Membrane protein clients of endoplasmic reticulum (ER)-associated degradation must be retrotranslocated from the ER membrane by the AAA-ATPase p97 for proteasomal degradation. Before direct engagement with p97, client transmembrane domains (TMDs) that have partially or fully crossed the membrane must be constantly shielded to avoid non-native interactions. How client TMDs are seamlessly escorted from the membrane to p97 is unknown. Here, we identified ER-anchored TMUB1 as a TMD-specific escortase. TMUB1 interacts with the TMD of clients within the membrane and holds ∼10–14 residues of a hydrophobic sequence that is exposed out of membrane, using its transmembrane and cytosolic regions, respectively. The ubiquitin-like domain of TMUB1 recruits p97, which can pull client TMDs from bound TMUB1 into the cytosol. The disruption of TMUB1 escortase activity impairs retrotranslocation and stabilizes retrotranslocating intermediates of client proteins within the ER membrane. Thus, TMUB1 promotes TMD segregation by safeguarding the TMD movement from the membrane to p97.

内质网 (ER) 相关降解的膜蛋白客户必须通过 AAA-ATPase p97 从 ER 膜逆向转位以进行蛋白酶体降解。在与 p97 直接接合之前，必须不断屏蔽已部分或完全穿过膜的客户跨膜结构域 (TMD)，以避免非天然相互作用。客户 TMD 如何从膜无缝护送到 p97 尚不清楚。在这里，我们将 ER 锚定的 TMUB1 鉴定为 TMD 特异性护送酶。TMUB1 与膜内客户的 TMD 相互作用，并分别使用其跨膜和细胞溶质区域，保持约 10-14 个暴露在膜外的疏水序列残基。TMUB1 的泛素样结构域招募 p97，它可以将客户 TMD 从结合的 TMUB1 拉入胞质溶胶。TMUB1 escortase 活性的破坏会损害逆向转运并稳定 ER 膜内客户蛋白的逆向转运中间体。因此，TMUB1 通过保护 TMD 从膜到 p97 的运动来促进 TMD 分离。