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.尾锚定蛋白引导导入（Guided Entry of Tail-anchored proteins, GET）通路负责将尾锚定（TA）蛋白精准递送至内质网（ER）。本研究阐明了酵母GET通路组分Get4/5如何介导Sgt2捕获TA蛋白：Sgt2可与TA序列结合，并将其递送至靶向组分Get3。Get4/5可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）的结合位点存在重叠；SRP是介导含内在跨膜结构域（Transmembrane domain, TM）蛋白共翻译靶向内质网的因子。新生肽链上的内在跨膜结构域在隧道出口处暴露后，可诱导SRP以高亲和力结合核糖体，进而阻断Get4/5与核糖体的结合。由此，靶向内质网的新生蛋白中跨膜结构域的位置，可直接在核糖体隧道出口处决定蛋白进入GET通路还是SRP通路。