Large-scale identification of ubiquitination sites on membrane-associated proteins in Arabidopsis thaliana seedlings.
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ABSTRACT:
SUBMITTER: Grubb LE
PROVIDER: S-EPMC8133621 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Protein phosphorylation and ubiquitination are two of the most abundant forms of post-translational modifications in eukaryotes, regulated by thousands of protein kinases, phosphatases, E3 ubiquitin ligases, and ubiquitin proteases. Although previous studies have catalogued several ubiquitinated proteins in plants (Walton et al., 2016), few membrane-localized proteins have been identified. Receptor kinases (RKs) initiate phosphorylation signal relays that regulate plant growth, development, and stress responses. While the regulatory role of phosphorylation on protein kinase function is well-documented (Couto and Zipfel, 2016), considerably less is known about the role of ubiquitination on protein kinase function, even though protein turnover is critical to their signaling competence and cellular homeostasis. Here we describe the large-scale identification of ubiquitination sites on Arabidopsis proteins associated with or integral to the plasma membrane, including over 100 protein kinases.
2021-02-01 | PXD021992 | Pride
Project description:Most mitochondrial proteins contain an N-terminal targeting signal that is removed by specific proteases following import. In plant mitochondria, only mitochondrial processing peptidase (MPP) has been characterized to date. Therefore, we sought to determine the substrates and cleavage sites of the Arabidopsis thaliana homologues to the yeast Icp55 and Oct1 proteins, using the newly developed ChaFRADIC method for N-terminal protein sequencing. We identified 88 and seven putative substrates for Arabidopsis ICP55 and OCT1, respectively. It was determined that the Arabidopsis ICP55 contains an almost identical cleavage site to that of Icp55 from yeast. However, it can also remove a far greater range of amino acids. The OCT1 substrates from Arabidopsis displayed no consensus cleavage motif, and do not contain the classical -10R motif identified in other eukaryotes. Arabidopsis OCT1 can also cleave presequences independently, without the prior cleavage of MPP. It was concluded that while both OCT1 and ICP55 were probably acquired early on in the evolution of mitochondria, their substrate profiles and cleavage sites have either remained very similar or diverged completely.
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Project description:The impact of fungal diseases on crop production negatively reflects on sustainable food production and overall economic health. Ergosterol is the major sterol component in fungal membranes and regarded as a general elicitor or microbe-associated molecular pattern (MAMP) molecule. Although plant responses to ergosterol have been reported, the perception mechanism is still unknown. Here, Arabidopsis thaliana protein fractions were used to identify those differentially regulated following ergosterol treatment; additionally, they were subjected to affinity-based chromatography enrichment strategies to capture and categorize ergosterol-interacting candidate proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Mature plants were treated with 250 nM ergosterol over a 24 h period, and plasma membrane-associated fractions were isolated. In addition, ergosterol was immobilized on two different affinity-based systems to capture interacting proteins/complexes. This resulted in the identification of defense-related proteins such as chitin elicitor receptor kinase (CERK), non-race specific disease resistance/harpin-induced (NDR1/HIN1)-like protein, Ras-related proteins, aquaporins, remorin protein, leucine-rich repeat (LRR)- receptor like kinases (RLKs), G-type lectin S-receptor-like serine/threonine-protein kinase (GsSRK), and glycosylphosphatidylinositol (GPI)-anchored protein. Furthermore, the results elucidated unknown signaling responses to this MAMP, including endocytosis, and other similarities to those previously reported for bacterial flagellin, lipopolysaccharides, and fungal chitin.
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Project description:Membrane protein crystallography is notoriously difficult due to challenges in protein expression and issues of degradation and structural stability. We have developed a novel method for large-scale screening of native sources for integral membrane proteins that have intrinsic biochemical properties favorable for crystallization. Highly expressed membrane proteins that are thermally stable and nonaggregating in detergent solutions were identified by mass spectrometry from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebrum. Many of the membrane proteins identified had been crystallized previously, supporting the promise of the approach. Most identified proteins have known functions and include high-value targets such as transporters and ATPases. To validate the method, we recombinantly expressed and purified the yeast protein, Yop1, which is responsible for endoplasmic reticulum curvature. We demonstrate that Yop1 can be purified with the detergent dodecylmaltoside without aggregating.
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Project description:DELLA proteins act as hubs that relay environmental information to the multiple transcriptional circuits that control growth and development through physical interaction with transcription factors from different families. We have analyzed the presence of one DELLA protein at the Arabidopsis genome by chromatin immunoprecipitation coupled to large-scale sequencing and we find that it binds at the promoters of multiple genes. Enrichment analysis shows a strong preference for cis elements recognized by specific transcription factor families. In particular, we demonstrate that DELLA proteins are recruited by type-B ARABIDOPSIS RESPONSE REGULATORS (ARR) to the promoters of cytokinin-regulated genes, where they act as transcriptional co-activators. The biological relevance of this mechanism is underpinned by the necessity of simultaneous presence of DELLAs and ARRs to restrict root meristem growth and to promote photomorphogenesis.
2015-07-23 | GSE59187 | GEO
Project description:Genetic markers such as single nucleotide polymorphisms (SNPs) are essential tools for positional cloning, association, or quantitative trait locus mapping and the determination of genetic relationships between individuals. We identified and characterized a genome-wide set of SNP markers by generating 10,706 expressed sequence tags (ESTs) from cDNA libraries derived from 6 different accessions, and by analysis of 606 sequence tagged sites (STS) from up to 12 accessions of the model flowering plant Arabidopsis thaliana. The cDNA libraries for EST sequencing were made from individuals that were stressed by various means to enrich for transcripts from genes expressed under such conditions. SNPs discovered in these sequences may be useful markers for mapping genes involved in interactions with the biotic and abiotic environment. The STS loci are distributed randomly over the genome. By comparison with the Col-0 genome sequence, we identified a total of 8051 SNPs and 637 insertion/deletion polymorphisms (InDel). Analysis of STS-derived SNPs shows that most SNPs are rare, but that it is possible to identify intermediate frequency framework markers that can be used for genetic mapping in many different combinations of accessions. A substantial proportion of SNPs located in ORFs caused a change of the encoded amino acid. A comparison of the density of our SNP markers among accessions in both the EST and STS datasets, revealed that Cvi-0 is the most divergent accession from Col-0 among the 12 accessions studied. All of these markers are freely available via the internet.
| S-EPMC403656 | biostudies-literature
Project description:The majority of proteins in eukaryotic cells are modified according to highly regulated mechanisms to fulfill specific functions and to achieve localization, stability, and transport. Protein ubiquitination is one of the major post-translational modifications occurring in eukaryotic cells. To obtain the proteomic dataset related to the ubiquitin (Ub)-dependent regulatory system in Arabidopsis, affinity purification with an anti-Ub antibody under native condition was performed. Using MS/MS analysis, 196 distinct proteins represented by 251 distinct genes were identified. The identified proteins were involved in metabolism (23.0%), stress response (21.4%), translation (16.8%), transport (6.7%), cell morphology (3.6%), and signal transduction (1.5%), in addition to proteolysis (16.8%) to which proteasome subunits (14.3%) is included. On the basis of potential ubiquitination-targeting signal motifs, in-gel mobilities, and previous reports, 78 of the identified proteins were classified as ubiquitinated proteins and the rest were speculated to be associated proteins of ubiquitinated proteins. The degradation of three proteins predicted to be ubiquitinated proteins was inhibited by a proteasome inhibitor, suggesting that the proteins were regulated by Ub/proteasome-dependent proteolysis.
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Project description:Cold stress is one of the major factors limiting global crop production. For survival at low temperatures, plants need to sense temperature changes in the surrounding environment. How plants sense and respond to the earliest drop in temperature is still not clearly understood. The plasma membrane and its adjacent extracellular and cytoplasmic sites are the first checkpoints for sensing temperature changes and the subsequent events, such as signal generation and solute transport. To understand how plants respond to early cold exposure, we used a mass spectrometry-based phosphoproteomic method to study the temporal changes in protein phosphorylation events in Arabidopsis membranes during 5 to 60 min of cold exposure. The results revealed that brief cold exposures led to rapid phosphorylation changes in the proteins involved in cellular ion homeostasis, solute and protein transport, cytoskeleton organization, vesical trafficking, protein modification, and signal transduction processes. The phosphorylation motif and kinase-substrate network analysis also revealed that multiple protein kinases, including RLKs, MAPKs, CDPKs, and their substrates, could be involved in early cold signaling. Taken together, our results provide a first look at the cold-responsive phosphoproteome changes of Arabidopsis membrane proteins that can be a significant resource to understand how plants respond to an early temperature drop.
| S-EPMC7696906 | biostudies-literature
Project description:One of the major goals of quantitative genetics is to unravel the complex interactions between molecular genetic factors and the environment. The effects of these genotype-by-environment interactions also affect and cause variation in gene expression. The regulatory loci responsible for this variation can be found by genetical genomics that involves the mapping of quantitative trait loci (QTLs) for gene expression traits also called expression-QTL (eQTLs). Most genetical genomics experiments published so far, are performed in a single environment and hence do not allow investigation of the role of genotype-by-environment interactions. Furthermore, most studies have been done in a steady state environment leading to acclimated expression patterns. However a response to the environment or change therein can be highly plastic and possibly lead to more and larger differences between genotypes. Here we present a genetical genomics study on 120 Arabidopsis thaliana, Landsberg erecta?×?Cape Verde Islands, recombinant inbred lines (RILs) in active response to the environment by treating them with 3?h of shade. The results of this experiment are compared to a previous study on seedlings of the same RILs from a steady state environment. The combination of two highly different conditions but exactly the same RILs with a fixed genetic variation showed the large role of genotype-by-environment interactions on gene expression levels. We found environment-dependent hotspots of transcript regulation. The major hotspot was confirmed by the expression profile of a near isogenic line. Our combined analysis leads us to propose CSN5A, a COP9 signalosome component, as a candidate regulator for the gene expression response to shade.
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Project description:BackgroundThe composition of the individual eukaryote's genome and its variation within a species remain poorly defined. Even for a sequenced genome such as that of the model plant Arabidopsis thaliana accession Col-0, the large arrays of heterochromatic repeats are incompletely sequenced, with gaps of uncertain size persisting in them.ResultsUsing geographically separate populations of A. thaliana, we assayed variation in the heterochromatic repeat arrays using two independent methods and identified significant polymorphism among them, with variation by as much as a factor of two in the centromeric 180 bp repeat, in the 45S rDNA arrays and in the Athila retroelements. In the accession with highest genome size as measured by flow cytometry, Loh-0, we found more than a two-fold increase in 5S RNA gene copies relative to Col-0; results from fluorescence in situ hybridization with 5S probes were consistent with the existence of size polymorphism between Loh-0 and Col-0 at the 5S loci. Comparative genomic hybridization results of Loh-0 and Col-0 did not support contiguous variation in copy number of protein-coding genes on the scale needed to explain their observed genome size difference. We developed a computational data model to test whether the variation we measured in the repeat fractions could account for the different genome sizes determined with flow cytometry, and found that this proposed relationship could account for about 50% of the variance in genome size among the accessions.ConclusionOur analyses are consistent with substantial repeat number polymorphism for 5S and 45S ribosomal genes among accession of A. thaliana. Differences are also suggested for centromeric and pericentromeric repeats. Our analysis also points to the difficulties in measuring the repeated fraction of the genome and suggests that independent validation of genome size should be sought in addition to flow cytometric measurements.
| S-EPMC2000876 | biostudies-literature