An Amphipathic Helix in Brl1 is Required for Nuclear Pore Complex Biogenesis in S. cerevisiae - KARMA analysis of Nup170 with/without Brl1 degradation (DIA)

This project was aimed at investigating nuclear pore complex biogenesis upon inducible degradation of Brl1 using the Kinetic Analysis of incorporation Rates in Macromolecular Assemblies (KARMA, Onischenko et al. 2020) method. In brief, the nuclear pore complex was purified through endogenously tagged Nup170 at several time points following auxin inducible degradation of Brl1. Simultaneously with the degradation of Brl1, newly synthesized proteins were metabolically labeled using a SILAC approach. The metabolic labeling of all nuclear pore complex constituents was assessed. Proteins that depend on Brl1 for their assembly show delayed metabolic labeling.