Structural Basis of Tail-Anchored Membrane Protein Biogenesis by the GET Insertase Complex
Structural Basis of Tail-Anchored Membrane Protein Biogenesis by the GET Insertase Complex
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DOI：
10.1016/j.molcel.2020.08.012
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发表时间：
2020-10-01
期刊：
影响因子：
16
通讯作者：
Sinning, Irmgard
中科院分区：
文献类型：
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作者：
McDowell, Melanie A.;Heimes, Michael;Sinning, Irmgard
膜蛋白生物发生面临着伴随疏水的跨膜螺旋以实现忠实的膜插入的挑战。尾部锚定蛋白(GET)的引导进入途径以尾部锚定(TA)蛋白为靶点，并通过插入酶(酵母Get1/get2或哺乳动物WRB/CAML)将尾部锚定(TA)蛋白插入内质网(ER)膜，该插入酶从细胞质伴侣蛋白(分别为Get3或TRC40)中捕获TA。在这里，我们介绍了人WRB/CAML/TRC40和酵母Get1/Get2/Get3复合体的冷冻电子显微镜重建、自然质谱学和基于结构的突变。Get3与膜插入酶的结合支持异四聚体的形成，而磷脂酰肌醇与异四聚体界面的结合稳定了插入酶在体内有效地插入TA。我们发现了一个与Get3/TRC40形成“门控”相互作用的Get2/CAML细胞质螺旋，Get3/TRC40对TA插入很重要。与YidC和内质网膜蛋白复合体(EMC)的结构同源性表明，利用亲水性沟槽为发散底物提供了一种进化上保守的插入机制。因此，我们提供了一个详细的结构和机制框架来理解TA膜插入。
Membrane protein biogenesis faces the challenge of chaperoning hydrophobic transmembrane helices for faithful membrane insertion. The guided entry of tail-anchored proteins (GET) pathway targets and inserts tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane with an insertase (yeast Get1/Get2 or mammalian WRB/CAML) that captures the TA from a cytoplasmic chaperone (Get3 or TRC40, respectively). Here, we present cryo-electron microscopy reconstructions, native mass spectrometry, and structure-based mutagenesis of human WRB/CAML/TRC40 and yeast Get1/Get2/Get3 complexes. Get3 binding to the membrane insertase supports heterotetramer formation, and phosphatidylinositol binding at the heterotetramer interface stabilizes the insertase for efficient TA insertion in vivo. We identify a Get2/CAML cytoplasmic helix that forms a "gating" interaction with Get3/TRC40 important for TA insertion. Structural homology with YidC and the ER membrane protein complex (EMC) implicates an evolutionarily conserved insertion mechanism for divergent substrates utilizing a hydrophilic groove. Thus, we provide a detailed structural and mechanistic framework to understand TA membrane insertion.