Fig. 2

Hek2-dependent translational repression and protein turnover define nucleoporin levels. a Transcriptome analysis of the hek2∆ mutant. The y-axis is the averaged log2 of the hek2∆/wt ratios calculated from two independent microarray hybridizations. The x-axis is the log2 of the averaged fluorescence intensities. mRNAs encoding NPCs components are colored depending on their association to Hek2 (from Fig. 1). b Single-molecule FISH was performed on wt and hek2∆ cells using set of probes specific for the indicated mRNAs. NSP1 and NUP100 probes were coupled to the Quasar570 fluorophore (red), and NUP133 probes to Quasar670 (far red). The z-projections are displayed, together with merged images with a nuclear staining (DAPI). Scale bar, 5 µm. c Polysome fractionation from wt and hek2∆ cells (W303 background). The absorbance at 254 nm (A₂₅₄) recorded during the collection of the fractions of the gradient is displayed. The positions of 40S, 60S, 80S ribosomal species are indicated, as well as the number of ribosomes per mRNA in polysomes fractions. d Relative distribution of the indicated mRNAs in polysome gradients from wt (black lines) and hek2∆ (red lines) cells. mRNAs amounts in each fraction were quantified by RT-qPCR, normalized to the sum of the fractions and to the distribution of a control spike RNA. Gray arrows indicate a decrease in the amounts of mRNAs found in the light fractions in hek2∆ cells, while red arrows point to an increase in the quantity of mRNAs found in the polysomes fractions. These results are representative of four independent experiments (two performed in the W303 background, two in the BY4742 background; see Supplementary Fig. 2). e Same as (d) for NUP133 and ACT1 control mRNAs. f Protein levels of the indicated nucleoporins (Nup116, Nup1, Nup133) and of a GFP-tagged version of Nup59 were scored in wt and hek2∆ cells treated with cycloheximide (CHX) for the indicated time (min). Top, Whole-cell extracts were analyzed by western blotting using anti-GFP, anti-GLFG, anti-FSFG or anti-Nup133 antibodies. Bottom, The relative amounts of the indicated proteins (mean and individual points; n = 3) were quantified over the time following CHX treatment and expressed relative to t = 0