Fig. 4: PPIA activity promotes expression of proteins enriched for IDRs.
a, Schematic of the pulsed SILAC experiment to evaluate protein synthesis. Pulse treatment for 24 h allowed for metabolic labelling of newly translated proteins. Protein synthesis was determined by the heavy to unlabelled ratio quantified by MS, as described in refs. 35,64. Two independent experiments were performed. b, Pulsed SILAC was performed and protein extracts from control or PPIA knockdown (Kd) cell lines were analysed to measure newly synthetized proteins. The heatmap represents the relative degree of protein synthesis (n = 345 overlapping proteins compared between the different cell types). c, Uptake of heavy amino acids by control or PPIA Kd 293T cells was quantified following a pulsed SILAC experiment. A value of 0.5 indicates the equal presence of light and heavy labelled peptides. *P < 0.05, **P < 0.01 and ****P < 0.0001; two-sided Wilcoxon rank-sum test (comparing 160 PPIA target proteins to 1,280 non-targets; similar findings were observed for HeLa cells). d, List of PPIA client proteins involved in protein phase separation based on PhaSepDB2.0. Proteins are listed by their official gene name. Nuclear bodies include nucleoli, Cajal bodies, nuclear speckles, paraspeckles, promyelocytic leukemia (PML) nuclear bodies and histone locus bodies. e, Immunoprecipitation of endogenous PPIA protein in HeLa cells followed by a western blot to detect the PPIA protein partners (poly(A)-binding protein 1 (PABPC1), DEAD-box helicase 6 (DDX6), Ras GTPase-activating protein-binding protein 1 (G3BP1) and nucleophosmin 1 (NPM1)). Data are representative of two independent experiments, confirmed by unbiased MS. f, PPIA activity is required for substrate binding. PPIA wild type, but not the G104A mutant, binds to PABPC1, DDX6 and NPM1 in IP–western experiments in HeLa cells. Data are representative of two independent experiments, confirmed by unbiased MS. g, Reduced expression of PPIA substrates following knockdown of the chaperone. HeLa cells were stably transduced with negative control or PPIA knockdown construct Kd1. Shown are representative immunoblots of n = 3 independent biological replicates. Right: a pairwise comparison shows significant reduction of PABPC1, DDX6 and NPM1 in PPIA knockdown cells by densitometry. GAPDH expression was used as a reference. Data are means ± s.d.; *P < 0.05, **P < 0.01; two-sided paired Student’s t-test. DMEM, Dulbecco’s modified Eagle medium.
