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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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Flow chart outlining our experimental strategy for protein classification.
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Figure 3: Flow chart outlining our experimental strategy for protein classification.

Mentions: Fig. 3 shows the design of a series of classification assays to determine which of the uncharacterized proteins were also nups. As discussed above, many proteins transiently associate with the NPC. We thus created an operational definition of a nup as a protein, the majority of which spends most of its time as a constituent of the octagonally symmetric NPC. For this study, proteins whose predominant localization is elsewhere, such as Sec13p (Siniossoglou et al. 1996) or nucleocytoplasmic transport factors (Rout et al. 1997), are not considered nups. Although such proteins may indeed behave as functional nups when associated with the NPC, the scope of our study precludes a detailed characterization of their behavior. According to our definition, a nup should colocalize with NPCs in the cell, and cofractionate with NPCs upon their isolation. Therefore, we tested each uncharacterized protein and (where necessary) proteins previously described as nups for these attributes. These proteins were genomically tagged at their COOH termini (ensuring normal expression levels) with a protein A (PrA) epitope in order to follow them during their characterization (Aitchison et al. 1995a). We also genomically tagged numerous proteins with the much smaller FLU moiety (Longtine et al. 1998) to check for tag-specific effects on protein localization. Our first screen involved localization of the epitope-tagged proteins in cells by immunofluorescence microscopy, because nups give a characteristic punctate nuclear rim staining pattern. However, as some nucleocytoplasmic transport factors and NE proteins also give a similar staining pattern, a second screen tested for colocalization with NPCs in a strain in which the NPCs themselves cluster into patches on the NE. Upon deletion of the gene encoding Nup120p the NPCs cluster to one side of the nuclear envelope (Fabre and Hurt 1997), which allows NE components to be distinguished from the coclustering NPC-associated nups and transport factors (Aitchison et al. 1996; Siniossoglou et al. 1996; Strambio-de-Castillia et al. 1999). Although most of the unknown ORFs gave immunostaining patterns typical for proteins of the ER, nucleus or nucleolus, 39 proteins showed associations with the NPCs (Fig. 4, Table ). In our third screen, the tagged proteins were followed by subcellular fractionation (Strambio-de-Castillia et al. 1995). We mainly used a preparation of NEs for this assay (Strambio-de-Castillia et al. 1999). While NE components and nups coenrich with the NE-containing fractions, nucleocytoplasmic transport factors and other proteins only partially associated with the NPC will be found mainly in the nucleoplasmic or cytoplasmic fractions (Fig. 5). Thus, the nups emerge as proteins that both cocluster with NPCs and preferentially cofractionate with the NEs.


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)

Flow chart outlining our experimental strategy for protein classification.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Flow chart outlining our experimental strategy for protein classification.
Mentions: Fig. 3 shows the design of a series of classification assays to determine which of the uncharacterized proteins were also nups. As discussed above, many proteins transiently associate with the NPC. We thus created an operational definition of a nup as a protein, the majority of which spends most of its time as a constituent of the octagonally symmetric NPC. For this study, proteins whose predominant localization is elsewhere, such as Sec13p (Siniossoglou et al. 1996) or nucleocytoplasmic transport factors (Rout et al. 1997), are not considered nups. Although such proteins may indeed behave as functional nups when associated with the NPC, the scope of our study precludes a detailed characterization of their behavior. According to our definition, a nup should colocalize with NPCs in the cell, and cofractionate with NPCs upon their isolation. Therefore, we tested each uncharacterized protein and (where necessary) proteins previously described as nups for these attributes. These proteins were genomically tagged at their COOH termini (ensuring normal expression levels) with a protein A (PrA) epitope in order to follow them during their characterization (Aitchison et al. 1995a). We also genomically tagged numerous proteins with the much smaller FLU moiety (Longtine et al. 1998) to check for tag-specific effects on protein localization. Our first screen involved localization of the epitope-tagged proteins in cells by immunofluorescence microscopy, because nups give a characteristic punctate nuclear rim staining pattern. However, as some nucleocytoplasmic transport factors and NE proteins also give a similar staining pattern, a second screen tested for colocalization with NPCs in a strain in which the NPCs themselves cluster into patches on the NE. Upon deletion of the gene encoding Nup120p the NPCs cluster to one side of the nuclear envelope (Fabre and Hurt 1997), which allows NE components to be distinguished from the coclustering NPC-associated nups and transport factors (Aitchison et al. 1996; Siniossoglou et al. 1996; Strambio-de-Castillia et al. 1999). Although most of the unknown ORFs gave immunostaining patterns typical for proteins of the ER, nucleus or nucleolus, 39 proteins showed associations with the NPCs (Fig. 4, Table ). In our third screen, the tagged proteins were followed by subcellular fractionation (Strambio-de-Castillia et al. 1995). We mainly used a preparation of NEs for this assay (Strambio-de-Castillia et al. 1999). While NE components and nups coenrich with the NE-containing fractions, nucleocytoplasmic transport factors and other proteins only partially associated with the NPC will be found mainly in the nucleoplasmic or cytoplasmic fractions (Fig. 5). Thus, the nups emerge as proteins that both cocluster with NPCs and preferentially cofractionate with the NEs.

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