Department of Molecular Medicine, Scripps Research, La Jolla, CA, USA.
Department of Molecular Medicine, Scripps Research, La Jolla, CA, USA
Life Sci Alliance. 2023 Jul 11;6(9). doi: 10.26508/lsa.202301998. Print 2023 Sep.
The nuclear envelope (NE) is a subdomain of the ER with prominent roles in nuclear organization, which are largely mediated by its distinctive protein composition. We developed methods to reveal low-abundance transmembrane (TM) proteins concentrated at the NE relative to the peripheral ER. Using label-free proteomics that compared isolated NEs with cytoplasmic membranes, we first identified proteins with apparent NE enrichment. In subsequent authentication, ectopically expressed candidates were analyzed by immunofluorescence microscopy to quantify their targeting to the NE in cultured cells. Ten proteins from a validation set were found to associate preferentially with the NE, including oxidoreductases, enzymes for lipid biosynthesis, and regulators of cell growth and survival. We determined that one of the validated candidates, the palmitoyltransferase Zdhhc6, modifies the NE oxidoreductase Tmx4 and thereby modulates its NE levels. This provides a functional rationale for the NE concentration of Zdhhc6. Overall, our methodology has revealed a group of previously unrecognized proteins concentrated at the NE and additional candidates. Future analysis of these can potentially unveil new mechanistic pathways associated with the NE.
核膜（NE）是内质网（ER）的一个亚域，在核组织中发挥着重要作用，这主要是由其独特的蛋白质组成介导的。我们开发了一些方法来揭示在细胞质 ER 中相对富集的低丰度跨膜（TM）蛋白。我们使用无标记蛋白质组学方法比较了分离的 NE 与细胞质膜，首次鉴定了具有明显 NE 富集的蛋白质。在随后的验证中，通过免疫荧光显微镜分析异位表达的候选蛋白，以量化它们在培养细胞中靶向 NE 的情况。从验证集中鉴定出的十个蛋白优先与 NE 相关，包括氧化还原酶、脂质生物合成酶以及细胞生长和存活的调节剂。我们确定了验证的候选蛋白之一，棕榈酰转移酶 Zdhhc6，修饰了 NE 氧化还原酶 Tmx4，从而调节其 NE 水平。这为 Zdhhc6 在 NE 中的集中提供了一个功能依据。总的来说，我们的方法揭示了一组以前未被识别的在 NE 中富集的蛋白质和其他候选蛋白。对这些蛋白的进一步分析可能会揭示与 NE 相关的新的机制途径。
