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. 2022 Aug 24;11:e78385. doi: 10.7554/eLife.78385

Figure 1. Brl1 preferentially binds young nuclear pore complexes (NPCs).

(A) Scheme of the NPC architecture. The colors indicate the assembly order as found in Onischenko et al., 2020. Nucleoporins (NUPs) that were reproducibly identified in Brl1 affinity purifications are shown in bold. (B) Schematic illustrating the transient binding of an NPC assembly factor during NPC assembly. (C) Enrichment of Brl1 in affinity pulldowns from Onischenko et al., 2020 using baits from the different assembly tiers. Early and intermediate tiers contain four different baits each; the late tier is represented by Mlp1 with three biological replicates for each bait. (D) Schematic representation of the recombination-induced tag exchange (RITE) strategy to visualize Brl1-mCherry co-localization with old or new NPCs marked by Nup170-yEGFP and the expected NE fluorescence intensity profiles. (E) Representative co-localization images of Brl1-mCherry with old or new Nup170-yEGFP marked NPCs using the RITE strategy described in (D). Cells were imaged ~30 min or ~5 hr after recombination induction, respectively. Fluorescence intensity profiles along the NE are displayed for cells denoted with an asterisk. (F) Pearson’s correlation between Nup170-yEGFP and Brl1-mCherry fluorescence intensity profiles along the NE in (E). Individual points reflect the average of a biological replicate with a minimum of 28 analyzed NE contours per condition. Two-tailed Student’s t-test (n = 5, p-value=0.00015).

Figure 1—source data 1. Label-free Brl1 intensities with 10 nucleoporin (NUP) baits from Onischenko et al., 2020; related to Figure 1C.
Figure 1—source data 2. Pearson’s correlation coefficients for Brl1-mCherry and new or old Nup170-yeGFP.

Figure 1.

Figure 1—figure supplement 1. Proteomic characterization of Brl1 nuclear pore complex (NPC) interactions.

Figure 1—figure supplement 1.

(A) All 1500 proteins co-purified in affinity pulldowns with 10 different nucleoporin (NUP) baits (Onischenko et al., 2020) were ranked by their fold enrichment difference between early and late tier baits. Mean ± SEM of three biological replicates. (B) Log10 abundance of NUPs belonging to the different assembly tiers in Brl1 affinity pulldowns (APs). Values for three biological replicates. (C) Reproducibility of the fractional labeling in KARMA (Kinetic Analysis of Incorporation Rates in Macromolecular Assemblies) assays with Brl1. Individual points correspond to the fractional labeling of a protein. (D) Heatmap showing the fractional labeling of NUPs in KARMA assays with Brl1 bait.
Figure 1—figure supplement 1—source data 1. Label-free nucleoporin/nuclear transport receptor (NUP/NTR) intensities with Brl1 bait (related to Figure 1—figure supplement 1B).