Interactome of the Nuclear Basket Proteins of the Yeast Nuclear Pore Complex

摘要

The nuclear basket, a discrete structure projecting from the nucleoplasmic face of the nuclear pore complex, makes multiple interactions with the nuclear envelope and nuclear periphery. Chief amongst its functions is the regulation of gene expression and RNA export. We have begun to explore the dynamic interactome of this structure through its two main components, Mlp1 and Mlp2. Genomically tagged versions of both Mlp1 and Mlp2 were affinity purified under conditions which retained many of their interacting partners. Purifications were also performed in yeast strains lacking key nuclear pore complex components in order to understand how the basket is attached to the nuclear pore complex and how in turn this affects the composition of the basket interactome. Following immunopurification, the isolated proteins were resolved by 1-D SDS-PAGE and each complete gel lane was subjected to MALDI MS analysis using a prOTOF2000 mass spectrometer (PerkinElmer). The XProteo computer algorithm was used to search the peptide fingerprint data in the NCBI database, and top candidate proteins were subjected to hypothesis-driven CID MS/MS with a vacuum-MALDI ion trap (vMALDI LTQ; Finnigan).