Molecular mechanisms of ESCRT-mediated autophagosome maturation in plants.

Diverse environmental stress factors affect the functionality of proteins and membrane compartments within cells causing potentially irremediable damage to the cell. A major process to eliminate nonfunctional molecular aggregates or damaged organelles under stress conditions is macroautophagy/autophagy, thus making its regulation critical for cellular adaptation and survival. The formation of autophagosomes is coordinated by a wide range of cellular factors and culminates in the closure of the cup-shaped double membrane or phagophore.

FYVE1/FREE1 is involved in glutamine-responsive TORC1 activation in plants.

Target of rapamycin complex 1 (TORC1) integrates nutrient availability, growth factors, and stress signals to regulate cellular metabolism according to its environment. Similar to mammals, amino acids have been shown to activate TORC1 in plants. However, as the Rag complex that controls amino acid-responsive TORC1 activation mechanisms in many eukaryotes is not conserved in plants, the amino acid-sensing mechanisms upstream of TORC1 in plants remain unknown.

Arabidopsis CaLB1 undergoes phase separation with the ESCRT protein ALIX and modulates autophagosome maturation.

Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require a complex choreography of numerous factors. The endosomal sorting complex required for transport (ESCRT) is implicated in the final step of autophagosomal maturation by sealing of the phagophore membrane.

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.

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.

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.

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.

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.

Membrane-specific and calcium-dependent binding of the Arabidopsis C2 domain protein CaLB revealed by ATR-FTIR spectroscopy.

C2 domain-containing proteins bind to cellular membranes and mediate diverse cellular processes. Although many of these membrane-interacting proteins have been identified, the molecular mechanisms of protein-membrane interactions and conformational dynamics are often poorly understood and remain to be investigated with appropriate methods. Here, we used attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy and biomimetic membrane systems to analyse CalB, a yet uncharacterized Arabidopsis C2 domain protein.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

Preparation of Clathrin-Coated Vesicles From Seedlings.

Clathrin coated vesicles (CCVs) mediate endocytosis of plasma membrane proteins and deliver their content to the endosomes for either subsequent recycling to the plasma membrane or transport to the vacuole for degradation. CCVs assemble also at the trans-Golgi network (TGN) and is responsible for the transport of proteins to other membranes. Oligomerization of clathrin and recruitment of adaptor protein complexes promote the budding and the release of CCVs.