Enrichment of nucleoporins from rat liver nuclei
To determine the composition of the mammalian NPC, we developed a fractionation procedure to highly enrich nucleoporins from rat liver nuclei. This procedure is a modification of one previously described (Dwyer and Blobel, 1976
) and entails the sequential solubilization of nuclear substructures. Until the final step, NPCs remain associated with the lamina and can be separated from solubilized proteins by centrifugation through a sucrose cushion. We have used EM, SDS-PAGE, and immunoblotting to confirm that NPCs remain intact and nucleoporins are not lost during fractionation.
The first step is a digestion with DNase and RNase in the presence of low concentrations of divalent cations. This solubilizes most intranuclear material, although some electron-dense aggregates remain associated with the inner nuclear membrane of the pelleted NEs ( A, arrows). A subsequent extraction with heparin clearly solubilizes these chromatin remnants (, compare A with B). In agreement with the EM data, SDS-PAGE analysis reveals that histones are partially solubilized by DNase/RNase and even more dramatically solubilized by heparin ( E). After the removal of chromatin, the nuclear membranes and their associated proteins are extracted by incubation in Triton X-100 and SDS, leaving NPCs embedded in the lamina. By negative staining ( C) it can be seen that the NPCs retain their characteristic eightfold symmetry and have a central transporter, indicating that they are largely intact. The final step is an incubation with the zwitterionic detergent Empigen BB, which selectively solubilizes the NPC as monomeric nucleoporins. The intact lamina ( D) is cleared from this solution of highly enriched nucleoporins by high-speed centrifugation. When no longer connected to the NPC, the lamina filaments appear to retract, resulting in gaps in the lamina network ( D). By SDS-PAGE it can be seen that the major proteins remaining in the Empigen BB pellet are the lamins ( E).
Figure 1. Fractionation of rat liver nuclei. (A–D) EM analysis of the fractionation of rat liver nuclei. Bar, 100 nm. (A) Thin-section EM of pelleted nuclear envelopes following digestion with DNase/RNase. Arrowheads indicate electron-dense aggregates associated (more ...)
Using a panel of anti-nucleoporin antibodies (mAb414 is shown as a representative example), we found that all tested nucleoporins fractionate in the Empigen BB supernatant ( F). Importantly, these include nucleoporins from distinct domains of the NPC, including the pore membrane (POM121, gp210), the central transporter (Nup62), and peripheral cytoplasmic (Nup214, Nup358) and nucleoplasmic (Nup153, Tpr) structures. The presence of nucleoporins from these diverse NPC structures further demonstrates that our purified NPCs are intact, strongly arguing that nucleoporins are not quantitatively lost during fractionation.
Identification and classification of proteins in the Empigen BB supernatant
Owing to the complexity of the SDS-PAGE profile of the Empigen BB supernatant, particularly in the 50–80-kD range (), we further separated these proteins by C4 reverse phase chromatography prior to SDS-PAGE (). We used a combination of single-step MS and tandem mass spectrometry (MS/MS) to identify proteins and/or ESTs in bands excised from both unseparated and C4-separated Empigen BB supernatant. C4 reverse phase separation facilitated the identification of less abundant proteins, as up to 100× more material could be analyzed. Reverse phase separation also simplified identification by decreasing the number of proteins in individual SDS-PAGE bands. Parallel analysis of bands from unseparated Empigen BB supernatant ensured that we identified proteins that could have been lost during chromatography, particularly late-eluting proteins that were recovered less efficiently from the C4 column. Proteins identified in unseparated Empigen BB supernatant () are shown in . Additional proteins identified only from the C4-separated fractions can be found in Table SI (available at http:www.jcb.org/cgi/content/full/jcb.200206106/DC1
). A total of 94 proteins were identified. Based on information in the literature and public databases, we initially classified 23 as nucleoporins, 18 as NPC-associated, 42 as non-NPC proteins, and 11 as uncharacterized ().
Figure 2. Identification of bands from unseparated Empigen BB supernatant. 10 U of Empigen BB supernatant were separated by SDS-PAGE and stained with Coomassie. Molecular weight markers are shown on the left and bands excised for mass spectrometric analysis are (more ...)
Figure 3. Chromatographic separation of the Empigen BB supernatant. ~1,000 U of C4-separated Empigen BB supernatant fractions were separated by SDS-PAGE and stained with Coomassie. Molecular weight markers are shown on the left and bands excised for mass (more ...)
Identification of proteins from unseparated Empigen BB supernatant
The 23 proteins classified as nucleoporins (, column 1) were all identified in unseparated Empigen BB supernatant and are, therefore, major components of the NPC fraction. Only ~1/2 of these nucleoporins were identified in rat databases, with the remaining proteins identified by searching the more complete mouse and human databases. This approach proved feasible because of the relatively high sequence conservation between rat, mouse, and human proteins and because of the large number of MS/MS measurements performed on peptides from each sample. We identified all previously described vertebrate nucleoporins with the exception of Gle1, whose association with the NPC may be dynamic (Watkins et al., 1998
Of the 18 proteins classified as NPC-associated proteins (, column 2), most were factors involved in nucleocytoplasmic transport, such as importin-α1, importin-β1, TAP, hnRNPs, Ran, RanGAP1, RCC1, and Hsc70. Lamins A, B, and C are components of the lamina which is intimately associated with the NPC and remains associated with it until the final step of the fractionation. The function of the other NPC-associated proteins in transport is not well understood, although all have been shown to localize, at least in part, to the NPC (Cai et al., 1997
; Saitoh et al., 1997
; Fontoura et al., 1999
; Hofmann et al., 2001
; Vasu et al., 2001
; Hang and Dasso, 2002
; Zhang et al., 2002
). Proteins classified as non-NPC proteins (, column 3) are known to function and/or localize at sites other than the NPC. Most non-NPC proteins were minor components since they were identified only in the C4-separated fractions. A number of these non-NPC proteins have been reported to be nuclear matrix- or chromatin-associated () and may, therefore, be connected directly or indirectly to the NPC/lamina. Other proteins may be contaminants, such as abundant transport substrates caught during translocation, or abundant liver or ER-associated enzymes. The final subset of proteins included those whose function and/or localization was unknown (, column 4). These were further divided into two subgroups: abundant proteins that were identified in unseparated Empigen BB supernatant and non-abundant proteins that were identified only in the C4-separated fractions. p42, p37, and p30 are preliminary names for proteins identified for the first time in this study.
To determine if any nucleoporins remained insoluble after the final fractionation step, we also identified proteins in the Empigen BB pellet (Fig. S1, available at http:www.jcb.org/cgi/content/full/jcb.200206106/DC1
and Table SII). Lamins A, B, and C were the major components of the Empigen BB pellet. Some nucleoporins (Nup214, gp210, Nup153, and Nup98) were also present, though only Nup98 was a major component. The uncharacterized proteins, p30 and MP-44, were also identified in the Empigen BB supernatant and pellet and will be discussed below.
Sequence analysis of uncharacterized proteins
Sequence analysis of the uncharacterized proteins revealed that three of the abundant proteins (FLJ12549, Sec13L, and MP-44) have homology to known nucleoporins. FLJ12549 has low-sequence homology to the Schizosaccharomyces pombe
homologue of Nup85p (19% identity and 37% similarity over its central ~400 amino acids). Although it displays no significant homology to Saccharomyces cerevisiae
Nup85p, this low level of conservation is frequently seen in nucleoporins and may still indicate functional conservation. The central ~150 amino acids of MP-44 are 21% identical (40% similar) to S. cerevisiae
Nup53p. Although this homology does not extend to the COOH-terminal amphipathic α-helix of Nup53p (Marelli et al., 1998
), there are predicted amphipathic regions in MP-44 which may be functionally homologous. Like Nup53p, MP-44 has a small number of scattered FG repeats but not the large domains seen in other nucleoporins. Sec13L is related to yeast Sec13p (30% identity and 47% similarity) but is more homologous to Seh1p (34% identity, 54% similarity). This unusually high degree of sequence conservation in a nucleoporin is largely due to the presence of six WD repeat motifs. The homology between Sec13L and Seh1p also extends to regions outside these repeats, making Sec13L the most likely candidate for the human homologue of Seh1p. Sec13R is the human homologue of Sec13p in both sequence (50% identity, 66% similarity) and function (Shaywitz et al., 1995
). The remaining four abundant proteins (ALADIN, also called Adracalin; p42; p37; and p30) are not homologous to any known nucleoporin and have no characterized homologues that could suggest a function. However, we did note that ALADIN, p42, and p37 all contain WD repeats (4, 5, and 4, respectively). Of the nonabundant proteins, none have homology to nucleoporins, although two have conserved sequence motifs. CoAA has two RNA recognition motif domains and HP1-BP74 has a domain found in linker histones H1 and H5 (Le Douarin et al., 1996
; Iwasaki et al., 2001
Subcellular localization of uncharacterized proteins
The uncharacterized proteins were further studied by examining the subcellular localization of transiently expressed GFP-tagged fusion proteins. Six of the abundant novel proteins (MP-44, Sec13L, FLJ12549, p37, p42, and ALADIN) showed punctate nuclear rim localization typical of nucleoporins (, A–F, left). This was particularly apparent in views of the nuclear surface (unpublished data). In each case the GFP signal colocalized with that of anti-nucleoporin antibody mAb414 (which recognizes Nup358, Nup214, Nup153, and Nup62), further confirming the NPC localization of these proteins (, A–F, middle and right). On the basis of their subcellular localization and, in some cases, homology to known nucleoporins, we propose that the five uncharacterized proteins (MP-44, Sec13L, FLJ12549, p37, and p42) be named Nup35, Seh1, Nup75, Nup37, and Nup43 (to avoid confusion with the unrelated yeast nucleoporin Nup42p). The remaining abundant novel protein (p30) was mostly nuclear with a significant enrichment at the nuclear periphery and around the nucleolus ( G, left). However, the GFP signal at the nuclear periphery did not colocalize with mAb414 ( G, middle and right) indicating that p30 is not a nucleoporin. We speculate that this protein may be involved in linking peripheral nuclear structures, such as the nuclear lamina, with the nuclear interior. A more detailed study of the localization of p30 will provide further insight into the functions of this novel protein.
Figure 4. Subcellular localization of uncharacterized proteins. GFP-tagged fusion proteins were transiently transfected into HeLa cells and their localization visualized by confocal microscopy 48 h posttransfection (green, left). Transfected cells were also labeled (more ...)
The nonabundant uncharacterized proteins (CoAA, c1orf28, KIAA1551, and HP1-BP74) were all nuclear with no obvious enrichment at the nuclear periphery (, H–K, left). In double labeling experiments with mAb414 (, H–K, middle and right), we saw no overlap between these proteins and the NPC. Therefore, these proteins are unlikely to be nucleoporins. Because many nonabundant proteins in the Empigen BB supernatant are nuclear matrix- or chromatin-associated, CoAA, c1orf28, KIAA1551, and HP1-BP74 may be similarly localized.
Mass estimation of the NPC
We estimated the relative abundance of nucleoporins in the Empigen BB supernatant by quantitation of SDS-PAGE band intensities (see Materials and methods). From their relative abundance we estimated the copy number per NPC of each nucleoporin, based on the assumption that nucleoporins would be present at a copy number of 8 or multiples of 8, owing to the rotational symmetry of the NPC (). From the nucleoporin copy number, we estimated NPC mass. This approach has certain limitations, such as nonquantitative dye-binding, stain saturation, and incomplete recovery of nucleoporins. Although these limitations were controlled for using different staining methods and different loading conditions, the results are an approximation. Overall, the estimated relative abundance of individual nucleoporins correlated well with that determined for their yeast homologues (Table III; Rout et al., 2000
) and resulted in an NPC mass estimate of ~60 MDa. The mass of the vertebrate NPC was previously estimated by STEM analysis of manually isolated Xenopus
oocyte nuclear envelopes (Reichelt et al., 1990
). The mass of 125 MDa reported in this study is a maximum since Xenopus
oocyte nuclear envelopes were isolated using mild conditions and probably retain many transport factors and cargo. By comparison, the yeast NPC has an estimated mass of 55–72 MDa (Rout and Blobel, 1993
; Yang et al., 1998
) but a calculated mass of only 44 MDa (Rout et al., 2000
). In vertebrates, the closely apposed lamina could also contribute to experimental mass measurements, as would posttranslational modification of nucleoporins by glycosylation and/or phosphorylation. Tissue and species differences may also contribute to differences between the estimated masses of the rat liver and Xenopus
oocyte NPCs. Overall, our analysis indicates that the mammalian NPC is composed of ~30 distinct nucleoporins that are present in similar ratios to their yeast counterparts.
Relative abundance of mammalian nucleoporins