Ubiquitin is conjugated by membrane ubiquitin ligase to three sites, including the N terminus, in transmembrane region of mammalian 3-hydroxy-3-methylglutaryl coenzyme A reductase - Implications for sterol-regulated enzyme degradation
Ubiquitin is conjugated by membrane ubiquitin ligase to three sites, including the N terminus, in transmembrane region of mammalian 3-hydroxy-3-methylglutaryl coenzyme A reductase - Implications for sterol-regulated enzyme degradation
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DOI：
10.1074/jbc.m405935200
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发表时间：
2004-09-10
影响因子：
4.8
通讯作者：
Roitelman, J
中科院分区：
文献类型：
--
作者：
Doolman, R;Leichner, GS;Roitelman, J
内质网（ER）糖蛋白3-羟基-3-甲基戊二酰辅酶A还原酶（HMGR）是胆固醇生物合成的关键酶，其稳定性受到甾醇的负调控。HMGR通过其n端区域锚定在内质网中，该区域横跨膜8次并包含一个甾醇感应域。我们之前已经确定哺乳动物HMGR的降解是由泛素-蛋白酶体系统介导的(Ravid， T., Doolman， R., Avner， R., Harats， D., and Roitelman， J. (2000) J. Biol。化学，275,35840 - 35847)。在这里，我们在HEK-293细胞中表达了ha标记的截断版本的HMGR，它包含所有8个跨膜跨度（350个n端残基）。与内源性HMGR类似，甾醇加速了HMG(350)-3HA蛋白的降解，验证了其作为研究HMGR周转的模型。HMG(240)-3HA缺乏最后两个跨膜跨越，但仍保留完整的甾醇感应结构域，其降解不再被甾醇加速。使用HMG(350)-3HA，我们证明了HMGR的跨膜区域以甾醇调节的方式泛素化。通过位点定向的Lys - b> Arg诱变，我们确定了Lys（248）和Lys（89）是泛素附着的内部赖氨酸，其中Lys（248）是泛素化的主要受体位点。此外，数据表明N端也被泛素化。Lys - b> Arg突变体的降解速率与其泛素化水平相关。值得注意的是，赖氨酸较少的HMG(350)-3HA的降解速度比野生型蛋白快，这表明赖氨酸（89）和赖氨酸（248）以外的赖氨酸减弱了后面残基的泛素化。在分离的微粒体中，atp依赖的HMGR泛素化需要E1作为唯一的胞质蛋白，这表明er结合的E2和E3酶催化了这种修饰。HMGR的泛素化与其从内质膜中提取有关，这一过程可能由细胞质p97/VCP/Cdc48p-Ufd1-Npl4复合物辅助，因为只有泛素化的HMGR才能降低p97。
The stability of the endoplasmic reticulum ( ER) glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the key enzyme in cholesterol biosynthesis, is negatively regulated by sterols. HMGR is anchored in the ER via its N-terminal region, which spans the membrane eight times and contains a sterol-sensing domain. We have previously established that degradation of mammalian HMGR is mediated by the ubiquitin-proteasome system (Ravid, T., Doolman, R., Avner, R., Harats, D., and Roitelman, J. ( 2000) J. Biol. Chem. 275, 35840 - 35847). Here we expressed in HEK-293 cells an HA-tagged-truncated version of HMGR that encompasses all eight transmembrane spans ( 350 N-terminal residues). Similar to endogenous HMGR, degradation of this HMG(350)-3HA protein was accelerated by sterols, validating it as a model to study HMGR turnover. The degradation of HMG(240)-3HA, which lacks the last two transmembrane spans yet retains an intact sterol-sensing domain, was no longer accelerated by sterols. Using HMG(350)-3HA, we demonstrate that transmembrane region of HMGR is ubiquitinated in a sterol-regulated fashion. Through site-directed Lys --> Arg mutagenesis, we pinpoint Lys(248) and Lys(89) as the internal lysines for ubiquitin attachment, with Lys(248) serving as the major acceptor site for polyubiquitination. Moreover, the data indicate that the N terminus is also ubiquitinated. The degradation rates of the Lys --> Arg mutants correlates with their level of ubiquitination. Notably, lysine-less HMG(350)-3HA is degraded faster than wild-type protein, suggesting that lysines other than Lys(89) and Lys(248) attenuate ubiquitination at the latter residues. The ATP-dependent ubiquitination of HMGR in isolated microsomes requires E1 as the sole cytosolic protein, indicating that ER-bound E2 and E3 enzymes catalyze this modification. Polyubiquitination of HMGR is correlated with its extraction from the ER membrane, a process likely to be assisted by cytosolic p97/VCP/Cdc48p-Ufd1-Npl4 complex, as only ubiquitinated HMGR pulls down p97.