Identification of new transmembrane proteins concentrated at the nuclear envelope using organellar proteomics of mesenchymal cells

Abstract

The nuclear envelope (NE) is an endoplasmic reticulum (ER) subdomain that contains characteristic components dedicated to nuclear functions. These include nuclear pore complexes (NPCs) -the channels for nucleocytoplasmic transport, and the nuclear lamina (NL) -a scaffold for NE and chromatin organization at the nuclear periphery. Since numerous human diseases associated with NE/NL proteins occur in mesenchyme-derived cells, a more comprehensive characterization of proteins concentrated at the NE in these cell types is warranted. Accordingly, we used proteomics to analyze NE and other subcellular fractions isolated from mesenchymal stem cells and from differentiated adipocytes and myocytes. We evaluated the proteomics datasets to calculate relative protein enrichment in the NE fraction, using a spectral abundance-based scoring system that accurately described most benchmark proteins. We then examined five high-scoring transmembrane proteins expressed in all three cell types that were not previously known to be enriched at the NE. Using quantitative immunofluorescence microscopy to track ectopically expressed proteins, we validated that all five of these components are substantially concentrated at the NE of multiple cell types. One (Itprip) is exposed to the outer nuclear membrane, a second (Smpd4) is enriched at the NPC, and the three others (Mfsd10, Tmx4, and Arl6ip6) are suggested to reside in the inner nuclear membrane. Considering their sequences and other features, these proteins provide new focal points for studying the functions and membrane dynamics of the NE. Our datasets should be useful for identifying additional NE-concentrated proteins, and for evaluating candidates that are identified in screening.

Proteomic mapping by rapamycin-dependent targeting of APEX2 identifies binding partners of VAPB at the inner nuclear membrane

“…In addition to these morphological studies, we also performed biochemical analyses. Obtaining pure INM-fractions is hardly possible, nevertheless, we subjected cell lysates to an established fractionation protocol (29). As shown in Fig.…”

Transcriptomic analyses suggest that mucopolysaccharidosis patients may be less susceptible to COVID‐19

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Comparative Interactome Analysis of Emerin, MAN1 and LEM2 Reveals a Unique Role for LEM2 in Nucleotide Excision Repair

“…In metazoan cells, the periphery of the nucleus is confined by the inner nuclear membrane (INM) and the underlying lamina, a filamentous meshwork of A-and B-type lamins [1]. The INM contains a diverse set of ubiquitous and tissue-specific integral membrane proteins, termed nuclear envelope transmembrane proteins (NETs) [2][3][4][5], but their functions are mostly elusive. The better characterized abundant NETs include a protein family defined by the presence of a LAP2-Emerin-MAN1 (LEM)-domain [6], which comprises seven members in mammals [7,8].…”