Cotransins target the Sec61 translocon and inhibit the biogenesis of an undefined subset of secretory and membrane proteins. Remarkably, cotransin inhibition depends on the unique signal peptide (SP) of each Sec61 client, which is required for cotranslational translocation into the endoplasmic reticulum. It remains unknown how an SP’s amino acid sequence and biophysical properties confer sensitivity to structurally distinct cotransins. Here we describe a fluorescence-based, pooled-cell screening platform to interrogate nearly all human SPs in parallel. We profiled two cotransins with distinct effects on cancer cells and discovered a small subset of SPs, including the oncoprotein human epidermal growth factor receptor 3 (HER3), with increased sensitivity to the more selective cotransin, KZR-9873. By comparing divergent mouse and human orthologs, we unveiled a position-dependent effect of arginine on SP sensitivity. Our multiplexed profiling platform reveals how cotransins can exploit subtle sequence differences to achieve SP discrimination.

Cotransins 以 Sec61 易位子为目标，抑制分泌蛋白和膜蛋白的未定义子集的生物发生。值得注意的是，共转蛋白抑制取决于每个 Sec61 客户的独特信号肽 (SP)，这是共翻译易位到内质网所必需的。目前尚不清楚 SP 的氨基酸序列和生物物理特性如何赋予其对结构不同的辅转蛋白的敏感性。在这里，我们描述了一个基于荧光的混合细胞筛选平台，可以并行询问几乎所有人类 SP。我们分析了两种对癌细胞具有不同作用的协同转运蛋白，并发现了一小部分 SP，包括癌蛋白人表皮生长因子受体 3 (HER3)，对更具选择性的协同转运蛋白 KZR-9873 具有更高的敏感性。通过比较不同的小鼠和人类直系同源物，我们揭示了精氨酸对 SP 敏感性的位置依赖性影响。我们的多重分析平台揭示了协同转运蛋白如何利用细微的序列差异来实现 SP 区分。