Limits...
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.

Show MeSH
Identification of proteins in a gel band by mass spectrometry. MALDI-TOF mass spectrum of the peptides produced by in-gel trypsin digestion of band no. 217 (molecular mass of ∼100 kD, fraction 35). The peak masses were used to identify proteins present in the band with the protein identification algorithm ProFound. The dominant protein component was Nup120p, which was identified with a probability 5 × 1022 higher than the next most probable protein. The peaks arising from Nup120p were subtracted from the spectrum and a new search was initiated, identifying Kap120p with a probability 3 × 109 higher than the next most probable protein. Finally, the peaks arising from Kap120p were subtracted from the spectrum and a new search was initiated identifying Pdr6p/Kap122p with a probability 1 × 108 higher than the next most probable protein. Colored markers indicate which peaks arise from each of the three identified proteins. The peaks labeled T arise from trypsin self-digestion. Unlabeled peaks likely originate from modified or incompletely digested peptides in Nup120p, Kap120p, and Kap122p, or from additional proteins.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169373&req=5

Figure 2: Identification of proteins in a gel band by mass spectrometry. MALDI-TOF mass spectrum of the peptides produced by in-gel trypsin digestion of band no. 217 (molecular mass of ∼100 kD, fraction 35). The peak masses were used to identify proteins present in the band with the protein identification algorithm ProFound. The dominant protein component was Nup120p, which was identified with a probability 5 × 1022 higher than the next most probable protein. The peaks arising from Nup120p were subtracted from the spectrum and a new search was initiated, identifying Kap120p with a probability 3 × 109 higher than the next most probable protein. Finally, the peaks arising from Kap120p were subtracted from the spectrum and a new search was initiated identifying Pdr6p/Kap122p with a probability 1 × 108 higher than the next most probable protein. Colored markers indicate which peaks arise from each of the three identified proteins. The peaks labeled T arise from trypsin self-digestion. Unlabeled peaks likely originate from modified or incompletely digested peptides in Nup120p, Kap120p, and Kap122p, or from additional proteins.

Mentions: To identify individual proteins associated with the NPC, we subjected the highly enriched NPC fractions to two-dimensional separations involving HPLC followed by SDS-PAGE. To obtain the highest resolution and yields, three different HPLC separation techniques were used. Fractions collected from each separation were then resolved by SDS-PAGE to generate a two-dimensional pattern of proteins. The pattern produced by hydroxyapatite HPLC/SDS-PAGE is shown in Fig. 1. Individual proteins in each band were identified by tryptic digestion followed by MALDI-TOF mass spectrometry. A sufficient number of tryptic peptide masses were determined from each band to unambiguously distinguish its corresponding open reading frame (ORF) in a genome database search (Kuster and Mann 1998). As an example, Fig. 2 shows the MALDI-TOF mass spectrum of the tryptic peptides obtained from a relatively weak band (no. 217). Three proteins were identified in the band: Nup120p is a known nup (Aitchison et al. 1995b; Heath et al. 1995), while Pdr6p/Kap120p and Ypl125p/Kap122p are both transport factors (see below). These three proteins were also detected in surrounding bands, providing independent verification of their presence in the NPC fraction. The MALDI-TOF peptide mapping procedure was used to analyze 465 bands from three independent hydroxyapatite HPLC/SDS-PAGE separations. In addition, 177 bands from the TFA-HPLC/SDS-PAGE separations were analyzed by MALDI-ion trap tandem mass spectrometry of the tryptic peptides (Qin et al. 1997). Finally, these data were combined with those obtained by the peptide microsequencing analysis of 49 prominent bands in the formic acid-HPLC/SDS-PAGE separation (Fernandez et al. 1994; Aitchison et al. 1995a,Aitchison et al. 1995b).


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)

Identification of proteins in a gel band by mass spectrometry. MALDI-TOF mass spectrum of the peptides produced by in-gel trypsin digestion of band no. 217 (molecular mass of ∼100 kD, fraction 35). The peak masses were used to identify proteins present in the band with the protein identification algorithm ProFound. The dominant protein component was Nup120p, which was identified with a probability 5 × 1022 higher than the next most probable protein. The peaks arising from Nup120p were subtracted from the spectrum and a new search was initiated, identifying Kap120p with a probability 3 × 109 higher than the next most probable protein. Finally, the peaks arising from Kap120p were subtracted from the spectrum and a new search was initiated identifying Pdr6p/Kap122p with a probability 1 × 108 higher than the next most probable protein. Colored markers indicate which peaks arise from each of the three identified proteins. The peaks labeled T arise from trypsin self-digestion. Unlabeled peaks likely originate from modified or incompletely digested peptides in Nup120p, Kap120p, and Kap122p, or from additional proteins.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Identification of proteins in a gel band by mass spectrometry. MALDI-TOF mass spectrum of the peptides produced by in-gel trypsin digestion of band no. 217 (molecular mass of ∼100 kD, fraction 35). The peak masses were used to identify proteins present in the band with the protein identification algorithm ProFound. The dominant protein component was Nup120p, which was identified with a probability 5 × 1022 higher than the next most probable protein. The peaks arising from Nup120p were subtracted from the spectrum and a new search was initiated, identifying Kap120p with a probability 3 × 109 higher than the next most probable protein. Finally, the peaks arising from Kap120p were subtracted from the spectrum and a new search was initiated identifying Pdr6p/Kap122p with a probability 1 × 108 higher than the next most probable protein. Colored markers indicate which peaks arise from each of the three identified proteins. The peaks labeled T arise from trypsin self-digestion. Unlabeled peaks likely originate from modified or incompletely digested peptides in Nup120p, Kap120p, and Kap122p, or from additional proteins.
Mentions: To identify individual proteins associated with the NPC, we subjected the highly enriched NPC fractions to two-dimensional separations involving HPLC followed by SDS-PAGE. To obtain the highest resolution and yields, three different HPLC separation techniques were used. Fractions collected from each separation were then resolved by SDS-PAGE to generate a two-dimensional pattern of proteins. The pattern produced by hydroxyapatite HPLC/SDS-PAGE is shown in Fig. 1. Individual proteins in each band were identified by tryptic digestion followed by MALDI-TOF mass spectrometry. A sufficient number of tryptic peptide masses were determined from each band to unambiguously distinguish its corresponding open reading frame (ORF) in a genome database search (Kuster and Mann 1998). As an example, Fig. 2 shows the MALDI-TOF mass spectrum of the tryptic peptides obtained from a relatively weak band (no. 217). Three proteins were identified in the band: Nup120p is a known nup (Aitchison et al. 1995b; Heath et al. 1995), while Pdr6p/Kap120p and Ypl125p/Kap122p are both transport factors (see below). These three proteins were also detected in surrounding bands, providing independent verification of their presence in the NPC fraction. The MALDI-TOF peptide mapping procedure was used to analyze 465 bands from three independent hydroxyapatite HPLC/SDS-PAGE separations. In addition, 177 bands from the TFA-HPLC/SDS-PAGE separations were analyzed by MALDI-ion trap tandem mass spectrometry of the tryptic peptides (Qin et al. 1997). Finally, these data were combined with those obtained by the peptide microsequencing analysis of 49 prominent bands in the formic acid-HPLC/SDS-PAGE separation (Fernandez et al. 1994; Aitchison et al. 1995a,Aitchison et al. 1995b).

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