Multifaceted disruption to cholesterol homeostasis upon loss of the mEMC

The mammalian Endoplasmic Reticulum Membrane protein Complex (mEMC) is an evolutionarily conserved hetero-oligomer whose function and mechanism of action remain elusive. Here we identify a multi-layered contribution of the mEMC to post-translational regulation of cholesterol homeostasis. Mammalian cells employ elaborate sensing and feedback mechanisms to balance free and stored cholesterol through biosynthesis, uptake, efflux and storage. We show that mEMC biogenesis is a prerequisite for optimal maturation of at least two key ER-resident sterol regulatory enzymes: squalene synthase (SQS) and sterol-O-acyltransferase (SOAT1). Without sufficient SQS, mEMC-deficient cells have attenuated cholesterol biosynthetic capability, and loss of SOAT1 significantly reduces the amount of stored cholesteryl esters (CEs). mEMC-deficient cells remain viable, maintaining free cholesterol levels with minimal transcriptional activation of cholesterogenic and uptake pathways by the sterol regulatory element binding protein (SREBP)-2. Consequently, cells lacking the mEMC were susceptible to changes in extracellular cholesterol availability and effectively were transformed into cholesterol auxotrophs. Supplementing mEMC-deficient cells with cholesterol or lanosterol failed to restore SQS or SOAT1. These data are consistent with a direct role for the mEMC in the biogenesis of SQS and SOAT1. The range of phenotypes observed previously with mEMC subunit depletion may result from its impact on fundamental cholesterol homeostatic mechanisms.哺乳动物内质网膜蛋白复合物（mammalian Endoplasmic Reticulum Membrane protein Complex, mEMC）是一类进化保守的异源寡聚体，其功能与作用机制至今仍不明晰。本研究揭示了mEMC在胆固醇稳态（cholesterol homeostasis）的翻译后调控中发挥的多层级作用。哺乳动物细胞依托精密的感知与反馈机制，通过胆固醇的生物合成、摄取、外排与储存过程，平衡游离胆固醇与储存胆固醇的水平。研究证实，mEMC的生物发生是至少两种关键内质网驻留固醇调节酶——鲨烯合酶（squalene synthase, SQS）与固醇O-酰基转移酶1（sterol-O-acyltransferase 1, SOAT1）——实现最优成熟的必要前提。当mEMC缺陷细胞缺乏足够的鲨烯合酶时，其胆固醇生物合成能力会被削弱；而固醇O-酰基转移酶1的缺失则会显著降低细胞内储存的胆固醇酯（cholesteryl esters, CEs）含量。mEMC缺陷细胞仍可维持存活，通过固醇调节元件结合蛋白2（sterol regulatory element binding protein 2, SREBP-2）介导的胆固醇合成与摄取通路的极低水平转录激活，维持游离胆固醇水平。因此，缺失mEMC的细胞对胞外胆固醇的可用性变化极为敏感，实质上转变为胆固醇营养缺陷型细胞。向mEMC缺陷细胞补充胆固醇或羊毛甾醇（lanosterol），无法恢复其鲨烯合酶与固醇O-酰基转移酶1的功能。上述数据表明，mEMC可直接参与鲨烯合酶与固醇O-酰基转移酶1的生物发生过程。此前在mEMC亚基敲低实验中观察到的多种表型，可能源于其对核心胆固醇稳态机制的调控作用。