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PUP-IT proximity-labeling of LST8-1 and RAPTOR1 interactors (transient expression in N. benthamiana)

ABSTRACT: PUP-IT proximity labeling of Arabidopsis Target of Rapamycin Complex (TORC) subunits LST8-1 and RAPTOR1, compared against a GFP control, via transient expression in Nicotiana benthamiana. The sample metadata, sequence database and FragPipe workflow are uploaded under the 'Metadata' tag. A short R script to format FragPipe TSV output for mzTab conversion is under 'Supplementary Files'.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Arabidopsis Thaliana (ncbitaxon:3702) Nicotiana Benthamiana (ncbitaxon:4100)

SUBMITTER: Staffan Persson

PROVIDER: MSV000096657 | MassIVE | Fri Dec 13 08:49:00 GMT 2024

SECONDARY ACCESSION(S): PXD058859

REPOSITORIES: MassIVE

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Project description:Pupylation is a posttranslational protein modification in bacteria resembling ubiquitination in eukaryotes. The prokaryotic ubiquitin-like protein Pup is covalently attached to other proteins via an isopeptide bond between its carboxyterminal glutamate residue and a lysine residue in the target. In mycobacteria, pupylation was shown to mark proteins for unfolding by the ATPase Mpa and subsequent degradation by the proteasome. However, the occurrence of pupylation in species without a proteasome like Corynebacterium glutamicum suggests that degradation may not be the only fate of pupylated proteins. The Îpup mutant senses a stronger iron limitation than the wild type. Among the 125 genes showing at least 2-fold changes in transcript levels in the Îpup mutant (p-value â¤ 0.05) were 54% of all genes known to be regulated by the master regulator of iron homeostasis DtxR (Brune et al., 2006; Wennerhold and Bott, 2006). Except for ftn, which is activated by DtxR and showed a 2-fold decreased mRNA level, the other DtxR target genes showed increased mRNA levels in the Îpup strain. These included ripA, encoding a transcriptional regulator of iron proteins, which represses a number of prominent iron-containing proteins under iron limitation, such as aconitase or succinate dehydrogenase (Wennerhold et al., 2005). 79% of the known RipA target genes showed decreased mRNA levels in the Îpup strain. DNA microarray analyses were performed to compare the mRNA levels of the C. glutamicum Îpup mutant and its parent wild type under iron-limited conditions. The two strains precultivated in CGXII medium with 4% (w/v) glucose and 1 ÂµM FeSO4 were inoculated into fresh medium to an OD600 of 1, cultured for 2 h, and harvested on ice by centrifugation (5 min at 4,000 g and 4Â°C). Please note that the GPL16989 array design comprises oligos for 4 different bacterial genomes. In the GPR-files in this study, all IDs/Names (oligonucleotides) which are not from the host C. glutamicum were replaced by the text EMPTY since only C. glutamicum expression was analyzed.

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PUP-IT proximity-labeling of LST8-1 and RAPTOR1 interactors (transient expression in N. benthamiana)

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