Ribosome-bound Get4/5 facilitate the capture of tail anchored proteins by Sgt2 in yeast. Ribosome-bound Get4/5 facilitate the capture of tail anchored proteins by Sgt2 in yeast

The guided entry of tail-anchored proteins (GET) pathway assists in the proper delivery of tail-anchored (TA) proteins to the ER. Here we uncover how the yeast GET pathway components Get4/5 mediate capture of TA proteins by Sgt2, which interacts with TA sequences and hands them over to the targeting component Get3. Get4/5 binds directly and with high affinity to ribosomes, positions Sgt2 close to the ribosomal tunnel exit, and facilitates the capture of TA proteins by Sgt2. The contact sites of Get4/5 on the ribosome overlap with those of SRP, the factor mediating cotranslational ER-targeting of proteins containing internal TM domains. Exposure of nascent, internal TM domains at the tunnel exit induces high-affinity ribosome-binding of SRP, which in turn prevents ribosome-binding of Get4/5. In this way, the position of TM domains within nascent ER-targeted proteins mediates partitioning into either the GET or SRP pathway directly at the ribosomal tunnel exit. Overall design: Wild-type BY4741 yeast cells or cells expressing genomically C-terminally His-Tev-ProtA-tagged Get4 were analysed by CRAC (Bohnsack et al., 2012).

尾锚定蛋白引导进入（Guided Entry of Tail-anchored proteins, GET）通路负责协助尾锚定（tail-anchored, TA）蛋白正确转运至内质网（Endoplasmic Reticulum, ER）。本研究阐明了酵母GET通路组分Get4/5介导Sgt2捕获TA蛋白的具体机制：Sgt2可结合TA序列并将其递送至靶向组分Get3。Get4/5能够直接以高亲和力结合核糖体，将Sgt2定位至核糖体隧道出口附近，并促进Sgt2对TA蛋白的捕获。Get4/5在核糖体上的结合位点与信号识别颗粒（Signal Recognition Particle, SRP）的结合位点重合，而SRP是介导含内部跨膜结构域（transmembrane domains, TM）蛋白共翻译靶向内质网的因子。当新生肽链的内部跨膜结构域在隧道出口暴露时，会诱导SRP与核糖体形成高亲和力结合，进而阻断Get4/5与核糖体的结合。借此机制，新生内质网靶向蛋白内部跨膜结构域的位置可直接在核糖体隧道出口处决定其分配进入GET通路还是SRP通路。实验整体设计：通过CRAC技术（Bohnsack等，2012）分析野生型BY4741酵母细胞，或基因组C端带有His-Tev-ProtA标签的Get4的酵母细胞。