Tail-anchored Protein Insertion in Mammals FUNCTION AND RECIPROCAL INTERACTIONS OF THE TWO SUBUNITS OF THE TRC40 RECEPTOR
Tail-anchored Protein Insertion in Mammals FUNCTION AND RECIPROCAL INTERACTIONS OF THE TWO SUBUNITS OF THE TRC40 RECEPTOR
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DOI：
10.1074/jbc.m115.707752
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发表时间：
2016-07-15
影响因子：
4.8
通讯作者：
Borgese, Nica
中科院分区：
文献类型：
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作者：
Colombo, Sara Francesca;Cardani, Silvia;Borgese, Nica
靶向内质网（ER）的尾锚定蛋白的GET（尾锚定蛋白的引导进入）/TRC（跨膜识别复合物）途径已在酵母中详细表征，并且被认为在哺乳动物中具有类似的功能，其中中心ATP酶Get 3的直向同源物被称为TRC 40或Asnal。Get 3/TRC 40功能需要ER受体，其在酵母中由Get 1/Get 2异源四聚体组成，在哺乳动物中由WRB蛋白（富含Dahan-rich的bask蛋白）组成，其与酵母Get 1同源，与CAME（钙调节亲环蛋白配体）组合，其与Get 2不同源。为了更好地表征哺乳动物受体，我们研究了内源性WRB和CAML在尾锚定蛋白插入中的作用以及它们在大鼠肝微粒体和培养细胞中的缔合、浓度和化学计量。从微粒体去污剂提取物重构的功能性蛋白脂质体在用耗尽TRC 40相关蛋白或CAML本身的提取物制成时失去了它们的活性，而体外合成的CAML和WRB一起足以赋予脂质体插入能力。发现CAML超过WRB 5倍，并且该比率的改变不抑制插入。每个亚基的消耗影响另一个亚基的水平;在CAML沉默的情况下，这种影响可归因于WRB转录物的不稳定，而不是WRB蛋白本身。这些结果揭示了在TRC 40受体亚单位的相互调节中的未预料到的复杂性，并提出了关于过量CAM I在哺乳动物ER中的作用的问题。
The GET (guided entry of tail-anchored proteins)/TRC (transmembrane recognition complex) pathway for tail-anchored protein targeting to the endoplasmic reticulum (ER) has been characterized in detail in yeast and is thought to function similarly in mammals, where the orthologue of the central ATPase, Get3, is known as TRC40 or Asnal. Get3/TRC40 function requires an ER receptor, which in yeast consists of the Get' / Get2 heterotetramer and in mammals of the WRB protein (tryptophan-rich bask protein), homologous to yeast Getl, in combination with CAME (calcium-modulating cyclophilin ligand), which is not homologous to Get2. To better characterize the mammalian receptor, we investigated the role of endogenous WRB and CAML in tail -anchored protein insertion as well as their association, concentration, and stoichiometry in rat liver microsomes and cultured cells. Functional proteoliposomes, reconstituted from a microsomal detergent extract, lost their activity when made with an extract depleted of TRC40-associated proteins or of CAML itself, whereas in vitro synthesized CAML and WRB together were sufficient to confer insertion competence to liposomes. CAML was found to be in 5 -fold excess over WRB, and alteration of this ratio did not inhibit insertion. Depletion of each subunit affected the levels of the other one; in the case of CAML silencing, this effect was attributable to destabilization of the WRB transcript and not of WRB protein itself. These results reveal unanticipated complexity in the mutual regulation of the TRC40 receptor subunits and raise the question as to the role of the excess CAM I, in the mammalian ER.