Figure 1. Combining different treatments and baits determines different components of the TORC interactome.
A) Scheme illustrating PUP-IT in the context of TOR signaling. PafA fused to the baits LST8-1, RAPTOR1, and ScFKBP labels individual and TOR complex associated protein interactors, while a GFP fusion acts as a control for unspecific interactions.
B–E) Volcano plots showing proteins significantly enriched in LST8-1 B,D) and RAPTOR1 C,E) baits using constitutive B,C) or inducible D,E) FLAG::PUP(E) expression.
F–G) Treatment-dependency of identified interactors using LST8-1 G) or RAPTOR1 H) baits and inducible FLAG::PUP(E) expression. Treatment-specific interactors are those only identified in one of the two treatments.
H) Rapamycin response of WT and ScFKBP-producing seedlings (n = 10 seedlings). Bar height indicates group median.
I) Volcano plot showing proteins significantly enriched in the ScFKBP bait using constitutive FLAG::PUP(E) expression.
In all volcano plots, fold changes are calculated from n = 3 replicates using MsqRob2, p-values are corrected for multiple comparisons using the Benjamini–Hochberg FDR method. Different letters in panel (H) indicate statistically significant differences between groups based on a one-way ANOVA followed by a Tukey-HSD test (𝛼 = 0.05).
Figure 2. Identification of phosphorylated direct interactors of the TOR complex and in silico corroboration using AlphaFold2.
A,B) Phosphorylated proteins enriched in LST8-1 and RAPTOR1 against the GFP control after 4 h A) and 24 h B) of sucrose treatment and FLAG::PUP(E) induction. Phosphorylation sites previously associated with TOR are indicated in blue, new sites in proteins previously reported to be phosphorylated by TOR in turquoise. Fold changes are calculated from n = 3 replicates using MsqRob2, p-values are corrected for multiple comparisons using the Benjamini–Hochberg FDR method.
C,D) The two significantly enriched motifs among the identified phosphorylation sites. The “SP” motif C) mirrors previous reports from TOR substrates, while the “RxxS” motif D) has previously been associated with TOR downstream interactor S6K1.
E) Interactions of 20 proteins from the four shown groups with the TORC components TOR, LST8-1, and RAPTOR1 were predicted using AlphaFold2 multimer. Vertical lines indicate the median of the local interaction score (LIS) distribution by group.
F) Predicted structures of the top-scoring interactions for each group: newly identified TORC interactor PANK2, TORC subunit RAPTOR1, senescence regulator S40-7, and KIN10 paralog KIN11.
Models are colored by predicted local distance difference test (pLDDT), with values above 70 indicating high confidence predictions.
Great work by Zheng et al. (2025) on how employing pupylation-based proximity labeling (PUP-IT) unravels a comprehensive #interactome of #Arabidopsis TOR complex.
Newly identified #PlantTOR interactors, like PANK2, were also supported by #AlphaFold2.
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