The generation of detergent-insoluble clipped fragments from an ERAD substrate in mammalian cells.

Proteostasis ensures the proper synthesis, folding, and trafficking of proteins and is required for cellular and organellar homeostasis. This network also oversees protein quality control within the cell and prevents accumulation of aberrant proteins, which can lead to cellular dysfunction and disease. For example, protein aggregates irreversibly disrupt proteostasis and can exert gain-of-function toxic effects. Although this process has been examined in detail for cytosolic proteins, how endoplasmic reticulum (ER)-tethered, aggregation-prone proteins are handled is ill-defined. To determine how a membrane protein with a cytoplasmic aggregation-prone domain is routed for ER-associated degradation (ERAD), we analyzed a new model substrate, TM-Ubc9ts. In yeast, we previously showed that TM-Ubc9ts ERAD requires Hsp104, which is absent in higher cells. In transient and stable HEK293 cells, we now report that TM-Ubc9ts degradation is largely proteasome-dependent, especially at elevated temperatures. In contrast to yeast, clipped TM-Ubc9ts polypeptides, which are stabilized upon proteasome inhibition, accumulate and are insoluble at elevated temperatures. TM-Ubc9ts cleavage is independent of the intramembrane protease RHBDL4, which clips other classes of ERAD substrates. These studies highlight an unappreciated mechanism underlying the degradation of aggregation-prone substrates in the ER and invite further work on other proteases that contribute to ERAD.

蛋白质稳态确保蛋白质的正确合成、折叠和运输，是细胞和细胞器内稳态所必需的。这个网络还监管细胞内的蛋白质质量控制，防止异常蛋白质的积累，因为异常蛋白质积累会导致细胞功能障碍和疾病。例如，蛋白质聚集体不可逆地破坏蛋白质稳态，并可能产生获得性功能毒性作用。尽管对胞质蛋白质的这一过程已经进行了详细研究，但内质网（ER）连接的、易聚集蛋白质是如何被处理的还不清楚。为了确定具有细胞质易聚集结构域的膜蛋白是如何被引导进行内质网相关降解（ERAD）的，我们分析了一种新的模型底物，TM - Ubc9ts。在酵母中，我们之前表明TM - Ubc9ts的ERAD需要Hsp104，而高等细胞中没有Hsp104。在瞬时和稳定的HEK293细胞中，我们现在报告TM - Ubc9ts的降解在很大程度上依赖蛋白酶体，特别是在高温下。与酵母不同，在蛋白酶体抑制时稳定的切割后的TM - Ubc9ts多肽在高温下积累且不溶。TM - Ubc9ts的切割不依赖于膜内蛋白酶RHBDL4，RHBDL4切割其他类别的ERAD底物。这些研究强调了内质网中易聚集底物降解的一种未被充分认识的机制，并促使进一步研究其他有助于ERAD的蛋白酶。