Endosomal trafficking and DNA damage checkpoint kinases dictate survival to replication stress by regulating amino acid uptake and protein synthesis

Atg6/Beclin 1 mediates autophagy and endosomal trafficking. We investigated how Atg6 influences replication stress. Combining genetic, genomic, metabolomic and proteomic approaches we found that the Vps34-Vps15-Atg6/Beclin 1-Vps38/UVRAG-phosphatydilinositol-3 phosphate (PtdIns(3)P) axis sensitizes cells to replication stress by favoring the degradation of plasma membrane amino acid (AA) transporters via endosomal trafficking and ESCRT proteins, while the PtdIns(3)P phosphatases Ymr1 and Inp53 promote survival to replication stress by reversing this process. An impaired AA uptake triggers activation of Gcn2, which attenuates protein synthesis by phosphorylating eIF2α. Mec1/Atr-Rad53/Chk1/Chk2 activation during replication stress further hinders translation efficiency by counteracting eIF2α dephosphorylation through Glc7/PP1. AA shortage-induced hyperphosphorylation of eIF2α inhibits the synthesis of 65 stress response proteins, thus resulting in cell sensitization to replication stress, while TORC1 promotes cell survival.
Our findings reveal an integrated network mediated by endosomal trafficking, translational control pathways and checkpoint kinases linking AA availability to the response to replication stress.

自噬相关蛋白6/Beclin 1（Atg6/Beclin 1）介导自噬与内体运输过程。本研究探究了Atg6对细胞复制应激的调控作用。通过整合遗传学、基因组学、代谢组学与蛋白质组学研究方法，我们发现Vps34-Vps15-Atg6/Beclin 1-Vps38/UVRAG-磷脂酰肌醇-3-磷酸（PtdIns(3)P）信号轴可通过内体运输途径与内体分选复合物（ESCRT）促进细胞膜氨基酸（amino acid, AA）转运蛋白的降解，从而使细胞对复制应激更为敏感；而磷脂酰肌醇-3-磷酸磷酸酶Ymr1与Inp53则可通过逆转该过程，提升细胞在复制应激下的存活率。氨基酸摄取受损会激活Gcn2激酶，后者通过磷酸化真核翻译起始因子2α（eIF2α）抑制蛋白质合成。复制应激过程中激活的Mec1/Atr-Rad53/Chk1/Chk2通路，可通过拮抗Glc7/蛋白磷酸酶1（PP1）对eIF2α的去磷酸化作用，进一步降低翻译效率。氨基酸匮乏引发的eIF2α过度磷酸化会抑制65种应激反应蛋白的合成，最终使细胞对复制应激更为敏感；而TOR复合物1（TORC1）则可促进细胞存活。
本研究揭示了一条由内体运输、翻译调控通路与检验点激酶介导的整合调控网络，该网络将氨基酸可用性与细胞对复制应激的响应联系起来。