The yeast nuclear pore complex: composition, architecture, and transport mechanism.
Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins).This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC.Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.
Affiliation: The Rockefeller University, New York, NY 10021, USA. firstname.lastname@example.org
An understanding of how the nuclear pore complex (NPC) mediates nucleocytoplasmic exchange requires a comprehensive inventory of the molecular components of the NPC and a knowledge of how each component contributes to the overall structure of this large molecular translocation machine. Therefore, we have taken a comprehensive approach to classify all components of the yeast NPC (nucleoporins). This involved identifying all the proteins present in a highly enriched NPC fraction, determining which of these proteins were nucleoporins, and localizing each nucleoporin within the NPC. Using these data, we present a map of the molecular architecture of the yeast NPC and provide evidence for a Brownian affinity gating mechanism for nucleocytoplasmic transport.
- Fungal Proteins/analysis*/chemistry/genetics
- Membrane Proteins/analysis*/chemistry/genetics
- Nuclear Envelope/chemistry*/genetics/metabolism*/ultrastructure
- Nuclear Proteins/analysis*/chemistry/genetics
- Saccharomyces cerevisiae/chemistry/metabolism/ultrastructure*
- Biological Transport
- Carrier Proteins/analysis/chemistry/genetics
- Chromatography, High Pressure Liquid
- Fluorescent Antibody Technique
- Microscopy, Immunoelectron
- Models, Biological
- Molecular Weight
- Recombinant Fusion Proteins/analysis/chemistry/genetics
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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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).
A systematic compositional analysis of the NPC has the potential to identify virtually all of the NPC components. However, an exhaustive analysis requires the isolation and identification of every detectable protein in a highly enriched preparation of intact NPCs. This has become feasible with the development of a method that produces large quantities of highly enriched yeast (Saccharomyces) NPCs (Rout and Blobel 1993), together with the completion of the yeast genome sequence and advances in protein identification techniques. This NPC fraction is the most highly enriched preparation available. The NPCs isolated by this mild procedure appear morphologically intact (Rout and Blobel 1993; Yang et al. 1998) and no significant loss has been found for any confirmed nup, including the most peripheral nups and all of the known pore membrane proteins (see Fig. 5, Table ). Indeed, many peripherally associated nucleocytoplasmic transport factors partially cofractionate with the NPCs during this procedure.