Fig. 6: Transient phosphorylation at S16 on PSMA5 is needed for interaction with PSMA1.

a Clearance profiles of PSMA5 peptides carrying S16 or S56 (yellow), as well as all unphosphorylated peptides (blue) are shown. b Structure of PSMA5 (in blue) in the mature proteasome. The neighboring alpha ring subunit PSMA1 is highlighted in red. S16 and S56 are highlighted as sticks. Both residues are unphosphorylated in the mature proteasome structure (PDB ID: 6MSB). c Pull-down under stringent buffer conditions (RIPA) of exogenously expressed PSMA5-GFP constructs in HeLa cells shows that the PSMA5-PSMA1 interaction is lost upon mutation of S16 and S56. y axis: unadjusted p value from a limma analysis. N = 2. See also Supplementary Fig. 7a. d Thermal stability of peptides phosphorylated at S16 and S56 is significantly lower than the PSMA5 median suggesting a different biophysical state for phosphorylated PSMA5. Data from ref. 27. t test (median-pS16: t = 4.75, df = 8.00; median-pS56: t: 6.88, df = 5.36). Error bars are SD. Number of individual replicates shown below. e Hypothesis for the role of S16 (and S56) phosphorylation. We hypothesize that transient S16 and S56 phosphorylation is required for PSMA5 maturation and its incorporation into the proteasome. f Phosphopeptides exhibiting differing clearance are enriched in protein complex subunits. Fisher’s exact test, two-sided. Protein complexes are from the CORUM core complex dataset⁴⁷. g Phosphopeptides with differing clearance exhibit altered thermal stability compared to the unmodified protein median suggesting altered molecular states such as protein–protein interactions. Proteome-wide thermal stability data from ref. 27. Two-sided Wilcoxon test. Boxplots consist of median line, box: upper and lower quartiles, whiskers: 1.5 times interquartile range. Outliers have been omitted for clarity. h Peptides with faster clearance are enriched in intrinsically-disordered protein regions. Disorder prediction from D2P2 (ref. 43). See “Methods” for details. Fisher’s exact test, two-sided. Source data are provided as a Source data file.