Figure 5.

Relative distribution of tagged proteins in subcellular fractions. (Top right) Cells from the Nup42p Flu-tagged strain were fractionated and the proteins from each fraction were separated by SDS-PAGE and visualized by amido black. Lanes 1–4 were loaded at one cell equivalent and lanes 5–7 were loaded at three cell equivalents. Fraction 1 contains cytoplasmic material, 2 and 3 contain membranes, and 4 contains mainly nuclei. The nuclei were subjected to a second round of fractionation to obtain the two chromatin fractions 5 and 6, and fraction 7 enriched in NEs. The majority of proteins, including the indicated abundant cytoplasmic markers (Pyk1p, Pdc1p, Eno2p, Tdh3p, and Gpm1p) identified by mass spectrometry did not coenrich with the nuclei or the NEs. The histone triplet at ∼14 kD coenriched with nuclei but not NEs. (Top left) Proteins that cofractionate with NEs. Fractions from the same enrichment procedure for various tagged strains were probed for the internal standard Pom152p (Ctrl) and the protein of interest, mostly through a protein A tag (PrA). In a few cases, a FLU tag (Flu) or a monospecific antibody (Ab) was used. As expected, known nups coenriched with the NE-containing fractions, as did the newly identified Nup60p, Nup192p, and Pom34p. Seh1p, Gle1p, Gle2p, and Nup42p/Rip1p also coenriched with NEs, securing the classification of these proteins as nups. (Middle right) Proteins that do not cofractionate with NEs. Mex67p coenriched mainly with nuclei and partially with NEs, but a significant amount remained in the nucleoplasm in agreement with its recent classification as an important new mRNA export factor (Katahira et al. 1999). Similarly, both Pdr6p/Kap122p and Ypl125p/Kap120p showed a partial association with the highly enriched NEs during fractionation which, taken together with the immunofluorescence microscopy data, confirmed them as karyopherins. Neither Yrb2p nor Nup2p, which are closely related to each other at the primary sequence level, cofractionated with the NPC-containing fractions. Yrb2p is now known not to be a nup (see Fig. 4), but the significant amount of Nup2p remaining with the NE fraction points to a strong association with the NPCs. As Npl4p did not cofractionate with NPCs (and showed no NPC association by immunofluorescence microscopy; Fig. 4), we concluded that it is not a strongly bound component of the NPC. (Bottom) Nuclei were separated into fractions containing spindle pole bodies (SPBs), crude NPCs, enriched NPCs, and highly enriched NPCs; fraction numbers are as previously described (Rout and Kilmartin 1990). Cdc31p, which coenriched with the Pom152p control in the NE preparation, also cofractionated with Nic96p and Nup159p, two previously known nucleoporins, in the highly enriched NPC preparation, which allowed us to characterize Cdc31p as a component of the NPC. However, unlike the controls, significant amounts of Cdc31p were found in the SPB fraction. Cdc31p had given SPB staining plus some peripheral nuclear signal by immunofluorescence microscopy (Biggins and Rose 1994). Thus, Cdc31p appears to be present in both NPCs and SPBs, behaving in a similar fashion to Ndc1p (Chial et al. 1998).