Total Proteome Analysis from Arabidopsis thaliana SUMO E3 Ligase Mutants
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)
TISSUE(S): Plant Cell, Rosette
SUBMITTER: 
Therese Rytz
LAB HEAD: Richard D Vierstra
PROVIDER: PXD009274 | Pride | 2018-04-23
REPOSITORIES: Pride
Publications
SUMOylome Profiling Reveals a Diverse Array of Nuclear Targets Modified by the SUMO Ligase SIZ1 during Heat Stress.
Rytz Thérèse C TC Miller Marcus J MJ McLoughlin Fionn F Augustine Robert C RC Marshall Richard S RS Juan Yu-Ting YT Charng Yee-Yung YY Scalf Mark M Smith Lloyd M LM Vierstra Richard D RD
The Plant cell 20180327 5
The posttranslational addition of small ubiquitin-like modifier (SUMO) is an essential protein modification in plants that provides protection against numerous environmental challenges. Ligation is accomplished by a small set of SUMO ligases, with the SAP-MIZ domain-containing SIZ1 and METHYL METHANESULFONATE-SENSITIVE21 (MMS21) ligases having critical roles in stress protection and DNA endoreduplication/repair, respectively. To help identify their corresponding targets in <i>Arabidopsis thalian ...[more]
Similar Datasets
Project description:Pulldown of an ER localized HPPase to identify putative ER microautophagy receptors.
2022-02-08 | PXD011039 | Pride
Project description:The turnover of cytoplasmic material via autophagic encapsulation and delivery to vacuoles is essential for recycling cellular constituents, especially under nutrient-limiting conditions. To determine how cells/tissues rely on autophagy, we applied in-depth multi-omic analyses to study maize (Zea mays) autophagy mutants grown after fixed carbon starvation. Surprisingly, broad alterations in the leaf metabolome were evident in plants missing the core autophagy component ATG12 even without stress, particularly affecting products of lipid turnover and secondary metabolites, which were underpinned by substantial changes in the transcriptome and/or proteome. Cross-comparison of mRNA and protein abundances allowed for the identification of organelles, protein complexes, and individual proteins targeted for selective autophagic clearance, and revealed several processes controlled by this catabolism. Fixed carbon starvation induced the proteins involved in alternative respiration together with the upregulation of various alternative catabolic processes. Collectively, we describe a facile proteomic strategy to survey autophagic substrates, and show that autophagy has a greater than expected influence in sculpting plant proteomes and membranes both before and during nitrogen stress.
2020-07-15 | PXD017077 | Pride
Project description:The turnover of cytoplasmic material via autophagic encapsulation and delivery to vacuoles is essential for recycling cellular constituents, especially under nutrient-limiting conditions. To determine how cells/tissues rely on autophagy, we applied in-depth multi-omic analyses to study maize (Zea mays) autophagy mutants grown under nitrogen-replete and starvation conditions. Surprisingly, broad alterations in the leaf metabolome were evident in plants missing the core autophagy component ATG12 even without stress, particularly affecting products of lipid turnover and secondary metabolites, which were underpinned by substantial changes in the transcriptome and/or proteome. Cross-comparison of mRNA and protein abundances allowed for the identification of organelles, protein complexes, and individual proteins targeted for selective autophagic clearance, and revealed several processes controlled by this catabolism. Collectively, we describe a facile proteomic strategy to survey autophagic substrates, and show that autophagy has a greater than expected influence in sculpting plant proteomes and membranes both before and during nitrogen stress.
2018-10-08 | PXD009627 | Pride
Project description:Duckweeds are a monophyletic group of rapidly reproducing aquatic monocots in the Lemnaceae family. Spirodela polyrhiza, the Greater Duckweed, has the largest body plan yet the smallest genome size in the family (1C = 150 Mb). Given their clonal, exponentially fast reproduction, a key question is whether genome structure is conserved across the species in the absence of meiotic recombination. We generated a highly contiguous, chromosome-scale assembly of Spirodela polyrhiza line Sp7498 using Oxford Nanopore plus Hi-C scaffolding (Sp7498_HiC) that is highly syntenic with a related line (Sp9509). Both the Sp7498_HiC and Sp9509 genome assemblies reveal large chromosomal misorientations in a recent PacBio assembly of Sp7498, highlighting the necessity of orthogonal long-range scaffolding techniques like Hi-C and BioNano optical mapping. Proteome analysis of Sp7498 verified the expression of nearly 2,250 proteins and revealed a high level of proteins involved in photosynthesis and carbohydrate metabolism among other functions. In addition, a strong increase in chloroplast proteins was observed that correlated to chloroplast density. This Sp7498_HiC genome was generated cheaply and quickly with a single Oxford Nanopore MinION flow cell and one Hi-C library in a classroom setting. Combining these data with a mass spectrometry-generated proteome, demonstrates that duckweed is a model for genomics- and proteomics-based education.
2022-10-26 | PXD017093 | Pride
Project description:The 5' untranslated region (UTR) sequence of eukaryotic mRNAs may contain upstream open reading frames (uORFs), which can regulate translation of the main open reading frame (mORF). The current model of translational regulation by uORFs posits that when a ribosome scans an mRNA and encounters a uORF, translation of that uORF can prevent ribosomes from reaching the mORF and cause decreased mORF translation. In this study, we first observed that rare variants in the 5' UTR dysregulate protein abundance. Upon further investigation, we found that rare variants near the start codon of uORFs can repress or derepress mORF translation, causing allelic changes in protein abundance. This finding holds for common variants as well, and common variants that modify uORF start codons also contribute disproportionately to metabolic and whole-plant phenotypes, suggesting that translational regulation by uORFs serves an adaptive function. These results provide evidence for the mechanisms by which natural sequence variation modulates gene expression, and ultimately, phenotype.
2021-11-02 | PXD026378 | Pride
Project description:Proteins that are vital to cell survival can become unfolded during heat stress, leading to the formation of toxic aggregates. Molecular chaperones, proteases and autophagic pathways are required to protect cells from the accumulation of protein aggregates. Small Heat Shock Proteins (sHSPs) form a first line of defense by binding to unfolding proteins to prevent irreversible protein aggregation and the complex is rapidly sequestered in stress granules during heat stress recovery. HSP101 subsequently accumulates on the surface of these insoluble foci to mediate protein disaggregation. The dynamics of this process is consistent between different cell types but indicates that protein homeostasis varies particularly between shoot and root cells. Immunoblot analysis revealed that a substantial portion of proteins present in these foci have ubiquitin moieties and protein disaggregation is necessary prior to proteasomal degradation. Co-immuno precipitation of HSP101 revealed an interaction with the 26S proteasome and localization studies revealed that HSP101 and RPN1 (Proteasome regulatory particle) initially accumulate in distinct cytosolic foci during heat stress but co-localize in the same foci during heat stress recovery which could spatiotemporal facilitate the degradation of the aggregated ubiquitinated proteins. To determine which proteins are degraded by the proteasome after disaggregation, we catalogued 620 proteins that are resolubilized by HSP101 during heat stress recovery. GO-annotation analysis revealed a significant enrichment for RNA-binding proteins, UBC13-MMS2 complex, transcription factors and ubiquitin-protein ligases. None of these proteins were preferentially targeted by the proteasome, indicating that ubiquitination occurs on a broad range of proteins and is important for the clearance of a diverse array of proteins.
2019-05-14 | PXD011483 | Pride
Project description:Autophagy or ‘self-eating’ is a conserved and essential route for recycling intracellular constituents in eukaryotes. For plants, autophagy supports robust crop performance by regulating traits such as nitrogen-use efficiency, carbon allocation, reproduction, grain fill, and protection against various environmental stresses. To define the autophagic mechanisms underlining nutrient partitioning, which are used to sustain plant fitness during rapid growth of sink tissues, we applied a comprehensive multi-omic approach using maize (Zea mays) mutants lacking the core autophagy component ATG12. A sink tissue of particular interest are seeds, which require massive investments of fixed carbon for development. The transcriptome, proteome, metabolome, and ionome of developing seeds from atg12 mutants at 8 and 18 days after pollination (DAP) were analyzed. Developing atg12 seeds exhibited pronounce transcriptional reprogramming at 8 DAP, which may contribute to the observed hyperaccumulation of proteins, metabolites, and ions at 18 DAP. Collectively, this multi-omic approach should provide new perspectives into the adaptive role(s) of autophagy in coordinating nutrient allocation.
2023-03-10 | PXD022863 | Pride
Project description:A GFP-mediated pulldown to identify autophagy targets of an autophagy receptor.
2019-03-13 | PXD011397 | Pride
Project description:Maintaining the integrity of nucleic acids is an essential feature of all organisms. Unwanted modification of DNA, if left unrepaired, is deleterious to cellular homeostasis and could have far-reaching consequences that include genomic instability and accumulation of mutations. Similarly, accumulation of damaged RNA has been correlated with various neurodegenerative diseases. Given their inherent reactivity, nucleic acids are susceptible to damage from both endogenous and exogenous reactive oxygen species (ROS) as well as alkylation agents. We have recently begun to address how some of these modifications on mRNA impact the function of the ribosome and in particular the decoding process. We find that most modifications severely change the speed and accuracy of translation. These observations revealed that oxidation of RNA is most likely to stall the ribosome and necessitates the presence of quality-control processes to handle damaged mRNA. To this end, we have uncovered a connection between the process of no-go decay (NGD), which degrades mRNAs that stall translation, and chemical insults. In the absence of key NGD factors in yeast, the levels of damaged mRNA significantly increase and cells are rendered sensitive to oxidizing and alkylation agents. Furthermore, these agents activate ribosome quality control (RQC) of nascent peptides. Deletion of the E3 ligase responsible for the ubiquitination of the nascent peptides resulted in the accumulation of protein aggregates in the presence of oxidizing and alkylating agents. These observations suggest that chemical damage stalls translation, activating NGD and RQC. Interestingly, the addition of nucleic-acids-damaging agents was also found to result in K63-linked ubiquitination of ribosomal proteins. This signaling-mode of ubiquitination precedes DNA-damage marks, indicating that cells respond to RNA damage due to stalled ribosomes before DNA damage. Collectively our data highlights the burden of chemically-damaged mRNA on cellular homeostasis and suggests that organisms evolved ribosome-based mRNA-surveillance processes to rapidly degrade it.
2019-11-07 | PXD013834 | Pride
Project description:Plants respond to mechanical stressors in a variety of ways, producing electrical and biochemical signals, altering gene expression profiles, and changing organellar organization and morphology. We used a combination of genetic and proteomic approaches to identify genes that modify signaling of MSL10, a mechanosensitive ion channel in Arabidopsis which promotes programmed cell death in response to the mechanical stress from osmotic cell swelling. Mass spectrometry was used to identify proteins that co-immunoprecipitate with MSL10-GFP or with MSL107xD-GFP, a phosphomimetic variant that cannot activate cell death signaling. Interestingly, proteomics identified many proteins that are found at ER-plasma membrane contact sites (EPCSs). Two of the most abundant proteins that co-precipitated with MSL10 are the EPCS proteins VAP27-1 and SYT1, both of which have been reported to relocalize into spherical membrane structures in response to mechanical stress, a phenomenon also observed with MSL10. SYT1 has previously been shown to promote resistance to mechanical stress. A suppressor screen of the gain-of-function msl10-3G allele yielded mutants with point mutations in synaptotagmin (SYT)5 and SYT7, proteins that form a functional complex with SYT1. Together, these results suggest that MSL10 interacts with SYT1, SYT5, and SYT7 at EPCSs and that its cell death signaling output is modulated by this interaction.
2022-10-26 | PXD018747 | Pride