Dataset Information

Proxiome at the 40S ribosomal head region according to proximity labeling MS experiments

ABSTRACT: The in vivo labeling technique Biotin identification (BioID, Roux et al., 2012) was used to capture proteins in the microenvironments of the ribosomal proteins Rps3 (uS3) and Rps20 (us10) in Saccharomyces cerevisiae. Biotinylated proteins were captured from cell lysates using affinity purification and subjected to SDS-PAGE followed by in-gel digestion with trypsin. Liquid chromatography-mass spectrometry (LC-MS) was performed to identify and relatively quantify peptides and thus proteins. Relative quantification of proteins was based on stable isotope labeling with amino acids in cell culture (SILAC). To account for putative differences in the expression levels of the enriched proteins, an input control was analyzed as well. To this end, an aliquot of the cell lysate was taken prior to protein enrichment and analyzed as well. Beyond the known and expected interaction partners of Rps3 and Rps20, we identified proteins not known to co-localize with them. Moreover, we could also observe changes in their environment when the WD40-repeat protein Asc1/RACK1 was absent from the head region.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive HF

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Oliver Valerius

LAB HEAD: Oliver Valerius

PROVIDER: PXD027267 | Pride | 2021-11-10

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 132

Publications

A Multi-Perspective Proximity View on the Dynamic Head Region of the Ribosomal 40S Subunit.

Schmitt Kerstin K Kraft Alina-Andrea AA Valerius Oliver O

International journal of molecular sciences 20211028 21

A comparison of overlapping proximity captures at the head region of the ribosomal 40S subunit (hr40S) in Saccharomyces cerevisiae from four adjacent perspectives, namely Asc1/RACK1, Rps2/uS5, Rps3/uS3, and Rps20/uS10, corroborates dynamic co-localization of proteins that control activity and fate of both ribosomes and mRNA. Co-locating factors that associate with the hr40S are involved in (i) (de)ubiquitination of ribosomal proteins (Hel2, Bre5-Ubp3), (ii) clamping of inact ...[more]

PMID: 34769086

Similar Datasets

Project description:The in vivo labeling technique Split-Biotin identification (Split-BioID, De Munter et al., 2017, Schopp et al., 2017, Cho et al., 2020) was used to capture the common microenvironment of Bre5 and the ribosomal protein Rps2 (uS5). Biotinylated proteins were enriched from cell lysates using affinity purification. After SDS-PAGE, proteins were digested in-gel with trypsin and the resulting peptides were analyzed by liquid chromatography-mass spectrometry (LC-MS) for identification and relative quantification against controls. An aliquot of the cell lysate that was used for the affinity purification was taken as an input control and analyzed as well by LC-MS to account for possible differences in the input abundance of enriched proteins. Stable isotope labeling with amino acids in cell culture (SILAC) was used for relative quantification of proteins according to the 2nSILAC approach (Dannenmaier et al., 2018). To evaluate the labeling efficiency, an aliquot of each culture was harvested separately prior to the pooling of the cells for the Split-BioID experiment. Cell lysates from these samples were used for SDS-PAGE, and a part of each gel lane was used for an in-gel digest of proteins. These peptides were also analyzed with LC-MS and the percentages of SILAC-labeled peptides (based on MSMS counts) were calculated. The captured proteins belong to a common cellular microenvironment of Bre5 and Rps2. References: Cho, K.F., Branon, T.C., Rajeev, S., Svinkina, T., Udeshi, N.D., Thoudam, T., Kwak, C., Rhee, H.W., Lee, I.K., Carr, S.A., Ting, A.Y. (2020). Split-TurboID enables contact-dependent proximity labeling in cells. Proc Natl Acad Sci U S A. 117, 12143-12154. Dannenmaier, S., Stiller, S.B., Morgenstern, M., Lübbert, P., Oeljeklaus, S., Wiedemann, N., Warscheid, B. (2018). Complete Native Stable Isotope Labeling by Amino Acids of Saccharomyces cerevisiae for Global Proteomic Analysis. Anal Chem 90, 10501-10509. De Munter, S., Görnemann, J., Derua, R., Lesage, B., Qian, J., Heroes, E., Waelkens, E., Van Eynde, A., Beullens, M., Bollen, M. (2017). Split-BioID: a proximity biotinylation assay for dimerization-dependent protein interactions. FEBS Lett 591, 415-424. Schopp, I.M., Amaya Ramirez, C.C., Debeljak, J., Kreibich, E., Skribbe, M., Wild, K., Béthune, J. (2017). Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes. Nat Commun 8,15690.

Project description:The plant pathogenic fungus Verticillium dahliae has homologs of the clock genes [Cascant-Lopez et al., 2020], which are well described in Neurospora crassa [Diernfellner et al., 2020]. In N. crassa, frequency (Frq) and the Frq-interacting RNA helicase (Frh) interact and form the Frq-Frh complex [Cheng et al., 2005]. Formation of this complex was described to be still possible but weakened by a single amino acid exchange of arginine 806 of Frh to histidine (Frh^R806H) [Shi et al., 2010; Conrad et al., 2016]. We found that both FRQ and wild-type FRH were required for enhanced conidiation and the light-dependent repression of microsclerotia formation in V. dahliae, but were dispensable for disease induction in tomato plants. Strikingly, the FRH^R806H mutant strain grew similar to the FRQ deletion strain, although transcript and protein levels of Frq were not significantly affected by the FRH^R806H mutation when compared with the wild-type. Therefore, we hypothesized that V. dahliae Frq and Frh form the Frq-Frh complex to regulate V. dahliae development, and that this complex formation is abolished upon the amino acid exchange in the Frh. By the use of protein pull-downs, potential interactions of Frh-GFP or Frh^R806H-GFP with Frq were investigated. While we found evidence for a direct interaction of Frh-GFP and Frq, no proteins were significantly co-enriched with Frh^R806H-GFP. References: Cascant-Lopez, E.; Crosthwaite, S.K.; Johnson, L.J.; Harrison, R.J. No evidence that homologs of key circadian clock genes direct circadian programs of development or mRNA abundance in Verticillium dahliae. Front Microbiol 2020, 11, 1977, doi:10.3389/fmicb.2020.01977. Diernfellner, A.C.R.; Brunner, M. Phosphorylation timers in the Neurospora crassa circadian clock. J Mol Biol 2020, 432, 3449–3465, doi:10.1016/j.jmb.2020.04.004. Cheng, P.; He, Q.; He, Q.; Wang, L.; Liu, Y. Regulation of the Neurospora circadian clock by an RNA helicase. Genes Dev 2005, 19, 234–241, doi:10.1101/gad.1266805. Shi, M.; Collett, M.; Loros, J.J.; Dunlap, J.C. FRQ-interacting RNA helicase mediates negative and positive feedback in the Neurospora circadian clock. Genetics 2010, 184, 351–361, doi:10.1534/genetics.109.111393. Conrad, K.S.; Hurley, J.M.; Widom, J.; Ringelberg, C.S.; Loros, J.J.; Dunlap, J.C.; Crane, B.R. Structure of the frequency‐interacting RNA helicase: a protein interaction hub for the circadian clock. EMBO J 2016, 35, 1707–1719, doi:10.15252/embj.201694327.

			Action	DRS
	KS_Q_1089.raw	Raw
	KS_Q_1090.raw	Raw
	KS_Q_1091.raw	Raw
	KS_Q_1092.raw	Raw
	KS_Q_1093.raw	Raw