we summarize the current knowledge on DUBs in plants, their cellular functions, and the regulatory mechanisms involved in the spatiotemporal regulation of plant DUBs.

Role of the dynamin-related protein 2 family and SH3P2 in clathrin-mediated endocytosis in Arabidopsis thaliana.

Clathrin-mediated endocytosis (CME) is vital for the regulation of plant growth and development through controlling plasma membrane protein composition and cargo uptake. CME relies on the precise recruitment of regulators for vesicle maturation and release. Homologues of components of mammalian vesicle scission are strong candidates to be part of the scission machinery in plants, but the precise roles of these proteins in this process are not fully understood. Here, we characterised the roles of the plant dynamin-related protein 2 (DRP2) family (hereafter DRP2s) and SH3-domain containing protein 2 (SH3P2), the plant homologue to recruiters of dynamins, such as endophilin and amphiphysin, in CME by combining high-resolution imaging of endocytic events in vivo and characterisation of the purified proteins in vitro. Although DRP2s and SH3P2 arrive similarly late during CME and physically interact, genetic analysis of the sh3p123 triple mutant and complementation assays with non-SH3P2-interacting DRP2 variants suggest that SH3P2 does not directly recruit DRP2s to the site of endocytosis. These observations imply that, despite the presence of many well-conserved endocytic components, plants have acquired a distinct mechanism for CME.

Deubiquitylating enzymes in Arabidopsis thaliana endocytic protein degradation.

The regulation of ubiquitylation is key for plant growth and development, in which the activities of ubiquitylating enzymes as well as deubiquitylating enzymes (DUBs) determine the stability or function of the modified proteins. In contrast with ubiquitylating enzymes, there are less numbers of DUBs. DUBs can be classified into seven protein families according to the amino acid sequence of their catalytic domains. The catalytic domains of animal and plant DUB families show high homology, whereas the regions outside of the catalytic site can vary a lot. By hydrolyzing the ubiquitin molecules from ubiquitylated proteins, DUBs control ubiquitin-dependent selective protein degradation pathways such as the proteasomal-, autophagic-, and endocytic degradation pathways. In the endocytic degradation pathway, DUBs can modulate the endocytic trafficking and thus the stability of plasma membrane proteins including receptors and transporters. To date, three DUB families were shown to control the endocytic degradation pathway namely associated molecule with the SH3 domain of STAM (AMSH) 3, ubiquitin-specific protease (UBP) 12 and UBP13, and ovarian tumor protease (OTU) 11 and OTU12. In this review we will summarize the activity, molecular functions, and target protein of these DUBs and how they contribute to the environmental response of plants.

Identification of promoter targets by Aureochrome 1a in the diatom Phaeodactylum tricornutum.

Aureochromes (AUREOs) are unique blue light receptors and transcription factors found only in stramenopile algae. While each of the four AUREOs identified in the diatom Phaeodactylum tricornutum may have a specific function, PtAUREO1a has been shown to have a strong impact on overall gene regulation, when light changes from red to blue light conditions. Despite its significance, the molecular mechanism of PtAUREO1a is largely unexplored. To comprehend the overall process of gene regulation by PtAUREO1a, we conducted a series of in vitro and in vivo experiments, including pull-down assays, yeast one-hybrid experiments, and phenotypical characterization using recombinant PtAUREOs and diatom mutant lines expressing a modified PtAureo1a gene. We describe the distinct light absorption properties of four PtAUREOs and the formation of all combinations of their potential dimers. We demonstrate the capability of PtAUREO1a and 1b to activate the genes, diatom-specific cyclin 2, PtAureo1a, and PtAureo1c under both light and dark conditions. Using mutant lines expressing a modified PtAUREO1a protein with a considerably reduced light absorption, we found novel evidence that PtAUREO1a regulates the expression of PtLHCF15, which is essential for red light acclimation. Based on current knowledge, we present a working model of PtAUREO1a gene regulation properties.

Arabidopsis thaliana B-GATA factors repress starch synthesis and gravitropic growth responses.

Plants perceive the direction of gravity during skotomorphogenic growth, and of gravity and light during photomorphogenic growth. Gravity perception occurs through the sedimentation of starch granules in shoot endodermal and root columella cells. In this study, we demonstrate that the Arabidopsis thaliana GATA factors GNC (GATA, NITRATE-INDUCIBLE, CARBON METABOLISM-INVOLVED) and GNL/CGA1 (GNC-LIKE/CYTOKININ-RESPONSIVE GATA1) repress starch granule growth and amyloplast differentiation in endodermal cells. In our comprehensive study, we analysed gravitropic responses in the shoot, root and hypocotyl. We performed an RNA-seq analysis, used advanced microscopy techniques to examine starch granule size, number and morphology and quantified transitory starch degradation patterns. Using transmission electron microscopy, we examined amyloplast development. Our results indicate that the altered gravitropic responses in hypocotyls, shoots and roots of gnc gnl mutants and GNL overexpressors are due to the differential accumulation of starch granules observed in the GATA genotypes. At the whole-plant level, GNC and GNL play a more complex role in starch synthesis, degradation and starch granule initiation. Our findings suggest that the light-regulated GNC and GNL help balance phototropic and gravitropic growth responses after the transition from skotomorphogenesis to photomorphogenesis by repressing the growth of starch granules.

Measuring the DUB Activity of Arabidopsis Deubiquitylating Enzymes.

Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here we show methods to analyze DUB activity using immunodetection, Coomassie brilliant blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

Lipid-mediated activation of plasma membrane-localized deubiquitylating enzymes modulate endosomal trafficking.

The abundance of plasma membrane-resident receptors and transporters has to be tightly regulated by ubiquitin-mediated endosomal degradation for the proper coordination of environmental stimuli and intracellular signaling. Arabidopsis OVARIAN TUMOR PROTEASE (OTU) 11 and OTU12 are plasma membrane-localized deubiquitylating enzymes (DUBs) that bind to phospholipids through a polybasic motif in the OTU domain. Here we show that the DUB activity of OTU11 and OTU12 towards K63-linked ubiquitin is stimulated by binding to lipid membranes containing anionic lipids. In addition, we show that the DUB activity of OTU11 against K6- and K11-linkages is also stimulated by anionic lipids, and that OTU11 and OTU12 can modulate the endosomal degradation of a model cargo and the auxin efflux transporter PIN2-GFP in vivo. Our results suggest that the catalytic activity of OTU11 and OTU12 is tightly connected to their ability to bind membranes and that OTU11 and OTU12 are involved in the fine-tuning of plasma membrane proteins in Arabidopsis.

Bacterial microcompartments for isethionate desulfonation in the taurine-degrading human-gut bacterium Bilophila wadsworthia.

BACKGROUND: Bilophila wadsworthia, a strictly anaerobic, sulfite-reducing bacterium and common member of the human gut microbiota, has been associated with diseases such as appendicitis and colitis. It is specialized on organosulfonate respiration for energy conservation, i.e., utilization of dietary and host-derived organosulfonates, such as taurine (2-aminoethansulfonate), as sulfite donors for sulfite respiration, producing hydrogen sulfide (H2S), an important intestinal metabolite that may have beneficial as well as detrimental effects on the colonic environment. Its taurine desulfonation pathway involves the glycyl radical enzyme (GRE) isethionate sulfite-lyase (IslAB), which cleaves isethionate (2-hydroxyethanesulfonate) into acetaldehyde and sulfite. RESULTS: We demonstrate that taurine metabolism in B. wadsworthia 3.1.6 involves bacterial microcompartments (BMCs). First, we confirmed taurine-inducible production of BMCs by proteomic, transcriptomic and ultra-thin sectioning and electron-microscopical analyses. Then, we isolated BMCs from taurine-grown cells by density-gradient ultracentrifugation and analyzed their composition by proteomics as well as by enzyme assays, which suggested that the GRE IslAB and acetaldehyde dehydrogenase are located inside of the BMCs. Finally, we are discussing the recycling of cofactors in the IslAB-BMCs and a potential shuttling of electrons across the BMC shell by a potential iron-sulfur (FeS) cluster-containing shell protein identified by sequence analysis. CONCLUSIONS: We characterized a novel subclass of BMCs and broadened the spectrum of reactions known to take place enclosed in BMCs, which is of biotechnological interest. We also provided more details on the energy metabolism of the opportunistic pathobiont B. wadsworthia and on microbial H2S production in the human gut.

MLP-PG1, a major latex-like protein identified in Cucurbita pepo, confers resistance through the induction of pathogenesis-related genes.

MAIN CONCLUSION: MLP-PG1, identified in Cucurbita pepo, plays a crucial role in resistance against fungal pathogens through the induction of pathogenesis-related genes. ASTRACT: MLP-PG1, a major latex-like protein (MLP) from zucchini (Cucurbita pepo), was identified as a transporting factor for hydrophobic organic pollutants. MLPs are members of the Bet v 1 family, similar to pathogenesis-related class 10 proteins (PR-10s). However, the biological functions of MLPs remain unclear. Herein, we show that MLP-PG1 induces the expression of pathogenesis-related (PR) genes and indirectly promotes resistance against pathogens. The activity of the MLP-PG1 promoter in leaves of transgenic tobacco plants was significantly enhanced by inoculation with Pseudomonas syringae pv. tabaci. However, MLP-PG1 did not induce direct resistance through RNase activity. Therefore, we examined the possibility that MLP-PG1 is indirectly involved in resistance; indeed, we found that MLP-PG1 induced the expression of defense-related genes. Overexpression of MLP-PG1 highly upregulated PR-2 and PR-5 and decreased the area of lesions caused by Botrytis cinerea in the leaves of transgenic tobacco plants. Our results demonstrate that MLP-PG1 is involved in indirect resistance against plant diseases, especially caused by fungal pathogens, through the induction of PR genes. This study is the first report to show the induction of PR genes by the expression of MLP from the RNA sequencing analysis and the involvement of MLP-PG1 in the resistance.

ESCRT Is a Great Sealer: Non-Endosomal Function of the ESCRT Machinery in Membrane Repair and Autophagy.

Components of the endosomal sorting complex required for transport (ESCRTs) were first identified in a genetic screen in budding yeast as factors interfering with vacuolar protein sorting. In the last three decades, intensive studies have revealed the subunit composition of ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III, their structure, the assembling mechanisms and their molecular and physiological functions. In plants, ESCRTs are essential for development, growth and stress responses. ESCRTs are best known for their function in endosomal trafficking, during which they are required for sorting ubiquitylated membrane proteins into intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs). The formation of ILVs requires the function of ESCRT-III, which has been shown to mediate the membrane scission. Although the function of plant ESCRTs has been predominantly discussed in the context of endosomal trafficking, recent studies in other model organisms revealed a versatile role of ESCRTs in diverse cellular events with broad physiological implications. The non-endosomal functions of ESCRTs include cytokinesis, viral budding, autophagy, nuclear envelope reformation and membrane repair, although many of these have not yet been studied in plants. In this review, recent findings on non-endosomal ESCRT functions in plant, yeast and animals are highlighted and discussed.

Knowing When to Self-Eat - Fine-Tuning Autophagy Through ATG8 Iso-forms in Plants.

Autophagy is a catabolic process that takes place under both normal and adverse conditions and is important for the degradation of various organelles and proteins that are no longer needed. Thus, it can be viewed as both a constitutive recycling machinery and an adaptation mechanism. Increase in the activity of autophagy can be caused by multiple biotic and abiotic stress factors. Though intensive research in the past decade has elucidated many molecular details of plant autophagy, the mechanisms of induction and regulation of the process remain understudied. Here, we discuss the role of ATG8 proteins in autophagic signaling and regulation with an emphasis on the significance of ATG8 diversification for adapting autophagy to the changing needs of plants.

Detection of Phosphorylation on Immunoprecipitates from Total Protein Extracts of Arabidopsis thaliana Seedlings.

Phosphorylation is a versatile posttranslational modification that can regulate the localization, stability, and conformation of proteins; protein-protein interactions; and enzyme activities. Phosphorylation of plasma membrane proteins, for example, can serve as recognition signals for ubiquitin ligases and hence can trigger its endocytic degradation. Key determinants of protein phosphorylation are kinases and phosphatases that are spatiotemporally regulated to phosphorylate or dephosphorylate specific target proteins. To understand the dynamics and regulatory mechanisms of protein phosphorylation, it is essential to analyze the phosphorylation status of the proteins and identify phosphorylation sites as well as the modifying enzymes. In this chapter, we describe methods that can be used for the detection of phosphoproteins that are immunoprecipitated from Arabidopsis total extracts.

Modified Enzymatic Assays for the Determination of Histamine in Fermented Foods.

ABSTRACT: Histamine is a biogenic amine, produced in spoiled fish and some fermented products, which causes a foodborne disease similar to an allergic reaction. Because regulatory levels on histamine in food have been set by many countries or organizations, a quick and accurate analysis of histamine is of great interest. An enzymatic histamine determination method on the basis of a colorimetric assay has been used to detect histamine for raw and canned tuna due to its simplicity and rapidity. However, note that some compounds in fermented foods interfere with assay results. In this study, the pretreatments and conditions of the assay for fermented foods were evaluated. Lowering the reaction temperature from 37 to 23°C was considerably effective in reducing the interference. As a result, histamine in salami and sauerkraut (≥5 to 10 mg/kg) could be determined with a 25-fold dilution, as in the manufacturer's instructions. Histamine in soy sauce (≥10 to 20 mg/L) could also be determined with a 100-fold dilution. Removing fat and protein in cheese samples by using perchloric acid with a resultant 25-fold dilution and removing polyphenol with polyvinylpolypyrrolidone for red wine with a fivefold dilution were feasible; the limits of quantification were 5 mg/kg and 1 mg/L, respectively. Good recovery rates, precision repeatability, and correlations with a high-performance liquid chromatography method were confirmed. These protocols are expected to be applicable for histamine determination in various foods and useful for preventing histamine food poisoning.

Ubiquitin recognition in endocytic trafficking - with or without ESCRT-0.

The ability to sense and adapt to the constantly changing environment is important for all organisms. Cell surface receptors and transporters are key for the fast response to extracellular stimuli and, thus, their abundance on the plasma membrane has to be strictly controlled. Heteromeric endosomal sorting complexes required for transport (ESCRTs) are responsible for mediating the post-translational degradation of endocytosed plasma membrane proteins in eukaryotes and are essential both in animals and plants. ESCRTs bind and sort ubiquitylated cargoes for vacuolar degradation. Although many components that comprise the multi-subunit ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III complexes are conserved in eukaryotes, plant and animal ESCRTs have diverged during the course of evolution. Homologues of ESCRT-0, which recognises ubiquitylated cargo, have emerged in metazoan and fungi but are not found in plants. Instead, the Arabidopsis genome encodes plant-specific ubiquitin adaptors and a greater number of target of Myb protein 1 (TOM1) homologues than in mammals. In this Review, we summarise and discuss recent findings on ubiquitin-binding proteins in Arabidopsis that could have equivalent functions to ESCRT-0. We further hypothesise that SH3 domain-containing proteins might serve as membrane curvature-sensing endophilin and amphiphysin homologues during plant endocytosis.

Transient Expression of ESCRT Components in Arabidopsis Root Cell Suspension Culture-Derived Protoplasts.

Localization studies are important to understand the function of diverse proteins. The endosomal trafficking pathway is very complex, and a lot of proteins function in this pathway, primarily the endosomal sorting complexes required for transport (ESCRTs). Some of the ESCRT-related proteins or mutant variants cannot be stably expressed in planta due to the toxicity of their expression. Therefore, a transient expression system is necessary to study their function. Transient expression in protoplasts from Arabidopsis root cell-derived culture serves as a fast and reliable method for the expression and cell biological and biochemical analyses of otherwise toxic constructs.

The Arabidopsis receptor kinase STRUBBELIG undergoes clathrin-dependent endocytosis.

Signaling mediated by cell surface receptor kinases is central to the coordination of growth patterns during organogenesis. Receptor kinase signaling is in part controlled through endocytosis and subcellular distribution of the respective receptor kinase. For the majority of plant cell surface receptors, the underlying trafficking mechanisms are not characterized. In Arabidopsis, tissue morphogenesis requires the atypical receptor kinase STRUBBELIG (SUB). Here, we studied the endocytic mechanism of SUB. Our data revealed that a functional SUB-enhanced green fluorescent protein (EGFP) fusion is ubiquitinated in vivo. We further showed that plasma membrane-bound SUB:EGFP becomes internalized in a clathrin-dependent fashion. We also found that SUB:EGFP associates with the trans-Golgi network and accumulates in multivesicular bodies and the vacuole. Co-immunoprecipitation experiments revealed that SUB:EGFP and clathrin are present within the same protein complex. Our genetic analysis showed that SUB and CLATHRIN HEAVY CHAIN (CHC) 2 regulate root hair patterning. By contrast, genetic reduction of CHC activity ameliorates the floral defects of sub mutants. Taken together, the data indicate that SUB undergoes clathrin-mediated endocytosis, that this process does not rely on stimulation of SUB signaling by an exogenous agent, and that SUB genetically interacts with clathrin-dependent pathways in a tissue-specific manner.