Dataset Information

Proteomic profiling of GFP-tagged PIN-LIKES (PILS2, PILS3, PILS6) in Arabidopsis root extracts reveals ERAD components as major interactors

ABSTRACT: This dataset describes a proteomic analysis of proteins co-immunoprecipitated with functional GFP-tagged PILS2, PILS3, and PILS6 fusion proteins in Arabidopsis thaliana roots. PILS (PIN-LIKES) proteins are auxin transport facilitators that localize to the endoplasmic reticulum (ER). To identify proteins that associate with PILS2, PILS3, and PILS6 under physiological conditions, we performed GFP-based immunoprecipitation (IP) followed by mass spectrometry (MS). Protein extracts were prepared from root tissues of Arabidopsis Col-0 (wild-type) and PILS2-, PILS3-, and PILS6-overexpressing transgenic lines expressing N- or C-terminal GFP fusion proteins. Plant material was ground in liquid nitrogen, and total proteins were extracted using aTris Buffer containing 1% CHAPS, 20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 15 mM EGTA, 75 mM NaCl, 1 mM DTT, 0.1% Tween-20, and protease inhibitors. Extracts were centrifuged, and the supernatants were quantified using the Bradford assay. 2mg of protein were subjected to immunoprecipitation using anti-GFP magnetic beads, Col-0 (non-tagged) extracts were used as negative controls. Co-immunoprecipitated proteins were then processed and analyzed by mass spectrometry.

INSTRUMENT(S):

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Root

SUBMITTER: Markus Hartl

LAB HEAD: Jürgen Kleine-Vehn

PROVIDER: PXD066215 | Pride | 2025-08-21

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20160308_QexHF2_RSLC8_Waidmann_KleineVehn_uni_ext_MFPL_onbead_Col_0_30per.raw	Raw
	20160308_QexHF2_RSLC8_Waidmann_KleineVehn_uni_ext_MFPL_onbead_PILS2_30per.raw	Raw
	20160308_QexHF2_RSLC8_Waidmann_KleineVehn_uni_ext_MFPL_onbead_PILS3_30per.raw	Raw
	201603_MaxQuant_result_txt.zip	Other
	201603_Perseus_result_1.5.015.zip	Other

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Project description:Many tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C. elegans, daf-16, promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16/FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p::gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p::gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p::gfp. Adult cell fate and the timing of col-19p::gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29. lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development lin-41 opposes adult cell fate by inhibiting the translation of lin-29, which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p::gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p::gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41, but does not require lin-29. Taken together, this work demonstrates a novel role for daf-16/FOXO as a heterochronic gene that promotes expression of lin-41/TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.

Dataset Information

Proteomic profiling of GFP-tagged PIN-LIKES (PILS2, PILS3, PILS6) in Arabidopsis root extracts reveals ERAD components as major interactors

Dataset's files

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