Ronzier Elsa, Satpute-Krishnan Prasanna
Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.
PLoS Biol. 2025 Apr 9;23(4):e3003084. doi: 10.1371/journal.pbio.3003084. eCollection 2025 Apr.
The p24-family member, TMED9, has recently emerged as a player in secretory pathway protein quality control (PQC) that influences the trafficking and degradation of misfolded proteins. Here, we show that TMED9 plays a central role in the PQC of GPI-anchored proteins (GPI-APs). Typically, upon release from the endoplasmic reticulum (ER)-resident chaperone calnexin, misfolded GPI-APs traffic to the Golgi by an ER-export pathway called Rapid ER stress-induced Export (RESET). From the Golgi, they access the plasma membrane where they are rapidly internalized for lysosomal degradation. We used biochemical and imaging approaches in cultured cells to demonstrate that at steady-state, the majority of misfolded GPI-APs reside in the ER in association with calnexin and TMED9. During RESET, they dissociate from calnexin and increase their association with TMED9. Inhibition of TMED9's function through siRNA-induced depletion or chemical inhibitor, BRD4780, blocked ER-export of misfolded GPI-APs. In contrast, TMED9-inhibition did not prevent ER-export of wild-type GPI-APs, indicating a specific role for TMED9 in GPI-AP PQC. Intriguingly, we discovered that acute treatment with BRD4780 induced a shift in TMED9 localization away from the ER to the downstream Golgi cisternae and blocked the RESET pathway. Upon removal of BRD4780 following acute treatment, TMED9 regained access to the ER where TMED9 was able to associate with the RESET substrate and restore the RESET pathway. These results suggest that TMED9 plays a requisite role in RESET by capturing misfolded GPI-APs that are released by calnexin within the ER and conveying them to the Golgi.
p24家族成员TMED9最近已成为分泌途径蛋白质质量控制（PQC）中的一个参与者，它会影响错误折叠蛋白质的运输和降解。在此，我们表明TMED9在糖基磷脂酰肌醇锚定蛋白（GPI-APs）的PQC中起核心作用。通常，错误折叠的GPI-APs从内质网（ER）驻留伴侣钙联蛋白释放后，通过一种称为快速内质网应激诱导输出（RESET）的ER输出途径运输到高尔基体。从高尔基体，它们进入质膜，在那里它们迅速被内化以便溶酶体降解。我们在培养细胞中使用生化和成像方法来证明，在稳态下，大多数错误折叠的GPI-APs与钙联蛋白和TMED9结合存在于内质网中。在RESET过程中，它们与钙联蛋白解离并增加与TMED9的结合。通过siRNA诱导的消耗或化学抑制剂BRD4780抑制TMED9的功能，会阻断错误折叠的GPI-APs的内质网输出。相比之下，抑制TMED9并不会阻止野生型GPI-APs的内质网输出，这表明TMED9在GPI-AP PQC中具有特定作用。有趣的是，我们发现用BRD4780进行急性处理会导致TMED9的定位从内质网转移到下游高尔基体池，并阻断RESET途径。急性处理后去除BRD4780，TMED9重新进入内质网，在那里TMED9能够与RESET底物结合并恢复RESET途径。这些结果表明，TMED9通过捕获在内质网中由钙联蛋白释放的错误折叠的GPI-APs并将它们运输到高尔基体，在RESET中发挥必要作用。
