SummaryIon fluxes mediated by glial cells are required for several physiological processes such as fluid homeostasis or the maintenance of low extracellular potassium during high neuronal activity. In mice, the disruption of the Cl− channel ClC-2 causes fluid accumulation leading to myelin vacuolation. A similar vacuolation phenotype is detected in humans affected with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a leukodystrophy which is caused by mutations in MLC1 or GLIALCAM. We here identify GlialCAM as a ClC-2 binding partner. GlialCAM and ClC-2 colocalize in Bergmann glia, in astrocyte-astrocyte junctions at astrocytic endfeet around blood vessels, and in myelinated fiber tracts. GlialCAM targets ClC-2 to cell junctions, increases ClC-2 mediated currents, and changes its functional properties. Disease-causing GLIALCAM mutations abolish the targeting of the channel to cell junctions. This work describes the first auxiliary subunit of ClC-2 and suggests that ClC-2 may play a role in the pathology of MLC disease.Video Abstract

Sun protein (Sun1 and Sun2) cDNAs were previously cloned based on the homology of their C-terminal regions (SUN (Sad1 and UNC) domain) with the Caenorhabditis elegans protein UNC-84 whose mutation disrupts nuclear migration/positioning. In this study, we raised an anti-Sun2 serum and identified Sun2 in mammalian cells. In HeLa cells, Sun2 displays a nuclear rim-like pattern typical for a nuclear envelope protein. The Sun2 antibody signal co-localizes with nuclear pore and INM markers signals. The rim-like pattern was also observed with the recombinant full-length Sun2 protein fused to either EGFP or V5 epitopes. In addition, we found that a recombinant truncated form of Sun2, extending from amino acids 26 to 339, is sufficient to specify the nuclear envelope localization. Biochemical analyses show that Sun2 is an 85-kDa protein that is partially insoluble in detergent with high salt concentration and in chaotropic agents. Furthermore, Sun2 is enriched in purified HeLa cell nuclei. Electron microscopy analysis shows that Sun2 localizes in the nuclear envelope with a subpopulation present in small clusters. Additionally, we show that the SUN domain of Sun2 is localized to the periplasmic space between the inner and the outer nuclear membranes. From our data, we conclude that Sun2 is a new mammalian inner nuclear membrane protein.Because the SUN domain is conserved from fission yeast to mammals, we suggest that Sun2 belongs to a new class of nuclear envelope proteins with potential relevance to nuclear membrane function in the context of the involvement of its components in an increasing spectrum of human diseases.In eukaryotic cells, the nuclear envelope, which separates the nucleoplasm from the cytoplasm, is composed of the inner and outer nuclear membranes (INM 1 and ONM, respectively), the latter membrane being continuous with the endoplasmic reticulum. The two membranes are separated by a thin lumen and are joined at nuclear pores (1-3). Underlying the INM is a meshwork of various lamin isoforms (4) that are in close contact with the INM and its resident proteins (5). Characterized INM proteins in mammalian cells include the lamin B receptor (6), lamin-associated polypeptides 1 and 2 (Lap1 and Lap2) (7), emerin (8), and Man1 (9). These proteins possess a hydrophilic N-terminal region that protrudes into the nucleoplasm as well as one or more hydrophobic regions leading to predicted single or multispanning transmembrane domains. INM proteins are immobilized in the nuclear envelope through their interaction with lamins and/or heterochromatin (10, 11).Beside their structural role, lamina components also exert additional functions through their ability to interact with effector proteins involved in various regulatory processes. For example, the lamin B receptor directly binds to HP1 (12), a heterochromatin protein involved in transcription repression. Lap2 (13,14), emerin (15), and Man1 (16) interact with the barrier-to-autointegration factor, a 10-kDa DNA-binding protein (17). The range of regulatory functions pe...

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.T he identification of predicted gene products at the protein level bridges the gap between genome sequencing data and protein function, and is referred to as ''functional genomics'' (1, 2). In this respect, the combination of subcellular fractionation and mass spectrometrical techniques (''subcellular proteomics'') is a powerful strategy for the initial identification of previously unknown protein components and for their assignment to particular subcellular structures (3, 4).The nuclei of eukaryotic cells contain several compartments defined by their morphological appearance in electron microscopy and by the distribution of a limited number of marker proteins (5). Because the structural and functional organization of nuclei seems to be intimately linked to the epigenetic control of gene expression, the characterization of such nuclear compartments at the molecular level is of great importance (6). The nuclear envelope (NE) is one of the least characterized compartments of the nucleus. It comprises an outer and inner nuclear membrane (ONM and INM, respectively), the pore membrane, the nuclear pore complexes, and the nuclear lamina (7). These subcompartments differ with respect to their protein components, but a thorough molecular characterization has not yet been achieved. Furthermore, a two-dimensional separation of NE membrane proteins by isoelectric focusing and SDS͞PAGE fails because the separation system discriminates against integral membrane proteins (8). Therefore, the characterization of the NE at the protein level has to overcome general analytical challenges; the NE con...

Barrier-to-autointegration factor (BAF) is a conserved 10-kDa chromatin protein essential in proliferating cells. BAF dimers bind double-stranded DNA, histone H3, histone H1.1, lamin A, and transcription regulators, plus emerin and other LEM-domain nuclear proteins. Two-dimensional gel analysis showed that endogenous human and Xenopus BAF are posttranslationally modified by phosphorylation and potentially other modifications and that they are hyperphosphorylated during mitosis. The invariant Ser-4 residue on BAF is a major site of phosphorylation during both interphase and mitosis. In HeLa cells that overexpressed the phosphomimetic BAF missense mutant S4E, but not S4A, emerin mislocalized from the nuclear envelope, suggesting Ser-4-nonphosphorylated BAF normally promotes emerin localization at the nuclear envelope. Supporting this model, wild-type BAF but not mutant S4E enhanced emerin binding to lamin A in vitro. Thus, Ser-4-unphosphorylated BAF has a positive role in localizing emerin; this role may be disease relevant because loss or mislocalization of emerin causes Emery-Dreifuss muscular dystrophy. Our findings further suggest Ser-4 phosphorylation inhibits BAF binding to emerin and lamin A, and thereby weakens emerin-lamin interactions during both mitosis and interphase.