Fig. 1: NatA and NatB diverging co-translational assembly pathways correlate with opposite stability propensities.

a S. cerevisiae NatA and NatB heterodimers. b NATs subunits co-translational assembly analysis by SeRP, revealing divergent co-translational assembly pathways. Showing the mean with experimental variation between replicates shaded (n = 2 biologically independent experiments). The ribosome position at which the enrichment stably crosses the twofold threshold is indicated. c NatA and NatB structures superimposed, demonstrating their high similarity as determined by TM-Align. d–h Thermostability analysis of NatA and NatB by MD simulations. d Complex vs. free thermostability comparison of the catalytic and auxiliary subunits. MD simulation at 100 °C, at a 300 ns timeframe; 30 overlaid frames, taken at 10 ns intervals. Highlighted are the regions prior to co-translational interaction initiation (minimal regions) of Naa15, and Naa20 and its equivalent in Naa10, and Naa25. The long IDRs of Naa10 are not shown. e, f RMSF of the free catalytic subunits (e) and auxiliary subunits (f) during 300 ns of MD simulation at 30 °C, 50 °C, and 100 °C. At all temperatures. The dashed line indicates the initiation of co-translational interaction. Shown are structurally conserved regions, as determined by TM-Align. g, h Catalytic subunits’ (g) and auxiliary subunits’ (h) RMSF boxplots of the structurally conserved regions, as in (e)–(f). Median is indicated with the box extending from the 25th to 75th percentiles and whiskers from minimum to maximum. Statistical analysis by unpaired two-sided t-test. n = 131 for Naa10, Naa20. n = 368 for Naa15, Naa25. i Solubility analysis of individual complex subunits tagged by GFP in vivo, in wildtype and in deletion strains expressing the tagged subunits orphaned from their partner subunit. A representative image is shown. j The fraction of cells displaying foci of GFP-tagged subunit. Data are mean ± SEM, n = 150 cells per sample from three biologically independent experiments, unpaired two-sided t-test (p value indicated). k Illustration of the assembly pathways of NatA and NatB, diverging in correlation with the subunits’ opposite tendency for instability (grey lines), with the independently stable subunits (Naa10 and Naa25) engaging their nascent partner, thus facilitating their stability. Created with BioRender.com.