Ksp1是酪蛋白II样激酶,其活性可防止酵母中营养丰富的条件下异常的巨自噬/自噬诱导。这里,我们描述了Ksp1作为一种新型自噬受体蛋白在Sn2/Med13中的非激酶依赖性作用,Sn2/Med13是一种已知的Snx4辅助转录因子自噬的货物.在这条道路上,保守转录调节因子的子集,Ssn2/Med13,Rim15和Msn2选择性地针对氮饥饿后的液泡蛋白水解,辅助分选nexin异二聚体Snx4-Atg20。在这里,我们显示吞噬细胞也吞没Ksp1及其货物进行液泡蛋白水解。在氮饥饿后,Ksp1在Ksp1中的Atg8家族相互作用基序(AIM)/LC3相互作用区(LIR)和Atg8中的LIR/AIM对接位点(LDS)的界面处与Atg8直接相关。LDS位点的突变可防止Ksp1的自噬降解。然而,C端经典AIM的缺失仍然允许Ssn2/Med13蛋白水解,这表明其他非规范AIM可能介导Ksp1-Atg8相互作用。Ksp1被三聚体支架复合物的成员Atg29募集到液泡周围吞噬团组装位点。这种相互作用与Atg8和Snx4无关,这表明Ksp1被早期招募到吞噬细胞中,Snx4随后交付ssn2/Med13。最后,长期氮饥饿后的正常细胞存活需要Ksp1。一起,这些研究定义了Ksp1作为介导Sn2/Med13降解的自噬受体蛋白的激酶非依赖性作用.他们还表明,由三聚体支架复合物构建的吞噬团能够进行受体介导的自噬。这些结果表明Ksp1的双重功能,其激酶活性防止自噬,同时发挥支持自噬降解的支架作用。
Ksp1 is a casein II-like kinase whose activity prevents aberrant macroautophagy/autophagy induction in nutrient-rich conditions in yeast. Here, we describe a kinase-independent role of Ksp1 as a novel autophagic receptor protein for Ssn2/Med13, a known cargo of Snx4-assisted autophagy of transcription factors. In this pathway, a subset of conserved transcriptional regulators, Ssn2/Med13, Rim15, and Msn2, are selectively targeted for vacuolar proteolysis following nitrogen starvation, assisted by the sorting nexin heterodimer Snx4-Atg20. Here we show that phagophores also engulf Ksp1 alongside its cargo for vacuolar proteolysis. Ksp1 directly associates with
Atg8 following nitrogen starvation at the interface of an
Atg8-family interacting motif (AIM)/LC3-interacting region (LIR) in Ksp1 and the LIR/AIM docking site (LDS) in
Atg8. Mutating the LDS site prevents the autophagic degradation of Ksp1. However, deletion of the C terminal canonical AIM still permitted Ssn2/Med13 proteolysis, suggesting that additional non-canonical AIMs may mediate the Ksp1-
Atg8 interaction. Ksp1 is recruited to the perivacuolar phagophore assembly site by Atg29, a member of the trimeric scaffold complex. This interaction is independent of
Atg8 and Snx4, suggesting that Ksp1 is recruited early to phagophores, with Snx4 delivering Ssn2/Med13 thereafter. Finally, normal cell survival following prolonged nitrogen starvation requires Ksp1. Together, these studies define a kinase-independent role for Ksp1 as an autophagic receptor protein mediating Ssn2/Med13 degradation. They also suggest that phagophores built by the trimeric scaffold complex are capable of receptor-mediated autophagy. These results demonstrate the dual functionality of Ksp1, whose kinase activity prevents autophagy while it plays a scaffolding role supporting autophagic degradation.Abbreviations: 3-AT: 3-aminotriazole; 17C: Atg17-Atg31-Atg29 trimeric scaffold complex; AIM:
Atg8-family interacting motif; ATG: autophagy related; CKM: CDK8 kinase module; Cvt: cytoplasm-to-vacuole targeting; IDR: intrinsically disordered region; LIR: LC3-interacting region; LDS: LIR/AIM docking site; MoRF: molecular recognition feature; NPC: nuclear pore complex; PAS: phagophore assembly site; PKA: protein kinase A; RBP: RNA-binding protein; UPS: ubiquitin-proteasome system. SAA-TF: Snx4-assisted autophagy of transcription factors; Y2H: yeast two-hybrid.