Nuclear Envelope Transmembrane Research Articles

High-Throughput Identification of Nuclear Envelope Protein Interactions in Schizosaccharomyces pombe Using an Arrayed Membrane Yeast-Two Hybrid Library.

The nuclear envelope (NE) contains a specialized set of integral membrane proteins that maintain nuclear shape and integrity and influence chromatin organization and gene expression. Advances in proteomics techniques and studies in model organisms have identified hundreds of proteins that localize to the NE. However, the function of many of these proteins at the NE remains unclear, in part due to a lack of understanding of the interactions that these proteins participate in at the NE membrane. To assist in the characterization of NE transmembrane protein interactions we developed an arrayed library of integral and peripheral membrane proteins from the fission yeast Schizosaccharomyces pombe for high-throughput screening using the split-ubiquitin based membrane yeast two -hybrid system. We used this approach to characterize protein interactions for three conserved proteins that localize to the inner nuclear membrane: Cut11/Ndc1, Lem2 and Ima1/Samp1/Net5. Additionally, we determined how the interaction network for Cut11 is altered in canonical temperature-sensitive cut11-ts mutants. This library and screening approach is readily applicable to characterizing the interactomes of integral membrane proteins localizing to various subcellular compartments.

Global profiling of plant nuclear membrane proteome in Arabidopsis.

The nuclear envelope (NE) is structurally and functionally vital for eukaryotic cells, yet its protein constituents and their functions are poorly understood in plants. Here, we combined subtractive proteomics and proximity-labelling technology coupled with quantitative mass spectrometry to understand the landscape of NE membrane proteins in Arabidopsis. We identified ~200 potential candidates for plant NE transmembrane (PNET) proteins, which unravelled the compositional diversity and uniqueness of the plant NE. One of the candidates, named PNET1, is a homologue of human TMEM209, a critical driver for lung cancer. A functional investigation revealed that PNET1 is a bona fide nucleoporin in plants. It displays both physical and genetic interactions with the nuclear pore complex (NPC) and is essential for embryo development and reproduction in different NPC contexts. Our study substantially enlarges the plant NE proteome and sheds new light on the membrane composition and function of the NPC.

P.34Nuclear envelope abnormalities in myotonic dystrophy primary myoblasts

Myotonic dystrophies (DM) are slowly progressing multisystemic diseases with a predominant muscular dystrophy - making DM the most frequent muscular dystrophy in adulthood. DM is caused by heterozygous DNA-repeat expansions in the DMPK gene (DM1) or the CNBP gene (DM2). The repeat-containing RNA accumulates in ribonuclear foci and splicing factors are sequestered to these foci, resulting in abnormal regulation of alternative splicing. In primary patient myoblast cell cultures we observed invaginations of the nuclear envelope (NE). These abnormalities correlate in DM1 with repeat length - which in turn correlates with disease severity. Searching for the reason of theses abnormalities we screened proteins of the nuclear lamina and NE transmembrane proteins (NETs). This revealed down-regulation of the lamins A and B1 as well as different isoforms of the NET nesprin 1. Lamins and nesprins are having functions in the mechanical stability of the nucleus; therefore their mis-regulation is likely directly involved in the observed nuclear abnormalities. This implies possible shared pathomechanism between DM1 and nuclear envelope linked disease.

The lamin code

Unicellular eukaryotes and metazoa present a nuclear envelope (NE) and metazoa express in it one or more lamins that give rise to the nuclear lamina. The expression of different types of lamins is related to the complexity of the organism and the expression of type-A lamins is related to the initial steps of tissue-specific cell differentiation. Several posttranslational modifications characterize the expression of lamin A in the course of cell differentiation, and the alteration of this expression pattern leads to impressive phenotypic diseases that are collectively referred to as laminopathies. This indicates a link between differential lamin A expression and tissue-specific cell commitment, and makes it conceivable that the lamin posttranslational modifications constitute a lamin code, utilized by metazoan cells to induce tissue-specific cell differentiation. Although the rules of this code are not yet deciphered, at the moment, the presence of adaptors, represented by NE transmembrane proteins (NETs), and of effectors, constituted by epigenetic repressors that modulate chromatin arrangement and gene expression, strongly supports the possibility that the rules of lamin modification represent one of the organic codes that characterize cell evolution.

Simple Separation of Functionally Distinct Populations of Lamin-Binding Proteins.

The inner membrane of the nuclear envelope (NE) is home to hundreds of integral membrane proteins (NE transmembrane proteins, "NETs") with conserved or tissue-specific roles in genome organization and nuclear function. Nearly all characterized NETs bind A- or B-type lamins directly. However, hundreds of NETs remain uncharacterized, collectively posing an enormous gap that must be bridged to understand nuclear function and genome biology. We provide technically simple protocols for the separation and recovery of functionally distinct populations of NETs and A-type lamins. This protocol was developed for emerin, an inner nuclear membrane protein that binds lamins and barrier-to-autointegration factor (BANF1) as a component of nuclear lamina structure, and has diverse roles in nuclear assembly, signaling, and gene regulation. This protocol separates easily solubilized ("easy") populations of nuclear lamina proteins (emerin, lamin A, BAF) from "sonication-dependent" populations. Depending on cell type, the "easy" and "sonication-dependent" fractions each contain up to about half the available emerin, A-type lamins, and BAF, whereas B-type lamins and histone H3 are predominantly sonication dependent. The two populations of emerin have distinct posttranslational modifications, and only one population associates with BAF. This method may be useful for functional screening or analysis of other lamin-associated proteins, including novel NETs emerging from proteomic studies.

NET37, a Nuclear Envelope Transmembrane Protein with Glycosidase Homology, Is Involved in Myoblast Differentiation

The nuclear lamina and its associated proteins are important for nuclear structure and chromatin organization and also have been implicated in the regulation of cell signaling and gene expression. In this study we demonstrate that the lamina-associated nuclear envelope transmembrane protein NET37 is required for myogenic differentiation of C2C12 cells. NET37, a member of glycosidase family 31, is highly expressed in mouse skeletal muscle and is strongly up-regulated during C2C12 differentiation. By protease mapping we show that its glycosidase homology domain is located in the lumen of the nuclear envelope/endoplasmic reticulum. When NET37 is depleted from proliferating myoblasts by RNAi, myogenic differentiation is significantly impaired, and there is a concomitant delay in up-regulation of the late myogenic transcription factor myogenin. We expressed silencing-resistant NET37 mutated at a conserved residue in the glycosidase domain and found that this predicted catalytically inactive protein is unable to support myogenesis in cells depleted of wild type NET37. Therefore, the enzymatic function of NET37 appears to be important for myogenic differentiation. C2C12 cells depleted of NET37 have reduced activation of Akt after shifting to differentiation medium and are defective in insulin like growth factor-II (IGF-II) secretion, an autocrine/paracrine factor involved in Akt activation. We also observed that pro-IGF-II co-immunoprecipitates with NET37. Based on our results, we propose that NET37 has a role in IGF-II maturation in the secretory pathway during myoblast differentiation. The localization of NET37 at the nuclear envelope raises the possibility that it may coordinate myogenic events between the nuclear interior and the endoplasmic reticulum lumen via transmembrane communication.