The Type II Hsp40 Sis1 Cooperates with Hsp70 and the E3 Ligase Ubr1 to Promote Degradation of Terminally Misfolded Cytosolic Protein

Mechanisms for cooperation between the cytosolic Hsp70 system and the ubiquitin proteasome system during protein triage are not clear. Herein, we identify new mechanisms for selection of misfolded cytosolic proteins for degradation via defining functional interactions between specific cytosolic Hsp70/Hsp40 pairs and quality control ubiquitin ligases. These studies revolved around the use of S. cerevisiae to elucidate the degradation pathway of a terminally misfolded reporter protein, short-lived GFP (slGFP). The Type I Hsp40 Ydj1 acts with Hsp70 to suppress slGFP aggregation. In contrast, the Type II Hsp40 Sis1 is required for proteasomal degradation of slGFP. Sis1 and Hsp70 operate sequentially with the quality control E3 ubiquitin ligase Ubr1 to target slGFP for degradation. Compromise of Sis1 or Ubr1 function leads slGFP to accumulate in a Triton X-100-soluble state with slGFP degradation intermediates being concentrated into perinuclear and peripheral puncta. Interestingly, when Sis1 activity is low the slGFP that is concentrated into puncta can be liberated from puncta and subsequently degraded. Conversely, in the absence of Ubr1, slGFP and the puncta that contain slGFP are relatively stable. Ubr1 mediates proteasomal degradation of slGFP that is released from cytosolic protein handling centers. Pathways for proteasomal degradation of misfolded cytosolic proteins involve functional interplay between Type II Hsp40/Hsp70 chaperone pairs, PQC E3 ligases, and storage depots for misfolded proteins.

蛋白质质量分拣过程中，胞质热休克蛋白70（cytosolic Hsp70）系统与泛素-蛋白酶体（ubiquitin proteasome）系统之间的协作机制尚不明确。本研究通过解析特定胞质Hsp70/Hsp40伴侣蛋白对与质控泛素连接酶之间的功能互作，鉴定出了错误折叠胞质蛋白的降解选择新机制。本研究以酿酒酵母（S. cerevisiae）为模型，阐明了终末错误折叠报告蛋白——短命绿色荧光蛋白（short-lived GFP, slGFP）的降解通路。I型Hsp40（Type I Hsp40）Ydj1可与Hsp70协同抑制slGFP聚集。与之相反，II型Hsp40（Type II Hsp40）Sis1是slGFP蛋白酶体降解所必需的因子。Sis1与Hsp70依次与质控E3泛素连接酶Ubr1（E3 ubiquitin ligase Ubr1）协同作用，将slGFP靶向降解。当Sis1或Ubr1功能受损时，slGFP会以Triton X-100可溶状态积累，且其降解中间体富集于核周及外周斑点中。有趣的是，当Sis1活性低下时，富集于斑点中的slGFP可从斑点中释放并随后被降解。反之，在Ubr1缺失的情况下，slGFP及其所在斑点相对稳定。Ubr1可介导从胞质蛋白质处理中心释放的slGFP的蛋白酶体降解。错误折叠胞质蛋白的蛋白酶体降解通路，涉及II型Hsp40/Hsp70伴侣蛋白对、质控E3连接酶以及错误折叠蛋白储存库之间的功能协同。