Figure 2. Mapping Brl1 association with nuclear pore complex (NPC) assembly intermediates using KARMA (Kinetic Analysis of Incorporation Rates in Macromolecular Assemblies).

(A) Principles of KARMA: newly synthesized proteins are pulse-labeled followed by the affinity purification of the nucleoporin (NUP) complexes through a tagged NPC-binding protein. The extent of metabolic labeling is then quantified by mass spectrometry and corresponds to the average protein age in the affinity-purified fraction. An assembly factor selectively binds young NPCs, thus leading to high metabolic labeling rates for NUPs present in the intermediates (1). This is not the case for proteins that join after the assembly factor completely or partially dissociates or when the process is probed with an NUP bait (2). (B) Comparison of the labeling rates for NUPs and nuclear transport receptors (NTRs) in KARMA assays with Brl1 bait (left, this study) and with 10 different NUP baits (right, Onischenko et al., 2020). Median of three biological replicates. (C) Inaccessible pool of NUPs in KARMA assays with Brl1 compared to NUP baits (Onischenko et al., 2020), evaluated using a three-state kinetic state model (KSM) (Onischenko et al., 2020). (D) Barplot depicting the extent of metabolic labeling for different NUPs in KARMA assays with Brl1 bait after 90 min. The dotted line indicates the median NTR labeling. Median ± SD of three biological replicates. (E) Fractional labeling values from 2D averaged for NPC subcomplexes and offset by NTR labeling projected onto the NPC scheme.