ESCRT-dependent protein sorting is required for the viability of yeast clathrin-mediated endocytosis mutants
ESCRT-dependent protein sorting is required for the viability of yeast clathrin-mediated endocytosis mutants
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DOI：
10.1111/tra.12731
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发表时间：
2020-06-01
期刊：
影响因子：
4.5
通讯作者：
Wendland, Beverly
中科院分区：
文献类型：
--
作者：
Hoban, Kyle;Lux, Samantha Y.;Wendland, Beverly
内吞作用调节许多过程，包括信号传导途径、营养吸收和蛋白质周转。在网格蛋白介导的内吞作用（CME）过程中，接头与跨膜货物蛋白的细胞质区域结合，并且许多内吞接头也直接参与网格蛋白的募集。该网格蛋白相关分选蛋白家族包括酵母菌蛋白酶 Ent1/2 和 AP180/PICALM 同源物 Yap1801/2。缺乏这四个接头但表达生存所需的 epsin N 端同源 (ENTH) 结构域 (4 Delta+ENTH) 的突变菌株表现出内吞缺陷，例如质膜 (PM) 处的货物积累。这种 CME 缺陷菌株提供了理想的敏化背景，用于揭示与网格蛋白接头相互作用的细胞成分。我们使用集落分区报告分析进行了诱变筛选，以鉴定 4 个 Delta+ENTH 细胞中致命的等位基因。通过互补分离候选合成致死基因后，我们证实 VPS4 突变导致 4 Delta+ENTH 菌株无法生存。 Vps4 介导运输 (ESCRT) 依赖性运输所需的内体分选复合物的最后一步，我们发现多个 ESCRT 在 4 Delta+ENTH 细胞中也是必需的，包括 Snf7、Snf8 和 Vps36。从另一种 CME 突变体 end3 Delta 菌株中删除 VPS4，同样会导致细胞死亡，并且上调不依赖于网格蛋白的内吞途径可挽救 4 个 Delta+ENTH vps4 Delta 细胞。由于内化蛋白到细胞表面的 Rcy1 依赖性循环增加，野生型背景中 Vps4 的损失导致多种货物在 PM 处积累。此外，vps4 Delta rcy1 Delta 突变体表现出有害的生长表型。总之，我们的研究结果揭示了 ESCRT 依赖性运输中断对内吞再循环和 PM 的影响，这一影响以前未被认识到。
Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin-mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin-associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N-terminal homology (ENTH) domain necessary for viability (4 Delta+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME-deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4 Delta+ENTH cells using a colony-sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4 Delta+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)-dependent trafficking, and we found that multiple ESCRTs are also essential in 4 Delta+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3 Delta strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin-independent endocytosis pathway rescued 4 Delta+ENTH vps4 Delta cells. Loss of Vps4 from an otherwise wild-type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1-dependent recycling of internalized protein to the cell surface. Additionally, vps4 Delta rcy1 Delta mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT-dependent trafficking on endocytic recycling and the PM.