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Evidence for a shared nuclear pore complex architecture that is conserved from the last common eukaryotic ancestor.

DeGrasse JA, DuBois KN, Devos D, Siegel TN, Sali A, Field MC, Rout MP, Chait BT - Mol. Cell Proteomics (2009)

Bottom Line: Thus, to gain a broad perspective on the origins and evolution of the NPC, we performed proteomics analyses of NPC-containing fractions from a divergent eukaryote (Trypanosoma brucei) and obtained a comprehensive inventory of its nucleoporins.Strikingly trypanosome nucleoporins clearly share with metazoa and yeast their fold type, domain organization, composition, and modularity.Overall these data provide conclusive evidence that the majority of NPC architecture is indeed conserved throughout the Eukaryota and was already established in the last common eukaryotic ancestor.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York 10065, USA.

ABSTRACT
The nuclear pore complex (NPC) is a macromolecular assembly embedded within the nuclear envelope that mediates bidirectional exchange of material between the nucleus and cytoplasm. Our recent work on the yeast NPC has revealed a simple modularity in its architecture and suggested a common evolutionary origin of the NPC and vesicle coating complexes in a progenitor protocoatomer. However, detailed compositional and structural information is currently only available for vertebrate and yeast NPCs, which are evolutionarily closely related. Hence our understanding of NPC composition in a full evolutionary context is sparse. Moreover despite the ubiquitous nature of the NPC, sequence searches in distant taxa have identified surprisingly few NPC components, suggesting that much of the NPC may not be conserved. Thus, to gain a broad perspective on the origins and evolution of the NPC, we performed proteomics analyses of NPC-containing fractions from a divergent eukaryote (Trypanosoma brucei) and obtained a comprehensive inventory of its nucleoporins. Strikingly trypanosome nucleoporins clearly share with metazoa and yeast their fold type, domain organization, composition, and modularity. Overall these data provide conclusive evidence that the majority of NPC architecture is indeed conserved throughout the Eukaryota and was already established in the last common eukaryotic ancestor. These findings strongly support the hypothesis that NPCs share a common ancestry with vesicle coating complexes and that both were established very early in eukaryotic evolution.

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Validation of candidate T. brucei Nups. A, one copy of open reading frame Tb11.03.0140 (TbNup158) was genomically tagged at the COOH terminus with GFP. A montage of 21 confocal planes from the analysis of a TbNup158-tagged trypanosome in late anaphase is shown; each z-slice is 150 nm thick. There are ∼150 puncta associated with the nuclear envelope in this example. B, fluorescent microscopy gallery of COOH-terminal genomically labeled TbNups and corresponding 4′,6-diamino-2-phenylindole dihydrochloride (DAPI) fluorescence to visualize the DNA. Apart from TbSec13, which was labeled using the 3×HA epitope and visualized with a mouse monoclonal anti-hemagglutinin antibody at 1:1000, all other open reading frames were tagged with GFP. Scale bars, 2 μm.
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Figure 2: Validation of candidate T. brucei Nups. A, one copy of open reading frame Tb11.03.0140 (TbNup158) was genomically tagged at the COOH terminus with GFP. A montage of 21 confocal planes from the analysis of a TbNup158-tagged trypanosome in late anaphase is shown; each z-slice is 150 nm thick. There are ∼150 puncta associated with the nuclear envelope in this example. B, fluorescent microscopy gallery of COOH-terminal genomically labeled TbNups and corresponding 4′,6-diamino-2-phenylindole dihydrochloride (DAPI) fluorescence to visualize the DNA. Apart from TbSec13, which was labeled using the 3×HA epitope and visualized with a mouse monoclonal anti-hemagglutinin antibody at 1:1000, all other open reading frames were tagged with GFP. Scale bars, 2 μm.

Mentions: The candidate TbNups were localized by genomic tagging and fluorescence microscopy (Table I and Figs. 2 and 3). Almost all the GFP-tagged candidate TbNups displayed a similar punctate decoration restricted to the rim of the nucleus (Fig. 2). The puncta displayed a relatively homogeneous intensity and distribution; the average density of fluorescent puncta was 5.1 puncta/μm2 (n = 10, σ = 0.8) with an average of 93 puncta (σ = 16) per nucleus (see Fig. 2A for an example). Such patterns are considered highly characteristic for Nups in all other eukaryotic taxa examined (49–53), and indeed all four of the annotated Nup homologs that we tested, Tb10.61.2630, Tb10.6k15.2350, Tb10.6k15.3670, and Tb11.03.0140, displayed this pattern. We confirmed using double labeling with a cross-reacting anti-Nup antibody that this pattern represents NPC localization (Fig. 3A) (48). In total, 20 of the 22 putative TbNups displayed such punctate rim staining, identifying them as bona fide TbNups (Fig. 2B). Multiple attempts to tag the two remaining candidate TbNups, Tb11.02.0270 and Tb927.4.5200, failed to generate positive clones. Seven additional proteins in the data set are not classified as TbNups because they localized as diffuse or speckled staining in the cytosol or nucleus (supplemental Fig. S2). Such localizations may be false negatives due to disrupted protein targeting upon carboxyl-terminal epitope tagging or alternatively may represent truly non-NPC-associated proteins.


Evidence for a shared nuclear pore complex architecture that is conserved from the last common eukaryotic ancestor.

DeGrasse JA, DuBois KN, Devos D, Siegel TN, Sali A, Field MC, Rout MP, Chait BT - Mol. Cell Proteomics (2009)

Validation of candidate T. brucei Nups. A, one copy of open reading frame Tb11.03.0140 (TbNup158) was genomically tagged at the COOH terminus with GFP. A montage of 21 confocal planes from the analysis of a TbNup158-tagged trypanosome in late anaphase is shown; each z-slice is 150 nm thick. There are ∼150 puncta associated with the nuclear envelope in this example. B, fluorescent microscopy gallery of COOH-terminal genomically labeled TbNups and corresponding 4′,6-diamino-2-phenylindole dihydrochloride (DAPI) fluorescence to visualize the DNA. Apart from TbSec13, which was labeled using the 3×HA epitope and visualized with a mouse monoclonal anti-hemagglutinin antibody at 1:1000, all other open reading frames were tagged with GFP. Scale bars, 2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2742445&req=5

Figure 2: Validation of candidate T. brucei Nups. A, one copy of open reading frame Tb11.03.0140 (TbNup158) was genomically tagged at the COOH terminus with GFP. A montage of 21 confocal planes from the analysis of a TbNup158-tagged trypanosome in late anaphase is shown; each z-slice is 150 nm thick. There are ∼150 puncta associated with the nuclear envelope in this example. B, fluorescent microscopy gallery of COOH-terminal genomically labeled TbNups and corresponding 4′,6-diamino-2-phenylindole dihydrochloride (DAPI) fluorescence to visualize the DNA. Apart from TbSec13, which was labeled using the 3×HA epitope and visualized with a mouse monoclonal anti-hemagglutinin antibody at 1:1000, all other open reading frames were tagged with GFP. Scale bars, 2 μm.
Mentions: The candidate TbNups were localized by genomic tagging and fluorescence microscopy (Table I and Figs. 2 and 3). Almost all the GFP-tagged candidate TbNups displayed a similar punctate decoration restricted to the rim of the nucleus (Fig. 2). The puncta displayed a relatively homogeneous intensity and distribution; the average density of fluorescent puncta was 5.1 puncta/μm2 (n = 10, σ = 0.8) with an average of 93 puncta (σ = 16) per nucleus (see Fig. 2A for an example). Such patterns are considered highly characteristic for Nups in all other eukaryotic taxa examined (49–53), and indeed all four of the annotated Nup homologs that we tested, Tb10.61.2630, Tb10.6k15.2350, Tb10.6k15.3670, and Tb11.03.0140, displayed this pattern. We confirmed using double labeling with a cross-reacting anti-Nup antibody that this pattern represents NPC localization (Fig. 3A) (48). In total, 20 of the 22 putative TbNups displayed such punctate rim staining, identifying them as bona fide TbNups (Fig. 2B). Multiple attempts to tag the two remaining candidate TbNups, Tb11.02.0270 and Tb927.4.5200, failed to generate positive clones. Seven additional proteins in the data set are not classified as TbNups because they localized as diffuse or speckled staining in the cytosol or nucleus (supplemental Fig. S2). Such localizations may be false negatives due to disrupted protein targeting upon carboxyl-terminal epitope tagging or alternatively may represent truly non-NPC-associated proteins.

Bottom Line: Thus, to gain a broad perspective on the origins and evolution of the NPC, we performed proteomics analyses of NPC-containing fractions from a divergent eukaryote (Trypanosoma brucei) and obtained a comprehensive inventory of its nucleoporins.Strikingly trypanosome nucleoporins clearly share with metazoa and yeast their fold type, domain organization, composition, and modularity.Overall these data provide conclusive evidence that the majority of NPC architecture is indeed conserved throughout the Eukaryota and was already established in the last common eukaryotic ancestor.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York 10065, USA.

ABSTRACT
The nuclear pore complex (NPC) is a macromolecular assembly embedded within the nuclear envelope that mediates bidirectional exchange of material between the nucleus and cytoplasm. Our recent work on the yeast NPC has revealed a simple modularity in its architecture and suggested a common evolutionary origin of the NPC and vesicle coating complexes in a progenitor protocoatomer. However, detailed compositional and structural information is currently only available for vertebrate and yeast NPCs, which are evolutionarily closely related. Hence our understanding of NPC composition in a full evolutionary context is sparse. Moreover despite the ubiquitous nature of the NPC, sequence searches in distant taxa have identified surprisingly few NPC components, suggesting that much of the NPC may not be conserved. Thus, to gain a broad perspective on the origins and evolution of the NPC, we performed proteomics analyses of NPC-containing fractions from a divergent eukaryote (Trypanosoma brucei) and obtained a comprehensive inventory of its nucleoporins. Strikingly trypanosome nucleoporins clearly share with metazoa and yeast their fold type, domain organization, composition, and modularity. Overall these data provide conclusive evidence that the majority of NPC architecture is indeed conserved throughout the Eukaryota and was already established in the last common eukaryotic ancestor. These findings strongly support the hypothesis that NPCs share a common ancestry with vesicle coating complexes and that both were established very early in eukaryotic evolution.

Show MeSH
Related in: MedlinePlus