RPTOR BioID2 reveals spatial association between mTORC1 and FAs. (A) Immunostaining of RPTOR-BioID2-expressing U2OS cells treated with 50 µM biotin overnight. Cells were stained for biotin and myc-tagged RPTR. Scale bars, 20 µm (insets, 10 µm). Nuclei were visualized with DAPI. (B) Representative immunoblots of streptavidin pull-downs following 50 µM biotin incubation overnight (left) and immunoprecipitation (IP) with anti-mTOR antibody (right). WCL, whole-cell lysate. (C) Unsupervised analysis of RPTOR BioID samples (BioID2-based proximity labeling) quantified by MS. Hierarchical cluster analysis of Spearman rank correlation coefficients of pairwise comparisons for all samples analyzed by MS (P < 2 × 10−127, Spearman’s test; left). Principal-component analysis of samples analyzed by MS (right). The first two principal components account for 88.6% of the total variance of the dataset. (D) Enrichment of noncontaminant proteins specifically enriched in RPTOR BioID samples (n = 4 independent experiments; P < 0.01, two-sided Welch’s t test with permutation-based FDR correction, artificial within groups variance = 2). Proteins enriched by at least 256-fold or with P < 10−6 (two-sided Welch’s t test) are labeled; enriched TOR signaling components (Gene Ontology accession 0031929) are labeled in bold. Putative contaminant proteins are indicated by gray crosses. (E) Interaction network analysis of TOR signaling components identified in U2OS cells by RPTOR BioID and MS. (Fand G) KEGG pathway gene set enrichment analysis and Gene Ontology biological process overrepresentation enrichment analysis of noncontaminant proteins specifically enriched in RPTOR BioID samples. Biological processes enriched with P < 10−9 (hypergeometric test with Benjamini–Hochberg correction; F) and pathways enriched with P < 0.0001 (Kolmogorov–Smirnov test with FDR correction; G) are shown. (H) Interaction network analysis of RPTOR-proximal adhesome components identified by RPTOR BioID. (I) Diagrammatic representation of mTORC1 and lysosomal proteins identified in meta-adhesome datasets. See Table S2 and Table S3 for more details. (J and K) Interaction network analysis of reported physical and predicted protein–protein interactions and pathway associations between mTOR signaling pathway components (J) and lysosome-associated proteins (K) detected in adhesion complex proteomes (meta-adhesome datasets). See Table S2 and Table S3 for additional details. (L) Identification of adhesion proteins significantly enriched in the RPTOR BioID2 dataset that were also identified in published BioID datasets of the FA proteins paxillin and kindlin-2 in mouse pancreatic fibroblasts (Chastney et al., 2020).
