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The yeast nuclear pore complex: composition, architecture, and transport mechanism.

Rout MP, Aitchison JD, Suprapto A, Hjertaas K, Zhao Y, Chait BT - J. Cell Biol. (2000)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Rockefeller University, New York, NY 10021, USA. rout@rockvax.rockefeller.edu

ABSTRACT
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.

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Relative abundance of nucleoporins in the NPC. Each data point represents the ratio of the signal from the tagged nucleoporin to the signal for the internal standard (Table ), which are proportional to the relative quantity of each protein in the NPC, averaged from at least two independent measurements for each nup (each generated from four different ratio measurements). The brackets indicate clusters of relative abundance containing the nups indicated at the right. Nups found exclusively on one side of the NPC are shown as open diamonds.
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Figure 9: Relative abundance of nucleoporins in the NPC. Each data point represents the ratio of the signal from the tagged nucleoporin to the signal for the internal standard (Table ), which are proportional to the relative quantity of each protein in the NPC, averaged from at least two independent measurements for each nup (each generated from four different ratio measurements). The brackets indicate clusters of relative abundance containing the nups indicated at the right. Nups found exclusively on one side of the NPC are shown as open diamonds.

Mentions: We generated aligned montages to show the distribution of labeling around the NPC for 27 nups (Fig. 7). The montages preserved the morphology and dimensions of individual NPCs, indicating that our alignments were accurate. Our results disagree with certain localizations performed using one particular technique (Fahrenkrog et al. 1998; Kosova et al. 1999; Strahm et al. 1999). That technique yields localizations that include a distribution of Gle1p throughout the cytoplasm, and Nup42p throughout the nucleoplasm; these seem highly unlikely, as both of these proteins cofractionate absolutely with the NPC-containing fractions (Fig. 5). Furthermore, the authors acknowledge potential problems with their methodology, including overexpression of the tagged protein and epitope inaccessibility, which we avoided. By contrast, considerable credence is given to our data by their consistency with results obtained by electron microscopy (Rout and Blobel 1993; Yang et al. 1998), with all other yeast nup localizations (Kraemer et al. 1995; Nehrbass et al. 1996; Hurwitz et al. 1998; Marelli et al. 1998; Wente, S.R., personal communication), with the proximity of nups deduced from the isolation of nup subcomplexes (Grandi et al. 1993, Grandi et al. 1995; Siniossoglou et al. 1996), and with our immunofluorescence localization, cofractionation, and quantitation data (see Fig. 4, Fig. 5, and Fig. 9).


The yeast nuclear pore complex: composition, architecture, and transport mechanism.

Rout MP, Aitchison JD, Suprapto A, Hjertaas K, Zhao Y, Chait BT - J. Cell Biol. (2000)

Relative abundance of nucleoporins in the NPC. Each data point represents the ratio of the signal from the tagged nucleoporin to the signal for the internal standard (Table ), which are proportional to the relative quantity of each protein in the NPC, averaged from at least two independent measurements for each nup (each generated from four different ratio measurements). The brackets indicate clusters of relative abundance containing the nups indicated at the right. Nups found exclusively on one side of the NPC are shown as open diamonds.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169373&req=5

Figure 9: Relative abundance of nucleoporins in the NPC. Each data point represents the ratio of the signal from the tagged nucleoporin to the signal for the internal standard (Table ), which are proportional to the relative quantity of each protein in the NPC, averaged from at least two independent measurements for each nup (each generated from four different ratio measurements). The brackets indicate clusters of relative abundance containing the nups indicated at the right. Nups found exclusively on one side of the NPC are shown as open diamonds.
Mentions: We generated aligned montages to show the distribution of labeling around the NPC for 27 nups (Fig. 7). The montages preserved the morphology and dimensions of individual NPCs, indicating that our alignments were accurate. Our results disagree with certain localizations performed using one particular technique (Fahrenkrog et al. 1998; Kosova et al. 1999; Strahm et al. 1999). That technique yields localizations that include a distribution of Gle1p throughout the cytoplasm, and Nup42p throughout the nucleoplasm; these seem highly unlikely, as both of these proteins cofractionate absolutely with the NPC-containing fractions (Fig. 5). Furthermore, the authors acknowledge potential problems with their methodology, including overexpression of the tagged protein and epitope inaccessibility, which we avoided. By contrast, considerable credence is given to our data by their consistency with results obtained by electron microscopy (Rout and Blobel 1993; Yang et al. 1998), with all other yeast nup localizations (Kraemer et al. 1995; Nehrbass et al. 1996; Hurwitz et al. 1998; Marelli et al. 1998; Wente, S.R., personal communication), with the proximity of nups deduced from the isolation of nup subcomplexes (Grandi et al. 1993, Grandi et al. 1995; Siniossoglou et al. 1996), and with our immunofluorescence localization, cofractionation, and quantitation data (see Fig. 4, Fig. 5, and Fig. 9).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Rockefeller University, New York, NY 10021, USA. rout@rockvax.rockefeller.edu

ABSTRACT
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.

Show MeSH