Proteomic analysis of the mammalian nuclear pore complex

Abstract

As the sole site of nucleocytoplasmic transport, the nuclear pore complex (NPC) has a vital cellular role. Nonetheless, much remains to be learned about many fundamental aspects of NPC function. To further understand the structure and function of the mammalian NPC, we have completed a proteomic analysis to identify and classify all of its protein components. We used mass spectrometry to identify all proteins present in a biochemically purified NPC fraction. Based on previous characterization, sequence homology, and subcellular localization, 29 of these proteins were classified as nucleoporins, and a further 18 were classified as NPC-associated proteins. Among the 29 nucleoporins were six previously undiscovered nucleoporins and a novel family of WD repeat nucleoporins. One of these WD repeat nucleoporins is ALADIN, the gene mutated in triple-A (or Allgrove) syndrome. Our analysis defines the proteome of the mammalian NPC for the first time and paves the way for a more detailed characterization of NPC structure and function.

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 with the inner nuclear membrane. (B) Thin-section EM of nuclear envelopes after extraction with heparin. (C) Negative staining EM of the pellet after extraction with Triton X-100/SDS. (D) Negative staining EM of the pellet after extraction with Empigen BB. (E) Silver stained SDS-PAGE analysis of supernatant (S) and pellet (P) from each step of the fractionation. Molecular weight markers are shown on the left. Histones (H) and lamins (L) are denoted on the right. (F) Immunoblot analysis of the pellet and supernatant from each step of the fractionation using mAb414 which recognizes the nucleoporins Nup358, Nup214, Nup153, and Nup62 (Davis and Blobel, 1986). Molecular weight markers are indicated on the left and nucleoporins are indicated on the right.

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 indicated on the right. Proteins identified in these bands are shown in Table I.

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 spectrometric analysis are indicated to the right of each band. Proteins identified in these bands are shown in Tables I and SI, available at http:www.jcb.org/cgi/content/full/jcb.200206106/DC1 (histones are not shown on this gel).

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 with mAb414 (red, middle). Merged images showing the extent of colocalization are shown in the right panel.