Lipid-anchored Proteasomes Control Membrane Protein Homeostasis

As the major protein degradation machinery of eukaryotic cells, the 26S proteasome is generally thought to localize in the nucleus and cytosol. A portion of proteasomes are known to associate with various membrane structures of the cell, the mechanism and biological meaning of which have been elusive. Here we show that N-myristoylation of the proteasome subunit Rpt2 is an evolutionarily conserved determinant of proteasome-membrane interaction. Loss of this modification leads to embryonic lethality in mice, significant reduction of migration ability in MEFs and profound changes in the membrane-associated proteome as determined by SILAC-MS, suggesting a key role of membrane-tethered proteasomes in carrying out compartmentalized protein degradation. And the tumorigenicity is reduced in the oncogene-transformed MEF without modification. Serendipitously, we found that the Rpt2-G2A mutation cell lines confers partial resistance to proteasome inhibitors, such as Bortezomib and MG132. Thus, N-myristoylation of Rpt2 determines the localization and activity of the proteasome at the membrane, which is critical for embryogenesis, cellular homeostasis and tumorigenesis. Overall design: Total RNA was isolated from immortalized MEFs or 3 pairs (WT and G2A littermates) of E12.5 fetal livers using the Trizol method. Next-generation sequencing was done by BGI (Shenzhen, China).

作为真核细胞的核心蛋白质降解机器，26S蛋白酶体（26S proteasome）通常被认为定位于细胞核与细胞质基质中。已有研究证实，部分蛋白酶体可与细胞的多种膜结构相结合，但其结合的分子机制与生物学意义长期以来尚未明确。
本研究发现，蛋白酶体亚基Rpt2的N-肉豆蔻酰化（N-myristoylation）是调控蛋白酶体与膜结构相互作用的进化保守决定因素。该修饰的缺失会导致小鼠胚胎致死，显著降低小鼠胚胎成纤维细胞（MEFs）的迁移能力，且通过稳定同位素标记氨基酸细胞培养质谱（SILAC-MS）分析发现，膜结合蛋白质组发生显著改变，这提示膜锚定蛋白酶体在执行区域化蛋白质降解过程中发挥关键作用。在缺失该修饰的癌基因转化型MEFs中，细胞的致瘤性同样有所降低。意外的是，本研究还发现Rpt2-G2A突变细胞系可使细胞对硼替佐米（Bortezomib）、MG132等蛋白酶体抑制剂产生部分耐药性。综上，Rpt2的N-肉豆蔻酰化决定了蛋白酶体在膜上的定位与活性，这对于胚胎发生、细胞稳态以及肿瘤发生均具有关键意义。
实验设计：采用Trizol法从永生化小鼠胚胎成纤维细胞，以及3对E12.5胎肝组织（野生型与G2A突变型同窝仔鼠配对样本）中提取总RNA。随后由中国深圳华大基因（BGI）完成下一代测序。