Acharya Nirbhik, Daniel Emily A, Dao Thuy P, Niblo Jessica K, Mulvey Erin, Sukenik Shahar, Kraut Daniel A, Roelofs Jeroen, Castañeda Carlos A
bioRxiv. 2025 May 13:2025.03.14.643327. doi: 10.1101/2025.03.14.643327.
Ubiquitin-binding shuttle proteins are important components of stress-induced biomolecular condensates in cells. Yeast Dsk2 scaffolds proteasome-containing condensates via multivalent interactions with proteasomes and ubiquitinated substrates under azide-induced mitochondrial stress or extended growth conditions. However, the molecular mechanisms underlying how these shuttle proteins work are unknown. Here, we identify that the middle chaperone-binding STI1 domain is the main driver of Dsk2 self-association and phase separation . Using NMR spectroscopy and computational simulations, we find that the STI1 domain interacts with three transient amphipathic helices within the intrinsically-disordered regions of Dsk2. Removal of either the STI1 domain or these helices significantly reduces the propensity for Dsk2 to phase separate. , removal of the STI1 domain in Dsk2 has the opposite effect, resulting in an increase of proteasome-containing condensates due to an accumulation of polyubiquitinated substrates. Modeling of STI1-helix interactions reveals a binding mode that is reminiscent of interactions between chaperone STI1/DP2 domains and client proteins containing amphipathic or transmembrane helices. Our findings support a model whereby STI1-helix interactions important for Dsk2 condensate formation can be replaced by STI1-client interactions for downstream chaperone or other protein quality control outcomes.
The intrinsically disordered regions of Dsk2 harbor transient helices that regulate protein properties via interactions with the STI1 domain. The STI1 domain is a significant driver of Dsk2 self-association and phase separation . Dsk2 colocalizes with ubiquitinated substrates and proteasome in reconstituted condensates.Absence of Dsk2 STI1 domain in stressed yeast cells promotes formation of proteasome condensates coupled with upregulation of polyubiquitinated substrates.
泛素结合穿梭蛋白是细胞应激诱导的生物分子凝聚物的重要组成部分。在叠氮化物诱导的线粒体应激或延长生长条件下，酵母Dsk2通过与蛋白酶体和泛素化底物的多价相互作用来构建含蛋白酶体的凝聚物。然而，这些穿梭蛋白发挥作用的分子机制尚不清楚。在这里，我们确定中间的分子伴侣结合STI1结构域是Dsk2自缔合和相分离的主要驱动因素。使用核磁共振光谱和计算模拟，我们发现STI1结构域与Dsk2内在无序区域内的三个瞬时两亲螺旋相互作用。去除STI1结构域或这些螺旋会显著降低Dsk2相分离的倾向。相反，去除Dsk2中的STI1结构域会产生相反的效果，由于多聚泛素化底物的积累，导致含蛋白酶体的凝聚物增加。STI1-螺旋相互作用的模型揭示了一种结合模式，让人联想到分子伴侣STI1/DP2结构域与含有两亲或跨膜螺旋的客户蛋白之间的相互作用。我们的研究结果支持了一种模型，即对Dsk2凝聚物形成重要的STI1-螺旋相互作用可以被用于下游分子伴侣或其他蛋白质质量控制结果的STI1-客户相互作用所取代。
Dsk2的内在无序区域含有瞬时螺旋，通过与STI1结构域的相互作用调节蛋白质特性。STI1结构域是Dsk2自缔合和相分离的重要驱动因素。在重组凝聚物中，Dsk2与泛素化底物和蛋白酶体共定位。应激酵母细胞中缺乏Dsk2 STI1结构域会促进蛋白酶体凝聚物的形成，并伴随着多聚泛素化底物的上调。
