Nuclear envelope proteomics: novel integral membrane proteins of the inner nuclear membrane

Abstract

The nuclear envelope (NE) is one of the least characterized structures of eukaryotic cells. The study of its functional roles is hampered by the small number of proteins known to be specifically located to it. Here, we present a comprehensive characterization of the NE proteome. We applied different fractionation procedures and isolated protein subsets derived from distinct NE compartments. We identified 148 different proteins by 16-benzyl dimethyl hexadecyl ammonium chloride (16-BAC) gel electrophoresis and matrix-assisted laser desorption ionization (MALDI) mass spectrometry; among them were 19 previously unknown or noncharacterized. The identification of known proteins in particular NE fractions enabled us to assign novel proteins to NE substructures. Thus, our subcellular proteomics approach retains the screening character of classical proteomic studies, but also allows a number of predictions about subcellular localization and interactions of previously noncharacterized proteins. We demonstrate this result by showing that two novel transmembrane proteins, a 100-kDa protein with similarity to Caenorhabditis elegans Unc-84A and an unrelated 45-kDa protein we named LUMA, reside in the inner nuclear membrane and likely interact with the nuclear lamina. The utility of our approach is not restricted to the investigation of the NE. Our approach should be applicable to the analysis of other complex membrane structures of the cell as well.

Figure 1

Preparation of NE subfractions. (A) Illustrates schematically the architecture of the nucleus of a eukaryotic cell with emphasis on the NE compartments. The frame indicates the substructures present in the NE preparation. (B) Fractionation scheme applied to the fractionation of the NE. (C) Distribution of the marker proteins calnexin (ONM/ER membrane), LAP 2β (INM), and lamin B1 (nuclear lamina) throughout the different NE subfractions. Calnexin is absent from the TX-100-resistant fraction; lamin B1 is almost absent from the chaotrope-resistant fraction.

Figure 2

Distribution of NE proteins on the different fractions. Selected proteins detected in the TX-100-resistant NE fraction (Tx) and in the chaotrope-resistant fraction (U/C) grouped according to their subcellular localization. See also Tables 5 and 6 for the identity of the proteins. INM, inner nuclear membrane; ER/ONM, endoplasmic reticulum/outer nuclear membrane; L/M, nuclear lamina and attached protein scaffold; NPC, nuclear pore complex; CS, cytoskeleton; Mito, mitochondria. Note the differences in the distribution of ER/ONM proteins, L/M proteins, and NPC proteins.

Figure 3

INM and novel NE proteins as separated by 16-BAC-SDS/PAGE. (A) Proteins of the TX-100-resistant fraction. (B) Proteins of the chaotrope-resistant fraction.

Figure 4

Identification of murine KIAA0810 and LUMA by MALDI-MS. (A) Peptide mass fingerprint of murine KIAA0810. Some of the peptides matching the predicted protein sequence are indicated. Peptides labeled by asterisks correspond to the amino acid sequence stretch present in the translated cDNA clone ID15962 of the FANTOM database, but missing in the putative protein PID 12852531. (B) PSD fragment ion spectrum of the peptide of MH⁺ = 1082.58 Da corresponding to the sequence F905RVHGEPIQ913 of murine KIAA0810. B-ions are N-terminal; Y′′-ions are C-terminal. These predominant ion types arise from backbone fragmentation within the amide bonds but still contain one terminus of the parent ion. Because of the presence of an internal arginine residue, strong signals of −17-Da satellites of B-ions (loss of ammonia) are observed and indicated as “ΔNH3.” (C) Peptide mass fingerprint of LUMA; some peptides matching the predicted amino acid sequence are indicated. (D) PSD fragment ion spectrum of the peptide of MH⁺ = 1359.68 Da corresponding to the sequence V17TSEPQPGFLER28 of LUMA. Proline-directed decay can be observed giving rise to a characteristic internal B-ion series starting with one of the prolines. Because of this decay, large B- or Y′′-ions exceeding the positions of the prolines are hardly detected.

Figure 5

Overexpressed KIAA0810 and LUMA give rise to a rim-like staining around the nucleus as visualized by indirect immunofluorescence: COS-7 cells were transfected with a V5-tagged KIAA0810 (A, B, and C) and LUMA (D, E, and F) cDNA plasmids. Immunofluorescence studies were carried out with permeabilized cells grown on glass cover slips, as described under Methods. Cells were treated with an anti-V5 monoclonal antibody and then incubated with a Cy2-linked goat anti-mouse IgG secondary antibody. Fluorescence images were obtained with a confocal laser scanning microscope. The overlay pictures (A and D) are composed of immunofluorescence (B and E) and differential interference contrast (C and F) images, which were recorded in parallel. Each picture is representative of at least three independent experiments.