What makes a yeast nuclear pore?

An established protocol was used to obtain nuclear fractions of yeast highly enriched in NPCs. Proteins contained in these fractions were separated by high performance liquid chromatography, and column fractions were separated by SDS gel electrophoresis and protein staining. Each distinct band was excised from the gel, digested with trypsin and analyzed by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) or MALDI ion trap tandem mass spectroscopy, or by microsequencing. Measured masses or sequences of peptides were used to identify corresponding open reading frames (ORFs) unambiguously from the yeast genome database. The authors claim that, by using large amounts of material and oversampling, they have identified all the proteins in the NPC fraction. To find the nups among the 174 identified proteins, the genes were first genomically epitope-tagged by insertion of a sequence within the genomic DNA that encodes a protein tag that can be identified later with an antibody. This ensured endogenous levels of expression and standardized detection in the subsequent characterization. Three criteria were used to determine whether the tagged proteins localized to the NPC: immunofluorescence localization to the nuclear rim; co-localization to the NPC in an NPC clustering assay; and biochemical fractionation to the nuclear envelope. This analysis identified 40 NPC proteins - 29 nups and 11 transport proteins, including three previously uncharacterized ORFs and four known proteins that had not been previously classified as nups. No motor proteins, ATPases, GTPases, or potential gate proteins were found. Further characterization of the nups localized the tagged proteins within the NPC by immunoelectron microscopic mapping onto a higher-resolution structure. Most nups were found on both the cytoplasmic and nuclear faces of the NPC. Two were exclusively nucleoplasmic, three exclusively cytoplasmic and four were found on both sides but showed a consistent bias